AIX-MARSEILLE UNIVERSITE

FACULTE DE MEDECINE DE MARSEILLE

ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes « URMITE ».

THESE DE DOCTORAT
présentée par

Bersissa Kumsa

MOLECULAR INVESTIGATION
OF ARTHROPODS AND VECTOR-
BORNE BACTERIA FROM ETHIOPIA

Soutenue le 02 Décembre 2014

pour obtenir le grade de Docteur d’Université d’Aix-Marseille

Spécialité : Maladies Infectieuses et Pathologies Humaines

Membres du Jury :

Prof. Philippe Parola (MD, PhD) Directeur de thèse

Prof. Sarah Bonnet (MD, PhD) Rapporteur
Prof. Pierre Marty (MD, PhD) Rapporteur
Prof. Pierre-Edouard Fournier (MD, PhD) Examinateur
 
Laboratoire d’accueil : 
Unité de Recherche sur les Maladies Infectieuses et Tropicales émergentes « URMITE » 
UM63, CNRS7278, IRD 498, Inserm 1095, Faculté de Médecine 
 
Avant Propos

Le format de présentation de cette thèse correspond à une

recommandation de la spécialité Maladies Infectieuses et
Microbiologie, à l‟intérieur du Master de Sciences de la Vie et de
la Santé qui dépend de l‟Ecole Doctorale des Sciences de la Vie de
Marseille. Le candidat est amené à respecter des règles qui lui sont
imposées et qui comportent un format de thèse utilisé dans le Nord
de l‟Europe permettant un meilleur rangement que les thèses
traditionnelles. Par ailleurs, la partie introduction et bibliographie
est remplacée par une revue envoyée dans un journal afin de
permettre une évaluation extérieure de la qualité de la revue et de
permettre à l‟étudiant de le commencer le plus tôt possible une
bibliographie exhaustive sur le domaine de cette thèse. Par
ailleurs, la thèse est présentée sur article publié, accepté ou soumis
associé d‟un bref commentaire donnant le sens général du travail.
Cette forme de présentation a paru plus en adéquation avec les
exigences de la compétition internationale et permet de se
concentrer sur des travaux qui bénéficieront d‟une diffusion
internationale.

Professeur Didier RAOULT

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 Acknowledgements
First and foremost I would like to express my sincere
gratitude to my PhD supervisor, Prof. Didier Raoult,
the Director of the laboratory and renowned scientist of
Clinical microbiology, for his continuous guidance,
suggestions and constructive comments throughout my
research. I also would like to thank him very much for
giving me a very important chance to do a PhD degree,
for his financial support (AP+HM) of my PhD research
at his laboratory.
I am very pleased and thankful to my PhD supervisor
Prof. Philippe Parola for his constant suggestions, lots
of good ideas, rigorous guidance, very professional
comments, great advice and mentor for my PhD
research.
I am greatly thankful to my 3rd PhD supervisor Dr.
Cristina Scolvoccshi for her constant suggestions, lots
of critically important comments and great advice
during my PhD research.
I would like to thank the reviewers of my PhD thesis
Prof. Sarah Bonnet and Prof Marthy Pierre for their
high quality professional advices and detailed review
during the preparation of the thesis. Their sincere
suggestions were critically important to improve the
quality of this PhD thesis.

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My heartfelt thanks go to the technicins of URMITE
laboratory particularily Annick Bernard, Laurence
Thomas, Veronique Brice, Stephanie Junoy, Marielle
Bedotto, Anne-Marie Gottrau and Jean-Michel
Berenger for their technical support during the whole of
my PhD research in the Laboratory.
Also my deepest heartfelt thanks go to the secretaries of
URMITE especially MS. Francine Simula and Valerie
Filosa for their very kind and honest help regarding
very important issues like accommodation and VISA
arrangements throughout my 3 years stay in Marseille
for PhD research at the laboratory.
I take this opportunity to thank my wife Tadelech Fite
and son Barecha Bersissa for their great love, moral
support and patience throughout my long absence from
the lovely home.
Lastly but not least my great thanks goes to my beloved
mother Dinkitu Abetu Morka, for her moral support,
great love and thinking about me for the entire of her
life.

5
TABLE OF CONTENTS

Acknowledgements 3 
 

INTRODUCTION 13
 
2. REVIEW 19 
2.1. Article 1  21 
Negelected Vector-borne Bacterial Zoonoses in East
Africa. Kumsa, B., Raoult, D., Parola, P.,
To be submitted to Acta Tropica
3. TICKS 121 
3.1. Article 2  127 
Spotted fever group rickettsiae in ixodid ticks in
Oromia, Ethiopia. Kumsa, B., Socolovschi, C., Raoult,
D., Parola, P., 2014. Ticks Tick Borne Dis
3.2. Article 3  137 
Occurrence and genotyping of Coxiella burnetii in
ixodid ticks in Oromia, Ethiopia. Kumsa, B.,
Socolovschi, C., Almeras, L., Raoult, D., Parola, P., To
be submitted to Plos Neglected tropical Diseases.
3.3. Article 4  165 
New Borrelia species detected in ixodid ticks in
Oromia, Ethiopia. Kumsa, B., Socolovschi, C., Raoult,
R., Parola, P., To be submitted to Ticks Tick Borne Dis.
3.4. Article 5  193 
Morphological, MALDI TOF Mass spectrometry and
molecular identification of ixodid tick species in
Oromia, Ethiopia. Kumsa, B., Almeras, L., Yussuf, A.,
Mediannikov, O., Raoult, D., Parola, P., To be
submitted to Veterinary Parasitollogy.

6
4. FLEAS 237 
4.1. Article 6  215 
Molecular detection of Rickettsia felis and Bartonella
henselae in dog and cat fleas in central Oromia,
Ethiopia. Kumsa, B., Parola, P., Raoult, D.,
Socolovschi, C., Am. J. Trop. Med. Hyg., 90(3), 2014,
pp. 457–462
 
5. LICE AND SHEEP KED 249 
5.1. Article 7  227 
Molecular detection of Acinetobacter species in the lice
and keds of domestic animals in Oromia Regional State,
Ethiopia. Kumsa, B., Socolovschi, C., Raoult, D.,
Parola, P., PLoS ONE 7(12): e52377.
doi:10.1371/journal.pone.0052377.
5.2. Article 8  241 
Bartonella melophagi in Melophagus ovinus (sheep
ked) collected from sheep in northern Oromia, Ethiopia.
Kumsa, B., Raoult, D., Parola, P., Socolovschi, C.,
Comparative Immunology, Microbiology and Infectious
Diseases 37 (2014) 69– 76.
 
6. CONCLUSIONS 249 
6.1. Conclusions and perspectives  251 

Bibliography 281 
 

7
Résumé
Les arthropodes comme les tiques, les puces, les poux et les moutons ked
se trouvent dans toutes les régions du monde et sont plus fréquents dans
les pays tropicaux. Les vecteurs arthropodes hématophages comme les
tiques, les puces, les poux et les moutons ked transmettent les bactéries
qui sont généralement associés aux maladies fébriles chez l'homme. Les
informations sur les bactéries transmises par les arthropodes est rare en
Ethiopie. Ainsi, l'objectif principal de cette thèse est d'accroître les
connaissances sur les bactéries à transmission vectorielle en Ethiopie par
l'exploration de leur présence dans plusieurs types d'arthropodes y compris
les tiques, les puces, les poux et les moutons ked prélevés sur des animaux
dans plusieurs départements du pays. En outre, nous avons fait une
expérience sur les nouveaux outils pour identifier les tiques par MALDI-TOF
MS protéines profilage et des méthodes moléculaires.
Notre étude visant à explorer les bactéries dans les ixodidae prélevés sur
des animaux domestiques en Éthiopie a révélé une prévalence globale
de 6% (46/767) des rickettsies de SFG, 3,8% (29/767) ADN de Borrelia et
6,4% (54/842) de C. burnetii dans différentes espèces de tiques. Africae
Rickettsia a été détecté dans Amblyommma variegatum, Am. gemma,
Am. cohaerens, Amblyomma spp. larves, nymphes Amblyomma,
Rhipicephalus (Boophilus) decoloratus et Rh. (Bo.) Nymphes de
decoloratu. La prévalence de R. africae était plus élevée dans le
département centrale et orientale que dans l'ouest. R. aeschlimannii a été
détecté dans Hyalomma rufipes marginatum et Hy. truncatum. Notre
étude est la première à détecter R. massiliae dans Rhipicephalus
Praetextatus en Ethiopie. Borrelia spp. a été détecté dans Am. cohaerens,
Am. variegatum, Am. gemma, les larves Amblyomma et les nymphes
Amblyomma. La nouvelle Borrelia spp. détectée dans les tiques
Amblyomma clustérise entre le groupe de la fièvre récurrente et le groupe
Borrelia de la maladie de Lyme alors que la Borrelia sp. détectée dans
Rhipicephalus tiques clustérise avec B. theileri / B. lonestari. Coxiella
burnetii a été détecté dans Am. gemma, Rh. pulchellus et dans d'autres
espèces de tiques, principalement en zone Borana en Oromia. Les 18
génotypes de tiques des départements du sud-est et les 20 génotypes de
tiques dans les départements centraux ont été déterminés par le typage
par séquençage multilocus (MST) de C. burneti. Après la création d'une
base de données de références fiables des spectres obtenus à partir des
jambes sur un total de 41 spécimens de tiques suivie par un test à
l'aveugle avec un total de 44 spécimens de tiques, nous avons identifié
11 espèces de tiques ixodidae par la méthode MALDI-TOF-MS. Les
méthodes d'identification morphologiques des tiques et MALDI-TOF ont

8
été confirmées par séquençage du gène de l'ARNr 12S d’où une
identification moléculaire des différentes espèces de tiques.
L'étude pour étudier les bactéries dans 303 puces prélevés sur des chiens
et des chats domestiques en Ethiopie qui ont été identifiés comme étant
morphologiquement Ctenocephalides felis felis, Ctenocephalides canis,
Pulex irritans et Echidnophaga gallinacé montré Rickettsia felis dans 21%
des puces, principalement dans Ctenocephalides felis, avec une
semblable prévalence dans les puces de chiens et de chats. Notre étude
est la première à signaler Bartonella henselae en C. felis de l'Ethiopie.
Notre étude sur les bactéries des poux et des moutons ked (Melophagus
ovinus) a révélé la présence d'Acinetobacter spp. dans M. ovinus,
Heterodoxus spiniger, Bovicola ovis et Linognathus vituli. La séquence du
gène rpoB partiel a révélé la présence de A. soli, A. lowffii, A. Pitti et
3 nouveaux Acinetobacter spp. dans les poux et Keds. L'identification
moléculaire des poux à l'aide d'une analyse du gène 18S a confirmé les
méthodes morphologiques d'identification des poux. Bartonella
melophagi a été identifié par une PCR standard, suivi par un séquençage
du fragment de la gltA et gène rpoB chez M. ovinus.
La présente étude a contribué à accroître les connaissances actuelles en
Ethiopie en donnant la répartition géographique des rickettsies du groupe
de la fièvre boutonneuse avec les espèces de tiques associées, la
première preuve moléculaire de détection de R. massiliae dans
Rhipicephalus Praetextatus, de B. henselae dans C. felis, B . melophagi
dans M. ovinus, nouvelle Borrelia spp. dans des tiques Amblyomma,
nouveau Acinetobacter spp. dans des poux et KED et première preuve de
génotypes de C. burnetii chez les tiques en Ethiopie et a introduit un
nouveau procédé d'identification des tiques par la technique MALDI TOF
MS.
Dans l'ensemble, nos résultats alerte les médecins en charge des patients
avec fièvre d'étiologie inconnue en Ethiopie et ceux qui se soucient de
voyageurs en provenance de l'Ethiopie à prendre en compte la présence
de plusieurs espèces zoonotiques à transmission vectorielle de bactéries, y
compris SFG rickettsies, C. burnetii, R. felis, B. henselae et B. melophagi
comme agents pathogènes potentiels. Cependant, les études futures sont
nécessaires pour traiter l'isolement, la culture et les génotypes de ces
bactéries provenant de différentes sources et de déterminer l'importance
en santé publique de ces bactéries et la compétence vectorielle des
arthropodes en Ethiopie.

Mots-clé: transmission vectorielle bactéries, les arthropodes, les

humains, les animaux domestiques, Oromia, en Ethiopie

9
Abstract

Arthropods such as ticks, fleas, lice and sheep ked are found in
every part of the world and are more common in tropical countries.
Haematophagous arthropod vectors such as ticks, fleas, lice and
sheep ked transmit bacteria that are usually associated with febrile
illnesses in humans. Information on vector-borne bacteria
transmitted by arthropods is scarce in Ethiopia. Thus, the main
objective of this thesis was to increase the present understanding on
vector-borne bacteria in Ethiopia by exploring their presence in
several types of arthropods including ticks, fleas, lice and sheep ked
collected from domestic animals in several districts in the country. In
addition, we made an experiment on emerging tools to identify
ticks by MALDI-TOF MS protein profiling and molecular methods.
Our study to explore bacteria in ixodid ticks collected from
domestic animals in Ethiopia revealed an overall prevalence of 6%
(46/767) SFG rickettsiae, 3.8% (29/767) Borrelia DNA and 6.4%
(54/842) C. burnetii in different tick species. Rickettsia africae was
detected in Amblyommma variegatum, Am. gemma, Am.
cohaerens, Amblyomma spp. larvae, Amblyomma nymphs,
Rhipicephalus (Boophilus) decoloratus and Rh. (Bo.) decoloratu
nymphs. The prevalence of R. africae was higher in the central and
eastern than in the western districts. R. aeschlimannii was detected
in Hyalomma marginatum rufipes and Hy. truncatum. Our study is
the first to detect R. massiliae in Rhipicephalus praetextatus in
Ethiopia. Borrelia spp. was detected in Am. cohaerens, Am.
variegatum, Am. gemma, Amblyomma larvae and Amblyomma
nymphs. The new Borrelia spp. detected in Amblyomma ticks cluster
in between the recurrent fever and the Lyme disease group
borreliae whereas the Borrelia sp. detected in Rhipicephalus ticks
clusters with B. theileri/B. lonestari. Coxiella burnetii was detected in
Am. gemma, Rh. pulchellus and in other tick species mainly in
Borana zone in Oromia. Genotype 18 in ticks from south-eastern
districts and genotype 20 in ticks from central districts were
determined by multi-spacer sequence typing (MST) of C. burneti.
After creating a reliable reference database of spectra obtained
from the legs of a total of 41 tick specimens followed by blind test
with a total of 44 tick specimens we correctly identified 11 ixodid
ticks species by MALDI-TOF-MS method. The morphological and

10
MALDI-TOF tick identification methods were confirmed by
sequencing of the 12S rRNA gene molecular identification of tick
species.
The study to investigate bacteria in 303 fleas collected from
domestic dogs and cats in Ethiopia that were morphologically
identified as Ctenocephalides felis felis, Ctenocephalides canis,
Pulex irritans and Echidnophaga gallinacean showed Rickettsia felis
in 21% of fleas, mainly in Ctenocephalides felis, with a similar
prevalence in fleas from dogs and cats. Our study is the first to
report Bartonella henselae in C. felis from Ethiopia.
The study to investigate bacteria in lice and sheep ked
(Melophagus ovinus) revealed Acinetobacter spp. in M. ovinus,
Heterodoxus spiniger, Bovicola ovis and Linognathus vituli. Partial
rpoB gene sequence revealed A. soli, A. lowffii, A. pitti and 3 new
Acinetobacter spp. in the lice and keds. Molecular identification of
lice using an 18S rRNA gene analysis confirmed the morphological
methods of lice identification. Bartonella melophagi was identified
by standard PCR followed by sequencing of fragments of the gltA
and rpoB genes in M. ovinus.
The present study contributed to increase the current knowledge in
Ethiopia by providing updated geographical distribution of spotted
fever group rickettsiae with associated tick species, provided first
molecular evidence of R. massiliae in Rhipicephalus praetextatus,
first report of B. henselae in C. felis, B. melophagi in M. ovinus, new
Borrelia spp. in Amblyomma ticks, new Acinetobacter spp. in lice
and ked of animals and first report of C. burnetii genotypes in ticks
in Ethiopia and introduces new method of tick identification by
MALDI TOF MS technique.
Overall, our findings alert physicians managing patients with fever of
unknown aetiology in Ethiopia and those who care for travellers
from Ethiopia to consider the presence of several vector-borne
zoonotic species of bacteria including SFG rickettsiae, C. burnetii,
R. felis, B. henselae and B. melophagi as potential causative agents.
However, future studies are needed to address the isolation, culture
and genotypes of these bacteria from different sources and
determine the public health significance of these bacteria and
vector competence of arthropods in Ethiopia.

Keyword: Vector-borne bacteria, arthropods, humans, domestic

animals, Oromia, Ethiopia

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INTRODUCTION

13
 
14
Introduction

Arthropods are one of the major causes of health problems in

both humans and animals worldwide (8,13,32). Ticks, fleas,

lice and sheep ked are found in every part of the world and

infest every class of wild and domestic animals and humans

(13,32). In tropical countries arthropods are very common

and widely distributed due to the presence of very conducive

climate and poor general hygiene and sanitation, lack of

prevention and control (41). Arthropods act as vectors and

reservoirs for several agents of emerging and re-emerging

infectious diseases of medical and veterinary importance

(3,6,7,27,34). Recent investigations indicate worldwide

escalating problems from arthropods and vector-borne

diseases caused by pathogens they transmit (8,9).

In Ethiopia the environment is very conducive for the

survival, development and reproduction of arthropods and

the country is endowed with diversified species of these

15
creatures (17). Arthropods such as ticks, fleas, lice and sheep

ked are common and widely distributed throughout all

regions in Ethiopia. Humans contact with arthropods is

favoured by several factors like 84% of the populations are

involved in agriculture and thus farmers and their family

members interact during their daily life activities with

ectoparasite-infested animals, wild animals and vegetation

which is the natural habitats of arthropods (18). Furthermore,

majority of health care institutions are poorly equipped and

thus confirmatory diagnosis isn‟t conducted for fever of

unknown aetiology which is the common symptom of most

vector-borne bacterial diseases thus misdiagnosis and

inappropriate treatment is a common problem in the country

(17).

Most of the previous studies on vector-borne bacterial

diseases in Ethiopia focus on infectious pathogens

transmitted by human body and head louse-borne diseases

16
like relapsing fever, trench fever and epidemic fever

(1,4,5,15,30,36,38,44,45). On the other hand other

arthropods such as ticks, fleas, sheep ked and other species

of arthropods have received less attention and consideration

by health professionals who don‟t have sufficient awareness

about these arthropods in the country (18). For instance,

most of the studies on the role of ixodid ticks as vectors of

human pathogens in Ethiopia were conducted before the

discovery of well-advanced molecular tools during the 1950s

and 60s (28,29) and generally ticks are considered to have

more veterinary significance than public health importance.

Also, the medical importance of other arthropods like fleas,

lice and sheep ked is not properly addressed in Ethiopia (17-

19). Furthermore, joint and collaborative works on common

issues for both veterinary and human health professionals to

mitigate vectorized zoonoses is left as unaddressed problem

in Ethiopia. All these aforementioned facts prompted the

17
investigators of the present study to explore the diversity of

bacteria that are able to be transmitted by blood feeding

arthropods such as ticks, fleas, lice and sheep ked collected

from domestic animals in Ethiopia. Thus, the main objective

of this study was to investigate vector-borne bacteria in ticks,

fleas, lice and sheep ked collected from animals in 9

different districts in Ethiopia using molecular tools. In

addition, we aimed to use emerging tools such as MALDI-

TOF MS protein profiling methods for identification of ticks

preserved in 70% ethanol in Ethiopia. In the first part of this

thesis a review on vector-borne bacterial diseases in east

Africa is presented. In this review we compiled the available

information about vector-borne bacterial diseases in east

African countries so as to update the current understanding

and the existing knowledge in Ethiopia as well as other

nearby east African countries.

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2. REVIEW

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20
2.1. Article 1
 

Negelected Vector-borne Bacterial

Zoonoses in East Africa.

Kumsa, B., Raoult, D., Parola, P.,

To be submitted to Acta Tropica

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22
 NEGLECTED VECTOR-BORNE

BACTERIAL ZOONOSES IN EAST AFRICA

Bersissa Kumsa, Didier Raoult, Philippe Parola

Aix Marseille Université,

URMITE, UM63, CNRS 7278, IRD 198,

Inserm 1095, 13005 Marseille, France.

URMITE, UMR CNRS 7278, IRD 198, INSERM U1095

Faculté de Médecine
27 Bd Jean Moulin
13385 Marseille cedex 5 France
Tel.: +33 (0)4 91 32 43 75
Fax: + 33 (0)4 91 38 77 72

To be submitted to Acta Tropica

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ABSTRACT
Vector-borne bacterial diseases cause significant morbidity
and mortality in both humans and animals around the world
and affect the global economy. They are transmitted during
the blood feeding activities of hematophagous arthropod
vectors such as ticks, fleas, lice and flies. Febrile illness is
one of the most common clinical symptoms associated with
vector-borne zoonotic bacterial infection and is the major
cause of morbidity and mortality in east African countries
but too often, tests are not available to determine the causes,
leading to misdiagnosis and inappropriate treatment. Due to
the widespread occurrence of malaria in east African
countries there is great confusion between malaria and
malaria mimicking treatable vector-borne bacterial infections
associated with febrile illnesses. Tick-borne bacterial
diseases (spotted fever rickettsiosis, typhus group
rickettsiosis, tick-borne relapsing fever borreliosis,
bartenolosis and Q fever), louse-borne bacterial diseases
(epidemic relapsing fever, trench fever, epidemic typhus and
Acinetobacter baumannii) and flea-borne bacterial zoonoses
(flea-borne spotted fever, murine typhus, cat scratch disease
and plague) are common in east African countries but poorly
investigated. Even though recent investigations indicate

25
escalating problems associated with arthropod vectors and
vector-borne diseases across other parts of the world
relatively little attention has been given to arthropod
transmitted zoonotic bacterial diseases in east African
countries. Thus the main objective of this review is to update
our current understanding on the geographical distribution,
epidemiology, clinical aspects, public health issues,
treatment and show research gaps on arthropod transmitted
zoonotic bacterial diseases in east African countries so as to
provide better understanding and draw the attention of
researchers, clinicians, microbiologists, scientific and
medical community working in this part of the world.

Keyword: Rickettsia, Borrelia, Bartonella, Coxiella burnetii

ticks, lice, fleas, humans, domestic animals, zoonotic
bacteria, vector-borne, east Africa

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TABLE OF CONTENTS
1. INTRODUCTION

2. Major Arthropod vectors of zoonotic bacteria in East

Africa

3. Tick-borne bacterial diseases

4. Louse-borne bacterial diseases

5. Flea-borne bacterial diseases

6. Mite-borne bacterial diseases

7. Other vector-borne bacterial diseases

8. Vector-brone bacterial diseases in travellers returning

from East Africa

9. Methods of detection of bacteria in Arthropods

10. Diagnosis of vector-borne bacteria in humans and

Animals

11. Treatment of vector-borne bacterial diseases

12. CONCLUSION

27
 
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1. INTRODUCTION

Arthropods such as ticks, fleas, lice and flies inhabit every

part of the world and infest every class of wild and domestic
animals and humans (Eisen and Gage, 2012; Jongejan and
Uilenberg, 2004). Arthropods are important for the
maintenance and transmission of many pathogens including
several species of bacteria, viruses, protozoa and helminths
causing diseases in humans, pets and domestic animals
worldwide (Billeter et al., 2008). Vector-borne diseases
cause significant morbidity and mortality in both humans
and animals around the world and affect the global economy,
representing approximately 17% of the burden of all
infectious diseases and also cause millions of dollars in
losses to the livestock industry (Dantas-Torres et al., 2012).
For instance, 7 of the 10 diseases targeted by the WHO
Special Programme for Research and Training in Tropical
Diseases, and 7 of the 17 diseases classified as neglected
tropical diseases (NTD) by WHO, are transmitted by
arthropods .
Recent investigations indicate escalating problems
associated with arthropod vectors and vector-borne diseases
caused by the pathogens they transmit across the countries of

29
the world due to expanding vector populations, global
warming, increased contacts between humans, animals and
vectors due to enhanced international commerce, increased
and more rapid global transport, human and animal
population dynamics, and emerging drug resistance in both
vectors and pathogens (Dantas-Torres et al., 2012; De la
Fuente and Estrada-Pena, 2012).
East Africa as defined by the United Nations comprises
countries including Djibouti, Eritrea, Ethiopia, Somalia,
South Sudan, Tanzania, Kenya, Uganda, Rwanda, Burundi,
Zambia, Zimbabwe, Malawi, Mozambique, Madagascar,
Mauritius, Comoros, Seychelles, Mayotte and Reunion
(Fig. 1). East Africa is characterized by very huge human,
domestic and wild animal populations and agriculture is the
backbone of the economy of the countries hence the daily
activities of the farming community favours close contact
between humans, animals and their ectoparasites (Cutler et
al., 2010a). The region is known for diverse vegetation and
climates very suitable and conducive for reproduction and
maintenance of all stages of arthropod vectors. Despite these
global facts, little information on vector-borne zoonotic
bacterial diseases is emanating in general from Africa and in
particular very scanty information from east African

30
countries (Mediannikov et al., 2013b; Parola, 2011).
Moreover, poor facility of health institutions that usually
lack resources for rigorous diagnostic tests to determine the
underlying cause of febrile illnesses is additional problem.
As a result misdiagnoses and mistreatment of common
diseases in east Africa is common due to great confusion
between malaria and malaria mimicking treatable vector-
borne bacterial infections that causes febrile illnesses in
infected patients (Mediannikov et al., 2013b). Recently,
there is growing number of studies reporting death of
humans in east African countries from vector-borne zoonotic
bacterial diseases due to misdiagnosis and mistreatment for
malaria and other mimicking diseases (Rutherford et al.,
2004). Most vector-borne zoonotic bacterial diseases are
commonly manifested by nonspecific febrile illnesses that
are difficult to diagnosis clinically, thus their clinical signs
and symptoms overlap with those of widely distributed
diseases such as malaria in east Africa, which may lead to
inaccurate diagnoses (Mediannikov et al., 2013b; Parola and
Raoult, 2001; Raoult and Roux, 1999). The majority of
recent research and reviews on vector-borne zoonotic
bacterial diseases focus and concentrate on USA, Europe,
and Asian countries, south, west and North Africa (Reif and

31
Macaluso, 2009). However, comparatively little attention has
been given to arthropod transmitted zoonotic bacteria in east
African countries (Cutler, 2010). Thus, the main objective of
this review is to update our present understanding on the
geographical distribution, epidemiology, clinical aspects,
public health issues, treatment and to show research gaps on
arthropod transmitted zoonotic bacterial diseases in east
African countries.

32
2.0. MAJOR ARTHROPOD VECTORS OF
ZOONOTIC BACTERIA IN EAST AFRICA

Arthropod vectors known to maintain and transmit zoonotic

bacteria are members of insects and arachnids and display
various types of association with their hosts such as obligate
to facultative, permanent to intermittent, superficial to
subcutaneous types of associations (Adler et al., 2011). The
association between the arthropod vectors and infested hosts
including humans results in a wide range of pathogenic
effects (Chomel et al., 2009). The feeding activities of
arthropods may cause direct damage to skin and other sub-
cutaneous tissues, inflammation, paralysis, exsanguinations
leading to significant blood loss, pruritis, erythema,
excoriation, papules, lichenification, scale and crusting and
self-trauma (Parola and Raoult, 2001). Wounds may be
subject to secondary infestation or bacterial infection. The
salivary and faecal antigens produced by arthropods during
feeding can stimulate immune responses in some individuals
leading to hypersensitivity (Liu and Bonnet, 2014).
Importantly, some arthropods also act as vectors and
reservoirs of bacteria, protozoa, viruses, cestodes and
nematodes (Liu and Bonnet, 2014; Mediannikov and

33
Fenollar, 2014). Close contact with domestic animals and
their ectoparasites is reported as the important risk of
infection with many vector-borne zoonotic bacteria for
humans (Angelakis and Raoult, 2010). The most common
arthropod vectors known to transmit zoonotic bacteria to
humans in east African countries are briefly described as
follows:

2.1. Ticks
Ticks are one of the most important arthropods responsible
for health problems in both humans and animals (Nicholson
et al., 2010; Parola and Raoult, 2001). They are vectors of
several agents of emerging and re-emerging infectious
diseases of medical and veterinary importance (Baneth,
2014). Ticks are second only after mosquitoes as vectors of
human infectious diseases (Jongejan and Uilenberg, 2004;
Parola and Raoult, 2001). Ticks are vectors and reservoirs of
zoonotic bacteria such as Borrelia spp., spotted fever and
typhus group Rickettsia spp., Ehrlichia spp., Coxiella
burnetii, Tularaemia spp. and Bartonella spp. which cause
emerging zoonoses in humans (Irwin and Jefferies, 2004;
Otranto et al., 2009; Pfaffle et al., 2013). Recent reports
show the growing health risks associated with ticks and tick-

34
borne diseases in both humans and animals (Dantas-Torres et
al., 2012; De la Fuente and Estrada-Pena, 2012; Piesman and
Eisen, 2008). To date, almost 900 tick species have been
described. These are divided into three families: the
Argasidae or soft ticks (191 species), the Ixodidae or hard
ticks (701 species) and the Nuttalliellidae consisting of only
one species, Nuttalliella namaqua (Barker and Murrell,
2004). The generic taxonomy of argasid ticks is currently
highly controversial with different authorities listing between
four and 10 valid genera. The genera Argas, Ornithodoros
and Carios, if recognized, are all of medical importance for
humans. For example, various relapsing fever Borrelia spp.
are transmitted by a variety of Ornithodoros spp. (Estrada-
Pena and Jongejan, 1999; Mediannikov and Fenollar, 2014).
The Ixodidae consist of 13 current genera of which the most
important for humans are Amblyomma, Dermacentor,
Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus
(Estrada-Pena and Jongejan, 1999; Parola and Raoult, 2001).
Ticks can be identified to the family, genus and species level
with use of numerous taxonomic keys that are available from
different regions of the world (Hoogstraal, 1956; Walker,
2003a). However, identification of larvae and nymphs of tick
is difficult and may require entomological skills. Molecular

35
methods such a mitochondrial markers (mitochondrial 12S
and 16S ribosomal DNAs, cytochrome oxidase subunit 1
[COX1], cytochrome b) are usually easy to amplify and
sequence, but the degree of intraspecies difference is rarely
enough to distinguish the phylogenetically close species but
are important to improve identification of ticks (Beati and
Keirans, 2001; Beati et al., 2012; Mediannikov and Fenollar,
2014). Nuclear markers (18S, 28S, internal transcribed
spacers 1 and 2) have also been used, but only entire
mitochondrial genome seems to be enough for phylogeneitc
studies (Burger et al., 2014). In addition to the technical and
logistical drawbacks of PCR assays, this approach is further
limited by the availability of gene sequences in genetic
databases. However, currently there is no PCR assay that can
distinguish tick species, and ideal PCR primer pairs that can
amplify the relevant gene fragments are not available but are
expected to be used more widely in the future particularly for
the differentiation of closely related species. Protein
profiling by matrix-assisted laser desorption ionization–time
of flight mass spectrometry (MALDI-TOF MS) is now
commonly used as routine identification method of
microorganisms in clinical microbiology (Karger et al.,
2012). There is growing reports on the use of MALDI-TOF

36
MS for the differentiation of arthropods from different
countries of the world. Recently, MALDI-TOF MS study has
been used to identify ticks in Germany (Karger et al., 2012)
and France (Yssouf et al., 2013) and is considered as
promising alternative method for the identification of ticks.
Even though molecular and MALDI-TOF MS studies on
ticks of east African countries are not available, the species
of ticks reported by using taxonomic keys and their
associated zoonotic bacterial diseases is compiled on table 1.
2.2. Lice
Lice are also one of the very important ectoparasites of
humans and animals worldwide. Lice are small, wingless
insects, obligate permanent ectoparasites and can‟t survive
more than few days off their hosts and are highly host
specific (Barker, 1994). Lice belong to the order Phthiraptera
which is divided into two major groups: Anoplura (the
hematophagous sucking lice of placental mammals) and
Mallophaga (the chewing or biting lice of birds, marsupials
and placental mammals) (Raoult and Roux, 1999). Although,
the Mallophaga group is likely paraphyletic, it is widely
agreed that sucking and chewing lice originated from a
common non-parasitic ancestral group closely related to the
order Psocoptera (book lice and bark lice) (Barker, 1994).

37
Humans can be infested with two types of lice (Anoplura):
Pediculus on the head (Pediculus capitis) and/or body
(Pediculus humanus) and Pthirus (in the pubic area Pthirus
pubis) (Raoult and Roux, 1999). Both anopluran and
mallophgan lice infest domestic farm animals and
companion animals; however, birds are infested only by
mallophagan lice species. Pediculus humanus (human body
louse) is the well established vector and known to transmit
Rickettsia prowazekii, Borrelia recurrentis, and Bartonella
quintana the causative agents of epidemic typhus, relapsing
fever, and trench fever, respectively (Badiaga and Brouqui,
2012). Recently Borrelia recurrentis and Bartonella
quintana are reported from Pediculus capitis in some
countries of the world including east African countries
(Badiaga and Brouqui, 2012; Boutellis et al., 2013).
Furthermore, Acinetobacter baumannii DNA have been
reported from both head and body lice in east African
countries (Badiaga and Brouqui, 2012; Gillespie et al.,
2009).
In domestic animals pediculosis (dermatitis due to lice) is
more common in cattle than any other species of domestic
animals (Kumsa et al., 2012a; Kumsa et al., 2012b). For
instance, Bovicola (Damalina) bovis) (chewing lice),

38
Linognathus vituli (the long-nosed cattle louse), Solenopotes
capillatus (the little blue cattle louse), Haematopinus
eurysternus (the short-nosed cattle louse), Haematopinus
quadripertusus (the cattle tail louse in tropics) and
Haematopinus tuberculatus (the buffalo louse) are the
species of lice known to infest cattle in different countries of
the world (Kumsa et al., 2012b). Likewise, 4 species of lice
on sheep (Kumsa et al., 2012a), 3 species on goats, 2 species
on equines, 1 species on pigs, 3 species in dogs and 1 species
on cats are documented on domestic animals around the
world (Kumsa et al., 2012a; Kumsa et al., 2012b). There is
little information on the role of lice of domestic animals as
vectors of zoonotic bacterial diseases from east African
countries. The only available information on vector-borne
bacteria in lice of domestic animals is the recent report of
Acinetobacter species in lice of cattle, sheep and dogs in
Ethiopia (Kumsa et al., 2012b). Details on the common
species of lice on humans, domestic animals and the
associated zoonotic bacteria is presented on table 2.

39
2.3. Fleas
Fleas (order Siphonaptera) are holometabolous obligate
blood-feeding ectoparasites of birds and mammals; the
immature stages (egg, larva and pupa) are found in burrows
or nests, whereas adult fleas are usually found on animal
hosts (Dobler and Pfeffer, 2011). Siphonaptera (fleas)
currently comprising 246 genera and approximately 2575
described taxa. Fleas are laterally compressed, wingless
insects that range from 1 to 10 mm in length (Traversa,
2013). They usually have small and shield shaped head.
Fleas are found in all continents including Antarctica and
they inhabit wide range of habitats and hosts, from equatorial
deserts through tropical rainforests to the arctic tundra
(Traversa, 2013).
Fleas are important vectors and reservoirs of several
emerging or re-emerging infectious diseases of great medical
and veterinary importance around the world (Dobler and
Pfeffer, 2011). The discovery that fleas were capable of
transmitting Yersinia pestis, the causative agent of plague
and later Rickettsia typhi, the causative agent of murine
typhus, stimulated a frenzy of flea studies in the early 20th
century (Eisen et al., 2012). Fleas have played a historic role
in human plagues, including the „Black Death‟ (bubonic

40
plague), which is transmitted by rodent fleas and is estimated
to have caused the deaths of a third of the world‟s population
during the Middle Ages (Eisen and Gage, 2012). Also
Ctenocephalides felis is a known vector for Bartonella
henselae, Bartonella clarridgeiae, and Rickettsia felis which
cause cat scratch disease, endocarditis and cat flea typhus in
humans, respectively. Xenosylla cheopis is the principal
vector of Rickettsia typhi the causative agent of murine
typhus and Yersinia pestis the causative agent of plague in
humans (Eisen and Gage, 2012).
Fleas are normally considered as ectoparasites of dogs and
cats, however, they infest and readily feed on other species
of domestic animals and humans (Dobler and Pfeffer, 2011).
Ctenocephalides felis felis (cat flea), Ctenocephalides canis
(dog flea), Pulex irritans (human flea), Echidnophaga
gallinacea (sticktight poultry flea) and Xenopsylla cheopis
(rat flea) are the most commonly reported species on dogs
and cats worldwide (Dobler and Pfeffer, 2011). Tunga
penetrans (called jigger or sand flea) is the species found
commonly in humans and also reported on dogs in all east
African countries (Franck et al., 2003). The females of the T.
penetrans penetrate the skin of the host to the basal layer of
the corium where they feed on blood and tissue exudates

41
produced by the host‟s inflammatory response. Only female
T. penetrans fleas cause tungiasis. The infestation of the skin
may cause severe damage by inflammatory response or by
bacterial super-infection (Mazigo et al., 2012). Domestic
dogs and cats play a peculiar role as bridging hosts for fleas
of different wild animals, domestic animals and humans, as
they will come into contact with different animals during
their hunting behaviour and therefore acquire the fleas of
different animals (Dobler and Pfeffer, 2011). Several flea
species are known to feed on humans and domestic animals
in east African countries (Kumsa and Mekonnen, 2011).

42
3. TICK-BORNE ZOONOTIC BACTERIA

3.1. Spotted fever group (SFG) Rickettsia species

Rickettsiae are small, obligate intracellular, short rod, Gram-

negative bacteria belonging to the genus Rickettsia, the
family Rickettsiaceae and the order Rickettsiales (Raoult and
Roux, 1997). Members of the genus Rickettsia could be
classified into spotted fever group (SFG) rickettsiae, typhus
group rickettsiae, the Rickettsia bellii group and the
Rickettsia canadensis group (Parola et al., 2013). The
number of species of the genus Rickettsia has kept increasing
in recent years due to the use of improved cell culture
techniques and molecular assays for isolation and
characterization of this bacterium (Parola et al., 2013).
Currently, the genus Rickettsia comprises 31 species that
cause diseases in vertebrate hosts, including humans,
domestic animals, birds, and wildlife (Merhej et al., 2014;
Parola et al., 2013). The SFG rickettsiae constitute species
which cluster into phylogenetically distinct clade and are
predominantly transmitted by ticks and cause tick-borne
rickettsial diseases in humans (Socolovschi et al., 2009). In
ticks Rickettsia species infect and colonize many organs
particularly the salivary glands and ovaries of adult females

43
(Parola et al., 2013; Raoult and Roux, 1997) as demonstrated
for some species. This favours transovarial and transstadial
transmission in some tick species and to transmit rickettsiae
to vertebrate hosts during blood feeding (Parola et al., 2013).
SFG rickettsiae are mainly associated with ticks, have an
optimal growth temperature of 32 ◦C, a guanosine plus
cytosine (G+C) content between 32% and 33%, can
polymerize actin and thereby enter the nuclei of host cells
and cause spotted fever in humans (Merhej and Raoult,
2011; Parola et al., 2013). In humans, spotted fever
rickettsiae induces symptoms that generally include fever,
headache, myalgia, rash, local lymphadenopathy, and an
eschar at the site of the tick bite, which might be useful in
diagnosis (Raoult and Roux, 1997).
The public health significance of rickettsial diseases among
indigenous east African populations is generally
underdiagnosed and frequently confused with malaria
(Mediannikov et al., 2013b; Parola, 2011). For instance,
infection with Rickettsia africae, the most common SFG
rickettsae in sub-Saharan Africa, is usually detected in
European and North American travellers returning home
from Africa than native nations (Parola et al., 2013). From
east African countries so far six Rickettsia species

44
pathogenic for human have been reported in ticks and /or
humans presented as follows.

3.1.1. Rickettsia africae

Rickettsia africae, the causative agent of African tick bite
fever (ATBF), was officially discovered in 1996 (Raoult and
Roux, 1997). R. africae was detected in Amblyomma
hebraeum a South African bont tick, which is a known as the
principal vector and reservoir of this bacterium in South
Africa (Parola et al., 2013). In other sub-Saharan African
countries Amblyomma variegatum, the tropical bont tick is
generally regarded as the principal vector and reservoir of
R. africae. Some studies reported very high infection rate
reaching 100% in Am. variegatum ticks in sub-Saharan
African countries (Parola et al., 2013). To date, R. africae
has been detected in ticks and/or humans in 22 sub-Saharan
African countries (Parola et al., 2005b; Parola et al., 2013).
Most of these reports were from west, central and North
African countries; however, this is despite the fact that
Amblyomma ticks are more common in east African
countries, for instance, Amblyomma species like
Am. cohaerens, Am. gemma and Am. lepidum are found only
in east African countries (Walker, 2003a). Recently,

45
R. africae has been detected in Rhipicephalus species ticks
but usually with very lower prevalence (Parola et al., 2013;
Raoult and Roux, 1997).
In east African countries R. africae has been detected in
5 Amblyomma species including in 15.8% Am. variegatum
(Macaluso et al., 2003) and in Am. variegatum,
Am. hebraeum and Am. gemma ticks (Mutai et al., 2013) in
Kenya, in 67% (26/39) (Lorusso et al., 2013) and 97.1%
(Nakao et al., 2013) Am. variegatum in Uganda, 1/13 Am.
variegatum Burundi (Parola et al., 2001), in 100% (10/10)
Am. variegatum collected from cattle in Juba in South Sudan
(Morita et al., 2004), in Amblyomma lepidum ticks (20%) in
Djibouti, in 10.17% Am . lepidum and ½ Am. variegatum
(Mura et al., 2008) and in 30.2% (16/53) Am. variegatum,
28.6% (12/42) Am. gemma , 0.8% (1/119) Am. cohaerens in
Ethiopia (Kumsa et al., 2014b) and in 90% (60/67) of
Amblyomma variegatum in the Union of the Comoros
(Yssouf et al., 2014). R. africae has also been detected in
Rhipicephalus species ticks with low prevalence in some east
African counties: in 0.7% (1/139) Rh. (Bo.) decoloratus and
25% (1/4) nymphs of Rh. (Bo.) decoloratus in Ethiopia
(Kumsa et al., 2014b) and in Rh. appendiculatus,
Rh. pulchellus and Rh. annulatus in Kenya (Macaluso et al.,

46
2003; Mutai et al., 2013) and in 1% (1/92) Rhipicephalus
appendiculatus and in 2.7% (8/296) of Rhipicephalus
(Boophilus) microplus in the Union of the Comoros (Yssouf
et al., 2014). However, the role of Rhipicephalus species
ticks in the epidemiology of ATBF is not yet determined and
it is suggested that the lower prevalence reflects the bacteria
is acquired during cofeeding with Amblyomma ticks or from
bacteraemic animals (Parola et al., 2013).
In humans SFG Rickettsia spp. was confirmed in 36/450
(8%) in hospitalized febrile patients in Tanzania (Prabhu et
al., 2011) and 2.9% (3/102) SFG Rickettsia DNA was
detected in dried thin blood smears prepared to test malaria
in children with febrile illnesses at Soddo Christian Hospital
in Wolaitta Soddo in Ethiopia (Aarsland et al., 2012), are the
available reports from east African countries. Despite the
very high abundance and prevalence of Amblyomma spp. and
other ticks and the highly suitable climates in east African
countries little information is available on R. africae
infection in humans (Parola and Raoult, 2001). However,
comparatively greater numbers of studies have been made in
west, central, south and North African countries on
R. africae in both ticks and humans than in eastern African
countries due to the involvement and construction of modern

47
laboratories by many European countries like France, UK
and Germany (Parola et al., 2013).

3.1.2. Rickettsia conorii subsp. conorii

Rickettsia conorii conorii, the agent of Mediterranean
spotted fever (MSF) (also called Boutonnneuse fever), is
endemic in North Africa and southern Europe and is one of
the oldest-recognized vector-borne infectious diseases.
R. conorii conorii was isolated in Tunisia in 1932 (Parola et
al., 2013). It is endemic in countries in the Mediterranean
region. Rhipicephalus sanguineus, the brown dog tick, is the
vector and possibly a reservoir of the pathogen (Parola et al.,
2005a). It has been commonly detected in ticks and human
blood in endemic north and West African countries like
Algeria, Tunisia, Morocco and Senegal. Dogs are considered
to be important transport hosts, capable of carrying infected
ticks close to their owners (Parola et al., 2013). In east
African countries it has been detected in Rhipicephalus spp.
(Rhipicephalus mushamae and Rhipicephalus simus) ticks
from dogs in Zimbabwe, Kenya and Somalia and in
Haemaphysalis punctaleachi ticks (1/9) from a dog in
Uganda and in human blood in one patient in Kenya. In
Zambia 16.7% seroprevalence of antibodies against

48
R. conorii was reported (Okabayashi et al., 1999). In human
patients the typical form of the disease is manifested by
clinical symptoms like high fever, flulike symptoms, a local
necrotic inflammation with a black crust called an eschar
(the “tache noire”) at the tick bite site and a maculopapular
rash (Parola et al., 2005a).

3.1.3. Rickettsia conorii subsp. israelensis

Rickettsia conorii subsp. israelensis, the causative agent of
the Israeli spotted fever (ISF), has been found in Southern
Europe, the Middle East and in Tunisia. In humans it
resembles the severe form of MSF (Raoult et al., 2001a). It
was first reported in 1946 in Israel. It is transmitted
transstadially in Rhipicephalus sanguineus from nymphs to
adults. Dogs have been suggested to be a competent
reservoir of R. conorii subsp. israelensis. The only report in
east African countries is the recent detection in Amblyomma
ticks in Kenya (Mutai et al., 2013).

3.1.4. Rickettsia aeschlimannii

Rickettsia aeschlimannii was first isolated in Morocco in
1992 from Hyalomma marginatum marginatum and was
characterized as a new SFG rickettsia in 1997 (Parola et al.,

49
2005a). The first disease in human was described in two
patients returning from Morocco to France showing MSF-
like illness. Hyalomma spp. ticks are considered as the main
vectors and reservoirs and transmit both transstadially and
transovarially to their offspring. It has been detected in
Hy. marginatum rufipes, Hy. marginatum marginatum,
Hy. aegyptium, Hy. dromedarii Hy. impeltatum and
Hy. truncatum mostly in north and western African countries
in Niger, Mali, Algeria, Egypt and Senegal (Parola et al.,
2013). To date, this bacterium has been detected in 8 sub-
Saharan African countries. In east African countries
R. aeschlimannii has been detected in Hyalomma spp. in
Kenya (Mutai et al., 2013), Sudan (Morita et al., 2004),
Zimbabwe and in 45.4% (5/11) Hyalomma marginatum
rufipes and 2.2% (1/46) Hy. truncatum in Ethiopia (Kumsa
et al., 2014). So far R. aeschlimannii has not reported in
humans from east African countries.

3.1.5. Rickettsia massiliae

Rickettsia massiliae was first isolated in 1992 from
Rh. sanguineus ticks collected near Marseille in France
(Beati and Raoult, 1993) and then detected in Rh. sanguineus
and Rh. turanicus in Algeria and Morocco. R. massiliae is

50
usually associated with the hard ticks of the genus
Rhipicephalus ticks (Rhipicephalus mushamae,
Rhipicephalus lunulatus, Rhipicephalus sulcatus and
Rhipicephalus guilhoni) mainly in the central, western and
north African countries in Central African Republic, Mali,
Algeria, Morocco. The first human case of infection with
R. massiliae was confirmed in 2005, 20 years after the
isolate was obtained from a 45-year-old male human patient
in Palermo (Italy) in 1985 (Vitale et al., 2006). R. massiliae
has been detected mainly in North and West African
countries predominantly in Rhipicephalus spp. including
8.1% (2/37) in Rh. muhsamae in Mali, 8.2% (5/61) in
Rh. senegalensis in Guinea and in 22.4% (11/49) in Rh.
guilhoni in Senegal (Parola et al., 2013). In east African
countries R. massiliae has been detected in Hy. truncatum in
Kenya (Mutai et al., 2013) and in 1.9% (1/52)
Rh. praetextatus in Ethiopia (Kumsa et al., 2014b). There is
no report on R. massiliae from humans in east African
countries.

3.1.6. Ricketttsia sibirica mongolitimonae

Ricketttsia sibirica mongolitimonae, the agent of
lymphangitis-associated rickettsiosis (LAR), is the

51
subspecies of R. sibirica first isolated in Beijing in China
from Hyalomma asiaticum ticks collected in Mongolia in
1991 (Parola et al., 2005b). R. sibirica mongolitimonae was
detected in Hy. truncatum (Parola et al., 2001). The first
human case of this Rickettsia was reported in South Africa
after PCR amplification from a biopsied eschar. Human
patients develop lymphangitis, fever, maculopapular rash,
engorged lymph nodes, and multiple eschars. R. sibirica
subsp. mongolitimonae has been detected in 14% of
H. truncatum ticks in Senegal. The only report in east
African countries is the recent detection in Am. gemma ticks
in Kenya (Mutai et al., 2013). There is no report on
R. sibirica subsp. mongolitimonae from humans from in east
African countries.

3.1.7. Candidatus Rickettsia hoogstraalii

The only report from east Africa on Candidatus Rickettsia
hoogstraalii, a new rickettsia with a Candidatus status and
unknown pathogenecity, has been detected in soft ticks
belonging to Ornithodoros moubata collected from Tanzania
(Cutler et al., 2006) and in Argas persicus ticks from
Ethiopia (Cutler et al., 2012). This Rickettsia has been
reported in soft ticks collected from bats and seabirds.

52
Likewise, a similar species has been detected in European
hard tick Haemaphysalis sulcata for the name Rickettsia
hoogstraalii was proposed. Phylogenetic studies of this
rickettsia clusters within the spotted fever group of
rickettsiae at the periphery of this group at location close to
R. akari and R. felis (Cutler et al., 2012).

3.2. Typhus Group (TG) Rickettsia species

Typhus group (TG) rickettsiae are mainly associated with
body lice (R. prowazekii) or fleas (R. typhi), have an optimal
growth temperature of 35 ◦C, a G+C content of 29%, are
only found in the cytoplasm of host cells and cause typhus in
humans (Merhej and Raoult, 2011). The isolation of
Rickettsia prowazekii, the agent of the louse-borne epidemic
typhus, from Amblyomma ticks collected from cattle in
Ethiopia at the Rocky Mountain Laboratory is the only
previous report from east African countries (Philip et al.,
1966b). However, this report still remains to be a mystery
and had never been confirmed by any other subsequent
studies conducted in Ethiopia. For instance, recent studies
(Kumsa et al., 2014b; Mediannikov et al., 2013a) didn‟t
found any typhus group rickettsiae in ixodid ticks collected

53
from cattle and other domestic animals from different
districts in Ethiopia.
On the other hand, investigators in Mexico (Medina-Sanchez
et al., 2005) detected R. prowazekii in Amblyomma ticks and
obtained an isolate of R. prowazekii from one of the ticks.
This finding again raises a question on natural reservoir of
epidemic typhus rickettsiae involving ixodid ticks and
challenges our current understanding that the human latent
infection-reactivation-louse-human cycle or the flying
squirrel-louse and/or flea-flying squirrel cycle is generally
believed to play important role in the origin and evolution of
R. prowazekii.

3.3. Tick-borne Relapsing Fever Borreliosis

In east Africa tick-borne relapsing fever (TBRF) is caused by
Borrelia duttonii which is transmitted by Ornithodoros
moubata (Cutler et al., 2010b). In ticks, the borreliae invade
all organs, including the salivary glands and excretory
organs. B. duttonii is transmitted to humans when the
feeding site is contaminated by either coxal secretions or
saliva during tick feeding. In Tanzania tick-borne relapsing
fever caused by B. duttonii is listed amongst the top 10 killer
diseases in children less than 5 years of age (Cutler, 2010).

54
The tick vector, Ornithodoros moubata, feeds for a short
time only (usually less than half an hour), then returns to the
earth floor or walls of the house. Humans are believed to be
the only natural reservoir for B. duttonii, unlike the situation
for B. crocidurae in West  Africa, where rodents are
reservoirs.
Recently, B. duttonii has been detected in the peripheral
blood collected from both chickens and pigs in Tanzania that
implicate the possibility of zoonotic importance of this
Borrelia species (Cutler et al., 2010b). In human patients it
causes symptoms like fever accompanied in more than 90%
of cases by tachycardia, headache, myalgia, arthralgia,
conjunctivitis, hepatomegaly and splenomegaly, along with
orange urine in a few cases (Cutler et al., 2006).
Recently, a new Borrelia species distant from both relapsing
fever group and Lyme borreliae was detected in 7.3% of the
Amblyomma cohaerens collected from cattle in southwest
parts of Ethiopia (Mediannikov et al., 2013a). Similarly, new
Borrelia species has been recently detected in 8/119 (6.7%)
Amblyomma cohaerens, 1/42 (2.4%) Amblyomma gemma,
3/53(5.7%) Ammblyomma variegatum, 5/22(22.7%)
Amblyomma larvae and 3/60(5%) Amblyomma nymphs
(Kumsa et al., 2014b) that cluster at intermediate position in

55
between the recurrent fever and the Lyme disease borreliae
group using 16S genus-specific qPCR. These Amblyomma
ticks were collected from domestic animals in different parts
of Ethiopia. However, the pathogenecity for humans of these
newly detected Borrelia species in Amblyomma ticks in
Ethiopia remains to be determined in the future.

3.4. Bartonellosis
Bartonella species are intracellular arthropod-borne small
hemotropic facultative Gram-negative bacilli or coccobacilli
belonging to the alpha 2 subgroup of Proteobacteria, family
Bartonellaceae (Billeter et al., 2008). Bartonella are aerobic
bacilli, oxidative negative, fastidious, slow growing and
highly adapted to their vertebrate hosts (Anderson and
Neuman, 1997; Breitschwerdt and Kordick, 2000; Guptill,
2010). The bacteria infect the erythrocytes and endothelial
cells of their mammalian hosts. In humans Bartonella spp.
are implicated in causing several emerging and remerging
diseases manifested by a wide variety of clinical signs
(Anderson and Neuman, 1997; Breitschwerdt and Kordick,
2000).
Many different species of haematophagous arthropods
including fleas, lice, sandflies and ticks are implicated to

56
mediate the transmission of Bartonella spp. between
reservoir hosts and humans (Anderson and Neuman, 1997;
Angelakis and Raoult, 2014). To date, the genus Bartonella
comprises more than 30 different species and subspecies that
infect a wide variety of mammalian hosts. In several
European countries, USA and Asia a number of tick species
(Ixodes spp., Dermacentor spp., Haemaphysalis spp. and
Rhipicephalus sanguineus,) have been shown to be positive
for Bartonella spp. based mainly on PCR and very rarely by
culture (Angelakis et al., 2010; Angelakis and Raoult, 2014).
On the contrary, few previous studies made to detect
Bartonella spp. in ixodid ticks from African countries
reported the absence of these pathogens (Kumsa et al.,
2014b; Mediannikov et al., 2013a). Review of the recent
literature also shows the presence of contradicting views on
the transmission of Bartonella spp. by ticks. For instance
Telford III and Wormser (Telford, III and Wormser, 2010)
concluded that tick transmission of any Bartonella spp. to
either animals or humans has not been established, however,
Angelakis et al. (Angelakis et al., 2010; Cotte et al., 2008;
Reis et al., 2011) (2010) reported the vector competence and
transmission of Bartonella species by different tick species.
As most Bartonella species are transmitted by lice and fleas

57
brief descriptions of some species are given on the respective
sections in this review.

3.5. Q fever
Coxiella burnetii, the causative agents of Q fever, is obligate
intracellular, small pleomorphic, nonmotile, rod (0.2–0.4mm
wide, 0.4–1.0mm long), Gram-negative bacteria belonging to
the genus Coxiella, the family Coxiellaceae, the order
Legionellales, the class Gammaproteobacteria, and the
phylum Proteobacteria.icks are one of the most important
arthropods known to act as reservoirs of C. burnetii. So far
C. burnetii was reported in more than 40 different tick
species in 12 genera in several countries of the world (Cutler
et al., 2007; Maurin and Raoult, 1999). Ticks acquire
infection during feeding on blood meal from infected
animals and can transmit to their offspring both
transovarially and transstadially (Arricau-Bouvery and
Rodolakis, 2005; Maurin and Raoult, 1999). Ticks play
important role in the epidemiology of Q fever by
contaminating the environment by excreting C. burnetii via
their faeces, saliva and coxal fluids. However, sufficient data
that shows transmission of C. burnetii to humans by blood
feeding ticks is not yet available (Porter et al., 2011).

58
In humans inhalation of aerosolized C. burnetii from the
contaminated environment is considered as the main means
of infection (Maurin and Raoult, 1999). In addition,
consumption of raw milk and milk-products, transplacental,
intradermal inoculation, blood transfusion, contact with
urine, faces and semen of infected animals (especially
ruminants) are also regarded as possible routes of infection
(Maurin and Raoult, 1999). Excretion of C. burnetii by
domestic ruminants is implicated as major cause of
environmental contamination and the source for human
infection. Infection in humans is often asymptomatic, but it
can manifest as an acute disease, such as pneumonia,
hepatitis or appear as self-limited flu-like syndrome (Cutler
et al., 2007). Q fever can also appear in a chronic form,
mainly involving endocarditis and vascular infection, as well
as hepatitis and chronic infection after pregnancy (Maurin
and Raoult, 1999). Generally the number of studies on
Q fever from African countries are lower than from the other
continents, in particular the number of reports on ticks,
animals and humans from east African countries are very
lower than North, south and western African countries
(Vanderburg et al., 2014).

59
From east African countries seroprevalence of antibodies to
C. burnetii in humans was reported in Tanzania (Prabhu et
al., 2011), in Zimbabwe (Kelly et al., 1993) , in Zambia
(Okabayashi et al., 1999), in Somalia (Botros et al., 1995)
and in Kenya (Knobel et al., 2013). In domestic ruminants
seroprevalence of antibodies to C. burnetii was reported in
Malawi (Staley et al., 1989), Tanzania (Hummel, 1976),
Zimbabwe (Kelly et al., 1993), Zambia (Qiu et al., 2013),
Ethiopia (Gumi et al., 2013) and in Kenya (Knobel et al.,
2013). In east African countries C. burnetii was detected by
molecular method in different species of ticks in Kenya
Knobel et al. (Knobel et al., 2013) and in Ethiopia in 5.3%
Amblyomma variegatum and 10.8% Hyalomma truncatum
ticks (Philip et al., 1966a) and 28.6% (14/49) Amblyomma
gemma, 25% (31/124) Rhipicephalus pulchellus, 7.1 %
(1/14) Hyalomma marignatum rufipes, 3.2 % (2/62)
Amblyomma variegatum, 3.1% (4/128) Amblyomma
cohaerens, 1.6% (1/63) Rhipicephalus praetextatus and
0.6% (1/153) Rhipicephalus (Booophilus) decoloratus
(Kumsa et al., 2014). As it can clearly be seen on Fig 2 very
fewer studies were made in east African countries than in the
North, west, middle and South African countries on
C. burnetii infection in both in humans and animals.

60
4. LOUSE-BORNE ZOONOTIC BACTERIA
4.1. Borrelia recurrentis
Borrelia recurrentis, transmitted by Pediculus humanus
corporis (human body lice), is the causative agent of
epidemic relapsing fever, that affected several million people
worldwide during the first half of the 20th century,
particularly during the world wars (Badiaga and Brouqui,
2012). Humans are the only known reservoir and host of
B. recurrentis. It was initially described in Ireland and was
one of the first louse-borne infectious diseases identified by
microscopy. Lice are infected upon ingestion of infected
blood meal from human hosts (Badiaga and Brouqui, 2012).
Then B. recurrentis passes from the gut to the coelomic
cavity and multiplies in the hemolymph. B. recurrentis don‟t
disseminate in tissues and hence don‟t found in the saliva or
the feces of lice. In lice infection last for the rest of entire
lifespan but there is no transovarial transmission of
B. recurrentis hence can‟t transmit borreliae to its progeny.
In humans the only possibility of infection by B. recurrentis
is by contamination when louse is usually crushed between
nails of fingers that releases and spreads B. recurrentis
which is highly infective bacterium and capable of

61
penetrating intact human mucosa and skin (Badiaga and
Brouqui, 2012; Raoult and Roux, 1999).
In infected humans clinical manifestations start abruptly with
a high-grade fever, pain, anorexia, dry coughs, and fatigue.
Complications include skin and mucosal haemorrhaging;
neurological, liver and renal involvement; and spleenic
rupture. Jaundice is a diagnostic clue that suggests relapsing
fever among louse-borne diseases (Raoult and Roux, 1999).
The disease is predominantly characterized by a series of
relapses that are less severe and shorter in duration, and
occur at intervals of 7 or 10 days. Common diagnostic
methods include the detection of Borrelia in Giemsa-stained
blood films and PCR assays (Brouqui et al., 2004; Raoult
and Roux, 1999). The death rate varies from 10% to 40% in
untreated patients, and from 2% to 4% in patients treated
with antibiotics such as doxycycline or erythromycin.
Louse-borne relapsing fever caused by B. recurrentis has
disappeared from many regions around the world (Raoult
and Roux, 1999). On the contrary this disease is still a major
public health problem in populations living in poor-hygiene
conditions where body louse infestations are prevalent in
east African countries including Ethiopia (Almaviva et al.,
1993; Borgnolo et al., 1993; Yimer et al., 2014a; Yimer et

62
al., 2014b) and the neighbouring countries like Sudan (de et
al., 1995), Eritrea, and Somalia. In the highland parts of
Ethiopia louse-borne relapsing fever is listed as the seventh
most common reason for hospital admission (2.5% of total
3777 cases) and the fifth most common cause of death (0.9%
of the total 42 cases and is associated with substantial illness
and death (Yimer et al., 2014a; Yimer et al., 2014b).
Recently, B. recurrentis DNA was detected in 23% of head
lice from patients with louse-borne relapsing fever in
Ethiopia, however, the role of head lice in the transmission
of this bacterium was not determined (Boutellis et al., 2013).

4.2. Bartonella quintana

Bartonella quintana, also transmitted by Pediculus humanus
corporis (human body lice), is the causative agent of trench
fever. B. quintana is a facultative intracellular Gram negative
bacteria, was first described during World War I (Badiaga
and Brouqui, 2012; Raoult and Roux, 1999). The name
„trench fever‟ was chosen because the disease was first
described in both Allied and German troops crowded into
trenches during the World War (Badiaga and Brouqui,
2012). It has been estimated that trench fever affected
several million people worldwide during the two world wars,

63
especially in Russia and on the Eastern, Central and Western
European fronts (Badiaga and Brouqui, 2012). The incidence
of trench fever dramatically decreased after World War II.
However, in the early 1990s, B. quintana was recognized as
a major re-emerging infectious disease in urban homeless
populations of developed countries in both Europe and the
United States who have poor living conditions (Raoult and
Roux, 1999). Even though, the natural reservoir of
B. quintana has not yet been fully established, infection with
B. quintana in lice lasts for the entire lifespan (Raoult and
Roux, 1997; Raoult and Roux, 1999). B. quintana multiplies
widely in louse intestines and passes with louse faeces.
Humans are infected by contamination of faeces containing
B. quintana from infected lice. B. quintana can survive for
long periods in the louse feces and can remain infectious for
up to 1 year (Badiaga and Brouqui, 2012; Raoult and Roux,
1999).
After an incubation period of 15 to 25 days infected persons
show signs like acute onset of a high-grade fever, headache,
dizziness, and characteristic pain in the legs. The first fever
episode can last for between 2 and 4 days. Occasionally, the
primary episode is followed by a relapse every 4-5 days
(Badiaga and Brouqui, 2012; Raoult and Roux, 1999). The

64
term “quintan fever” refers to the recurring 5-day attacks.
B. quintana causes trench fever, bacillary angiomatosis,
endocarditis, chronic bacteremia, and chronic
lymphadenopathy (Badiaga and Brouqui, 2012; Raoult and
Roux, 1999). Although trench fever often results in
prolonged disability, no deaths have been reported.
In east African countries B. quintana is commonly detected
in human body lice in Ethiopia, Zimbabwe, Burundi and
Rwanda (Fournier et al., 2002; Raoult et al., 1997; Roux and
Raoult, 1999). B. quintana was detected in head lice in
Ethiopia, however, the role of head lice was not determined
in the transmission of this bacterium (Sangare et al., 2014).

4.3. Rickettsia prowazekii

Rickettsia prowazekii also transmitted by human body lice is
the causative agent of epidemic typhus (louse-borne typhus)
(Raoult and Roux, 1999). Epidemic typhus has caused more
deaths than all of the wars in history. R. prowazekii is a
Gram negative, obligate intracellular bacterium from the α2
group of proteobacteria was first described during World
War I (Badiaga and Brouqui, 2012). In most countries
humans are considered as the main reservoir of the
bacterium, however, in the United States flying squirrel has

65
been suggested as a possible reservoir host of this bacterium.
Infected humans retain some rickettsiae for the rest of their
lives and under certain stressful conditions, they may relapse
in a relatively a milder bacteremic form illness called Brill–
Zinsser disease (Gillespie et al., 2009). These bacteraemic
humans are considered as the source of infection for lice and
to start a new epidemic. In untreated cases the mortality rate
of epidemic typhus varies from 0.7% to 60%. After an
incubation period of 10–14 days, patients usually experience
1–3 days of malaise associated with fever and multiple
painful symptoms, which are then followed by the
development of rashes (20–40% of patients), neurological
manifestations (80%), respiratory manifestations (38–70%),
and shock (7%) (Raoult and Roux, 1999).
Lice are infected upon ingestion of blood meal containing
rickettsiae from infected persons and then the bacteria infect
the midgut epithelial cells of the louse and multiply rapidly
(Badiaga and Brouqui, 2012; Raoult and Roux, 1999).
Excessive multiplication and growth of R. prowazekii causes
enlargement and eventually bursting of infected epithelial
cells releasing the rickettsiae into the gut lumen. Very large
quantities of rickettsiae are discharged with the feces of lice
and can remain infective for up to 100 days (Badiaga and

66
Brouqui, 2012). All stages of the louse life cycle can acquire
infection. Infection with R. prowazekii causes death of the
louse within 1 week due to the ruptured epithelial cells are
not replaced and this causes the blood to pass through the
intestine and the louse becomes red. Infected red lice die
shortly thereafter thus giving the named “red louse disease
Typhus (Raoult and Roux, 1999).
Ethiopia and Burundi are considered the endemic foci
countries of this disease in Africa and in east African
countries R. prowazekii was detected in human body lice in
Ethiopia, Burundi and Rwanda (Fournier et al., 2002; Raoult
et al., 1997).

4.4. Acinetobacter baumannii

Acinetobacter species are Gram-negative coccobacilli
bacteria commonly found in water, soil, mud, living
organisms, vegetables, as well as in the feces, urine and skin
of humans and animals (Kumsa et al., 2012b). In humans
members of the genus are emerged as opportunistic
pathogens and are frequently implicated in various types of
infections, especially in immunocompromised individuals
and intensive health care units throughout the world
(Brouqui et al., 2004). In tropical countries, they are

67
associated with severe community-acquired infections. They
have the capacity to survive for prolonged periods under a
wide range of environmental conditions. Acinetobacter
baumannii is described as the most common species of the
genus frequently associated with outbreaks and has been
repeatedly reported to develop a high level of resistance
against all of the available classes of antimicrobial drugs in
many parts of the world (Badiaga and Brouqui, 2012). In
east African countries Acinetobacter baumannii was detected
in 54 head (47%) and 77 body (71%) lice in Ethiopia
(Kempf et al., 2012). Although A. baumannii infections are
not confirmed to be transmitted by body lice, the role of
human lice in the transmission of this bacterium remains to
be seen in the future. Furthermore, other Acinetobacter
species such as A. pitti in Heterodoxus spiniger of dogs and
A. soli DNA in Linognathus vituli of cattle were detected in
animal lice in Ethiopia (Kumsa et al., 2012b). A. pittii is
reported as the second most commonly isolated
Acinetobacter sp. after A. baumannii in human patients
around the world. More recently other less known species
such as A. soli have been regularly associated with serious
infections and considered as emerging pathogens. For
instance A. soli outbreak as a cause of infection in neonatal

68
intensive health care unit in Korea and nosocomial
bloodstream infection due to A. pittii in United States were
reported (Kumsa et al., 2012b).

5. FLEA-BORNE ZOONOTIC BACTERIA

5.1. Rickettsia felis
Rickettsia felis (formerly known as the „ELB agent‟ named
for the original source of the fleas at Elward Laboratory) is
the etiological agent of an emerging flea-borne rickettsiosis
also known as flea-borne spotted fever and cat flea typhus
(Parola, 2011). Rickettsia felis is an obligate intracellular
Gram-negative bacterium belonging to the spotted fever
group (SFG) of rickettsiae. It was first detected in European
cat fleas (Ctenocephalides felis felis) and has been definitely
described in 2002 (Raoult et al., 2001c). Laboratory studies
have indicated that R. felis is maintained in C. felis fleas by
vertical and horizontal transmission and can persist for life
without causing any damage (Reif and Macaluso, 2009).
R. felis is transmitted to humans by fleabites and contact
with flea feces (Reif and Macaluso, 2009). In humans
infected with R. felis the common clinical signs are fever,
headache, and rash. Other signs include marked fatigue,
myalgia, photophobia, conjunctivitis, abdominal pain,

69
vomiting, and diarrhoea, as well as solitary, black crusted
skin lesions, muscle pain, in some cases local
lymphadenopathy and a characteristic inoculation eschar at
the site of the flea bite (Parola, 2011). Thus infection by
R. felis may be misdiagnosed due to similar clinical signs
with other arthropod-borne rickettsios such as murine typhus
or some members of the SFG, as well as other infectious
diseases like dengue, malaria, brucellosis, leptospirosis, or
even other clinical conditions like Kawasaki disease
especially in countries with poor diagnostic facilities (Parola,
2011). Hence only advanced molecular techniques can
currently distinguish the infections which are very rare
methods of diagnosis in Africa in general and in particular in
east Africa (Brouqui et al., 2004).
Although C. felis is considered to be the only known
biological vector of R. felis, R. felis has recently been
detected in 12 species of fleas (C. canis, C. orientis,
Anomiopsyllus nudata, Archaeopsylla erinacei,
Ctenophthalmus sp., Xenopsylla cheopis X. rasilliensis,
Tunga penetrans, Ceratophyllus gallinae, Spilospsyllus
cuniculi and Echidnophaga gallinacean); 8 species of ticks
(Haemaphysalis flava, Rhipicephalus sanguineus, Ixodes
ovatus, and Carios capensis); and 3 species of mites (chigger

70
mites in South Korea and mesostigmata mites in Taiwan) in
different countries around the world (Eisen and Gage, 2012;
Hirunkanokpun et al., 2011; Parola, 2011; Reif and
Macaluso, 2009). The reported hosts for these vectors were
mainly cats, dogs and rodents, and more rarely opossums,
hedgehogs, horses, sheep, goats, gerbils and monkeys
(Dobler and Pfeffer, 2011; Eisen and Gage, 2012; Reif and
Macaluso, 2009). Even though, few confirmed human cases
have been described infection by R. felis is considered as
worldwide disease (Parola, 2011). It is more common in
tropical countries with warm climatic conditions.
In east African countries R. felis has recently been reported
in C. felis and P. irritans specimens collected from human
dwellings in the southwestern regions of the country in
Ethiopia, Rickettsia felis was detected in 21% of C. felis,
11% P. irritans and 6% C. canis collected from dogs and
cats in Ethiopia (Kumsa et al., 2013). In Kenya R. felis has
been reported in C. felis, C. canis, and P. irritans (Jiang et
al., 2013).
Human infection with R. felis in east African country has
been reported only in Kenyan in 3.4% of the afebrile patients
in western Kenya were found to be positive for R. felis using
molecular methods (Maina et al., 2012) and also Recently,

71
R. felis was detected in patients suffering from fever of
unknown cause with a prevalence of 3.7-7.2% in Kenya
(Richards et al., 2010). In eastern Africa countries R. felis is
considered as the second cause of fever in rural areas after
malaria (Mediannikov et al., 2013b). However, in many
eastern African countries studies on R. felis have not yet
been conducted and some authors argue that R. felis infection
should be suspected as one of the causes of fevers of
unknown etiologic agent in malaria endemic east African
countries (Mediannikov et al., 2013b; Richards et al., 2010).

5.2. Rickettsia typhi

Rickettsia typhi (formerly Rickettsia mooseri) is the etiologic
agent of murine typhus and also known as fleaborne, rat,
urban and endemic typhus) which is a worldwide zoonosis
and is one of the most widely distributed arthropod-borne
infections (Eisen and Gage, 2012). Rickettsia typhi is an
obligate intracellular Gram-negative bacterium belonging to
the typhus group (TG) rickettsiae. Xenopsylla cheopis is the
main vector of R. typhi. The classical life cycle of R. typhi
involves rats mainly Rattus rattus and R. norvegicus and the
rat flea, Xenopsylla cheopis (Eisen and Gage, 2012; Gillespie
et al., 2009). In many parts of the world murine typhus

72
infection is intimately associated with the introduction of
commensal rodents, such as R. rattus, R. norvegicus, and
Mus musculus, and their ectoparasites, particularly fleas
(Azad, 1990). R. typhi is transmitted to reservoir host rats
and humans by the contamination of the bite site or skin
abrasions with Rickettsia-containing flea feces during or
after blood feeding, as well as via the flea bite. R. typhi do
not have any harmful effects on the health and activity of
either the Xenopsylla cheopis vector or the reservoir host
rats. R. typhi infects endothelial cells in mammalian hosts
and midgut epithelial cells in the flea host (Azad, 1990;
Bitam, 2012; Bitam et al., 2010; Eisen and Gage, 2012).
R. typhi is transferred from a rodent reservoir by an
arthropod (often X. cheopis) to humans. After incubation
periods of six to 14 days it causes a relatively mild febrile
illness and clinical sings similar to caused by other infectious
diseases and thus cases may be overlooked without a
laboratory confirmed diagnosis (Azad, 1990; Eisen and
Gage, 2012). The most common clinical manifestations are
high fever, severe headache, chills, myalgia, weakness, and
nausea (Civen and Ngo, 2008). Murine typhus patients
usually present with undifferentiated febrile illness. The
pathognomonic rash is described as macular (49%),

73
maculopapular (29%), papular (14%), petechial (6%), and
morbiliform (3%), usually centrally distributed on the trunk,
but also found on the extremities (Azad, 1990; Eisen and
Gage, 2012). Physicians practicing in or near R. typhi-
endemic areas need to consider murine typhus in the
differential diagnosis of a febrile illness of unknown
aetiology (Azad, 1990; Eisen and Gage, 2012).
In east African countries R. typhi has been reported in 62%
of R. rattus collected from buildings in Addis Ababa in
Ethiopia (Azad and Beard, 1998) and is also endemic in
other east African countries like Kenya, Uganda, Tanzania,
Madagascar, Malawi, Zambia, Zimbabwe and Mozambique
(Azad, 1990; Azad and Beard, 1998; Azad et al., 1990).
In humans an overall seroprevalence of 8% by (Prabhu et al.,
2011) and 9.3% by (Dill et al., 2013) of typhus group
rickettsiae was reported in Tanzania and also 5.0%
seroprevalence of antibodies against R. typhi was reported in
Zamia (Okabayashi et al., 1999).

5.3. Bartonella henselae

Bartonella henselae is the causative agent of cat-scratch
disease (CSD), also known as cat-scratch fever or
bartonellosis an extremely common worldwide zoonosis

74
(McElroy et al., 2010; Mosbacher et al., 2010). B. henselae
belongs to the genus Bartonella which are hemotropic
facultative Gram-negative intracellular bacteria that infects
primarily red blood cells but can also be found associated
with host endothelial, dendritic, and CD34+ cells, which
include lymphopoietic stem and progenitor cells, small-
vessel endothelial cells, and embryonic fibroblasts (McElroy
et al., 2010). Ctenocephalides felis (cat flea) is reported as
the primary arthropod vector for B. henselae (Breitschwerdt
et al., 2010; Chomel and Kasten, 2010). Fleas are infected up
on feeding blood meal during the periods of bacteraemia
when the bacteria circulate in the blood of an infected animal
and then infected fleas shed infectious organisms in their
feces (Eisen and Gage, 2012; McElroy et al., 2010). The
domestic cat is the primary animal reservoir of B. henselae
and transmit the pathogen to humans via scratches or bites,
though naturally infected cats are asymptomatic (Dobler and
Pfeffer, 2011; Eisen and Gage, 2012). Previous studies show
that B. hensealae is more common in stray cats than pet cats
and also cats younger than one year old are important
sources of the bacterium (Eisen and Gage, 2012). In
addition, dogs and other animals have also been implicated
as sources of human infection (Chomel et al., 2006; Dobler

75
and Pfeffer, 2011). B. henselae is transmitted between
animals, and from animals to humans, by inoculation with
contaminated flea feces on an open wound, such as a scratch
or bite (Eisen and Gage, 2012).
In infected humans the main clinical symptoms of cat scratch
disease are benign lymphadenopathy, low-grade fever,
malaise and aching, headache, anorexia, and splenomegaly
(Breitschwerdt et al., 2010; Chomel et al., 2006; Chomel and
Kasten, 2010; Eisen and Gage, 2012). Batonella spp. are
more frequently associated with several symptoms
particularity in ocular infections and endocarditis
(Breitschwerdt et al., 2010; Chomel et al., 2006; Chomel and
Kasten, 2010). In immunocompromized humans including
HIV-positive persons B. henselae typically causes systemic
bartonellosis which manifests as bacillary angiomatosis
(tumor-like growth of blood vessels on the external skin
surface) and bacillary parenchymous peliosis (hepatitis).
These syndromes are uncommon in immunocompetent
individuals (Breitschwerdt et al., 2010; Chomel et al., 2006;
Eisen and Gage, 2012).
Among the very few reports available from eastern African
countries include an overall percentage of 6% (2/34)
B. henselae DNA in C. felis collected from cats in Oromia

76
(Kumsa et al., 2013), Ethiopia and 11% (5/46) prevalence of
antibodies against Bartonella spp. in cats from Addis Ababa
and Bartonella spp. related to B. elizabethae in small
mammals in northern Ethiopia (Meheretu et al., 2013) and
Bartonella spp. in bats in Kenya are the only reports so far
made revealing huge information gap on bartonellosis from
East Africa (Kosoy et al., 2010).

5.4. Yersinia pestis

Yersinia pestis, an etiological agent of plague, is a Gram-
negative, nonmotile, non-spore-forming, coccobacillus
bacteria belongs to the genus Yersinia and the family
Enterobacteriaceae and has worldwide distribution (Morelli
et al., 2010; Raoult et al., 2013). It is historically associated
with rats (Rattus rattus and R. norvegicus) (Bitam et al.,
2010; Eisen and Gage, 2012; McElroy et al., 2010). Rodent
fleas, including Xenopsylla cheopis and Ropsylla montana,
are the principal reservoir animals important for transmission
among rodents and to humans (Eisen and Gage, 2012;
McElroy et al., 2010). Fleas acquire Y. pestis from an
infected blood meal (Perry and Fetherston, 1997). Infection
in the flea is restricted to the alimentary canal, and is not
transmitted transovarially.

77
Humans are infected with Y. pestis by direct transmission via
infectious droplets or by contact with contaminated fluid or
tissue or indirectly through flea bites by inoculation of the
bacteria with contaminated flea feces on an open wound
(Eisen and Gage, 2012; McElroy et al., 2010). The human
flea (Pulex irritans) is reported to play an important role in
spreading plague by human-to-human transmission (Eisen
and Gage, 2012). Humans are extremely susceptible to
Y. pestis infection and can be infected by bites of infected
fleas, direct contact with infected animal tissues, secretions
or respiratory droplets, by consumption of raw or
insufficiently cooked meat products, inhalation of
aerosolized respiratory excreta of animals or patients with
the pneumonic form of infection (Eisen and Gage, 2012;
Perry and Fetherston, 1997). Cats are highly susceptible to
plague and can transmit infection directly to humans. In
addition, cats can disseminate the bacteria by transporting
infected fleas or from their hunting behaviour rodent
carcasses into the residential environment (McElroy et al.,
2010).
Plague is a serious illness in humans that is likely to be fatal
if left untreated and occurs in the three major forms in
humans (McElroy et al., 2010). The first type is the bubonic

78
plague is the most common type and it accounts for 80–90%
of all reported plague cases worldwide (Eisen and Gage,
2012; McElroy et al., 2010; Perry and Fetherston, 1997).
Bubonic plague is transmitted through the bite of an infected
flea, or direct contact between infectious host body fluids
and open skin lesions or abrasions on human skin. Bubonic
plague is characterized by a painful, severely inflamed
lymph node (regional lymphadenopathy) known as a bubo
accompanied by fever, headache, chills, malaise and extreme
exhaustion. Illness usually begins 2–10 days following
exposure (Eisen and Gage, 2012; McElroy et al., 2010; Perry
and Fetherston, 1997). The second type is septicemic plague
which can be primary septicemic plague, which is often
attributed to cutaneous exposure to Y. pestis, is less common
and is characterized by fever and sepsis without regional
lymphadenopathy. Septicemic plague also occurs
secondarily to bubonic plague (Eisen and Gage, 2012;
McElroy et al., 2010; Perry and Fetherston, 1997). The third
type is the pneumonic form (primary or secondary to
bacteraemia) of plague which can result from untreated
bubonic plague infection can spread through the bloodstream
to the lungs, resulting in secondary pneumonic plague is
acquired through inhalation of plague bacteria contained in

79
respiratory droplets or other materials, is the least common,
but the most severe and rapidly progressing form (Eisen and
Gage, 2012; McElroy et al., 2010; Perry and Fetherston,
1997). Pneumonic plague can spread from person-to-person
through respiratory droplets (Eisen and Gage, 2012; Perry
and Fetherston, 1997). Persons infected through respiratory
exposure develop primary pneumonic plague. Both primary
and secondary pneumonic plague is fulminant illnesses, with
rapid progression to hypoxia, hemoptysis, and shock.
Without prompt appropriate treatment, pneumonic plague is
usually fatal within 3–4 days (Eisen and Gage, 2012;
McElroy et al., 2010; Perry and Fetherston, 1997).
Plague has recently been recognized as a re-emerging
disease of humans in East African countries in remote areas
where the disease is endemic and where persons live in
unsanitary environmental conditions in close contact with rat
infested with fleas in Kenya, Uganda, Tanzania (Laudisoit et
al., 2007), Madagascar (Ratovonjato et al., 2014), Malawi,
Zambia, Zimbabwe and Mozambique east African countries
(Neerinckx et al., 2010). In northwest Uganda, which has
had recent plague outbreaks, cat fleas (Ctenocephalides felis)
have been reported as the most common fleas in the home
environment, which is suspected to be a major exposure site

80
for human plague in this country (Amatre et al., 2009;
Neerinckx et al., 2010). Very recently, on 4 November 2014,
WHO has reported an outbreak of plague in 16 districts in
seven regions in Madagascar in which a total of 119 cases of
plague have been confirmed, including 40 deaths. About 2%
of reported cases are of the pneumonic form of plague
(http://www.who.int/csr/don/21-november-2014-plague/en/).

6. Mite-borne bacterial diseases

Mites are one of the most common ectoparasites of both
humans and animals, with 45,000 recognized species
(Fischer and Walton, 2014). Liponyssoides sanguineus is a
hematophagous biting mite of the rat, mouse, and other
domestic rodents. The house mouse mite (Liponyssoides
sanguineus) is the vector of R. akari. This mite can bites
humans and is responsible for the transmission of Rickettsia
akari, the etiological agent of rickettsial pox. Rickettsial pox
is relatively a mild disease, characterized by fever and
exanthema and was first described in New York in 1940.
This disease has been reported in countries in Europe, USA
and Asia. In African countries this disease has been reported
from the Republic of Central Africa and South Africa.
However, despite the presence of variety of mites in east

81
African countries there is no any information about this
disease (Bitam, 2012; Parola et al., 2013).

7. Other vector-borne bacterial diseases

7.1. Bartonella melophagi

Bartonella melophagi is the member of the genus Bartonella

which are small hemotropic, facultative, Gram-negative and
arthropod-borne intracellular alphaproteobacteria that belong
to the family Bartonellaceae (Anderson and Neuman, 1997;
Guptill, 2010). Recently, ruminant-associated Bartonella
spp., including B. chomeli, B. bovis, B. schoenbuchensis and
B. capreoli, have been reported to infect cattle and domestic
ruminants in some European countries and the USA (Chang
et al., 2000; Cherry et al., 2009; Raoult et al., 2005; Reeves
et al., 2006). In addition, recently several Bartonella spp.
have been detected using culture and PCR methods in
several species of biting flies including Horn flies
(Haematobia spp.), stable flies (Stomoxys spp.), deer flies
(Chrysops spp.), and horse flies (Tabanus spp.) in several
countries of the world (Billeter et al., 2008; Chomel et al.,
2009; Dehio et al., 2004; Gutierrez et al., 2014; Halos et al.,
2004). Detail information on Bartonella spp. in biting flies is

82
reviewed by Billeter et al. (Billeter et al., 2008) and Tsai et
al. (Tsai et al., 2011). B. melophagi is one of the ruminant
associated Bartonella spp. recently isolated from sheep
blood in the USA (Bemis and Kania, 2007) and the same
bacteria were isolated from the blood of two female patients
with pericarditis and skin lesions in the USA (Maggi et al.,
2009). Furthermore, Kosoy et al., (2007) have deposited the
rpoB gene sequence of B. melophagi amplified from
M. ovinus in GenBank (accession number: EF605288) as
unpublished data that can‟t be accessible using PubMed
search engine. Even though much information about this
bacterium is not available, recently Kumsa et al. (Kumsa et
al., 2014a) reported an overall prevalence of 88.6%
B. melophagi DNA in Melophagus ovinus (sheep ked)
collected from sheep in Ethiopia. This is the only report from
Africa and thus further research is needed on this bacterium.

7.2. Q fever
There are several reports on the presence of Coxiella burnetii
in biting flies from many countries around the world (Nelder
et al., 2008). However, there is no information on the
occurrence of this bacterium in flies in African countries.
Studies are required to clarify the presence or absence of this

83
bacterium in flies in Africa including east Africa which
inhabits several species of haemtophagous biting flies.

7.3. Rickettsia felis

Recently in west African counties Rickettsia felis has been
detected by molecular tools in Aedes albopictus (Asian tiger
mosquitoes) in Gabon (Socolovschi et al., 2012c) and in
Anopheles gambiae (the major vector in malaria infection) in
Côte d‟Ivoire (Socolovschi et al., 2012b). In addition, a
novel Rickettsia species that is closely related to R. felis was
detected in An. gambiae and in Anopheles melas in Senegal
(Mediannikov et al., 2013b). In this part of Africa R. felis
infections were frequently reported in febrile patients in
malaria-endemic regions (Mediannikov et al., 2013b). These
authors argue the possibility that mosquitoes may play some
type of role in the transmission of R. felis to humans and thus
R. felis infection should be suspected in regions where
malaria is endemic. However, despite the fact that east
African countries are endemic for malaria and home to a
great diverse species of mosquitoes, information on the
occurrence of R. felis in mosquitoes is not available.
Obviously, studies are required to address the presence or

84
absence of this bacterium in mosquitoes in east Africa
countries.

7.4. Acinetobacter species

Recently Acinetobacter lwoffii and another new
Acinetobacter sp. has been detected by molecular tools in
Melophagus ovinus (sheep ked) collected from sheep in
Ethiopia (Kumsa et al., 2012b). Despite the great sheep
population and a very high prevalence of this ectoparasite in
this region of Africa this is the only report in sheep ked from
Africa. More recently other less known Acinetobacter
species such as A. lwoffii have been regularly associated with
serious infections and considered as emerging pathogens.
For instance A. lwoffii associated with acute gastroenteritis in
USA and multidrug resistant A. lwoffii in southern Thailand
were reported (Badiaga and Brouqui, 2012).

85
8. Vector-borne bacterial diseases in travellers returning
from East Africa
The diverse geography, ecosystems and climate of Africa
attracts huge population of international travellers for
various reasons including as a tourist destination, for
international aid or voluntary work, research or education,
family or friend visit and for business travel. The number of
international travellers to sub-Saharan Africa had increased
by 4% (1.3 million travellers) in the year 2011 (Mendelson
et al., 2014).These travellers from different countries of the
world during their stay in Africa are exposed to various types
of endemic infectious diseases in Africa including vector-
borne bacteria (Walker, 2003b). In international travellers
systemic febrile illness, a common syndrome for vector-
borne bacteria, was reported as the leading symptoms in sub-
Saharan Africa (Hunziker et al., 2012; Mendelson et al.,
2014). For instance systemic febrile illness was diagnosed in
27% (1,474) of travellers returning from east African
countries to their home country (Mendelson et al., 2014).
To mention some of the individual reports diagnosed in
home returning travellers from east African countries
R. africae has been molecularly confirmed in a 62 year old
French man returning after 2 months stay in western part of

86
Ethiopia (Stephany et al., 2009) and also R. africae was
diagnosed in Italian, USA and Canadian citizens human
patients returning from Zimbabuwae, Tanzania and Kenya
respectively (Raoult et al., 2001b). A case of relapsing fever
borreliosis with cutaneous eschar and radiculopathy in a 77
year old French woman traveller returning from Ethiopia
transmitted was reported to be transmitted by hard ticks
using molecular tools (Socolovschi et al., 2012a). In another
situation a fatal case of spotted fever group rickettsial
infection was reported in a missionary woman from the
United States who lived in a rural town in the central district
of Kenya approximately 70 km north of Nairob who had a
history of tick exposure and the patient initially
misdiagnosed and treated for malaria and then for the second
time was treated with sulfa-containing antibiotic that is
believed to exacerbated the clinical severity of rickettsial
infections in this patient (Rutherford et al., 2004). In USA
African tick bite fever was confirmed in 48-year-old woman
who returned with influenza like illness and inoculation
eschar after 2 weeks of returning from a 13-day trip where
she had spent time at a game farm in Zimbabwe (Owen et
al., 2006). Also recently Murine typhus was confirmed using
molecular tools in returned two patients that were infected in

87
Madagascar (Walter et al., 2012). Spotted fever group
rickettsial infections was confirmed at the Centers for
Disease Control and Prevention in USA in returned travellers
from east African countries in Zimbabwe, Rwanda,
Mozambique, Zambia and Tanzania (McQuiston et al.,
2004). Of the total 197 SFG and 1 TG rickettsiosis, 5 acute
Q fever and 1 bartonellosis cases confirmed in returned
European travellers from Sub-Saharan Africa from east
African countries SFG rickettsioses was diagnosed in
patients who travelled to Zimbabwe (n = 13) and Tanzania (n
= 7) and 2 patients infected with acute Q fever in Tanzania
(Jensenius et al., 2009). Furthermore, Ta et al. (Ta et al.,
2008) also reported Q fever in three febrile patient travellers
who returned home to Spain after short period stay in sub-
Saharan Africa and Mozambique.
In conclusion zoonotic vector-borne bacteria have emerged
as one of the important causes of diseases in home returning
international travellers from east African countries and there
is a possibility of misdiagnosis and mistreatment due to the
high prevalence of common symptoms like systemic febrile
illness associated with infections by many bacterial species
(Mendelson et al., 2014). Thus vector-borne zoonotic

88
bacteria need to be suspected in febrile patients returning
from east Africa (Mediannikov et al., 2013b).

9. Detection of bacteria in Arthropods

Bacteria can be detected in blood-feeding arthropods like
ticks, lice, mites, fleas, flies and mosquitoes by several
methods like staining, immunodetection, culture methods
and molecular tools (Murrell et al., 2003). Gimenez or
Giemsa staining readily shows the presence of spotted fever
group rickettsiae, C. burnetii, and ehrlichiae (Murrell et al.,
2003). Borreliae could be seen by the use of dark-field
microscopy (Brouqui et al., 2004). When the bacteria invade
and multiply in all organs and fluids of arthropod as in the
case of Rickettsia species in ticks detection of bacteria in the
hemolymph or salivary glands can be performed (Brouqui et
al., 2004; Parola and Raoult, 2001). Hemolymph test is
performed under aseptical conditions on live ticks by
amputating the distal portion of the leg. Hemolymph from
the site of the cut leg is used to make smear onto a
microscope slide that is stained and examined under a
microscope for the presence of bacteria (Brouqui et al., 2004;
Parola and Raoult, 2001). In addition impression smears
could be made from salivary glands or ovaries removed from

89
dissected ticks, or the organs may be used for histological
testing. Immunodetection methods may also be used to
detect bacteria in haemolymph or organ smears. Slides are
air-dried and fixed in acetone before being treated with
polyclonal or monoclonal antibodies conjugated with
immunofluorescent labels (Brouqui et al., 2004; Parola et al.,
2013).
The gold standard for diagnosis of vector-borne bacterial
disease is by cultivation and identification of the etiologic
agent from an appropriate specimen (Brouqui et al., 2004).
Bacteria can be cultured from dead, frozen or live arthropods
after disinfecting with iodinated alcohol and rinsing in sterile
water, and drying on sterile paper in a laminar flow cabinet
the arthropod is macerated in 1 mL of cell culture medium
and inoculated into culturing medium for the target bacteria.
For instance a drop of hemolymph is used for cell culture
systems and shell vial technique used for culture of
Rickettsia species from live ticks (Brouqui et al., 2004;
Parola et al., 2013; Parola and Raoult, 2001; Raoult and
Roux, 1999). Appropriate antimicrobial agent that is not
effective against the target bacteria is added to the culture
media to prevent contamination of the cultures with gut
bacteria and fungi of the crushed arthropods. Bacteria like

90
Rickettsia, C. burnetii, and ehrlichiae can also be isolated by
use of the shell vial technique whereas borreliae are best
isolated and grown on the specialized growth medium BSK
II, which is a complex broth that contains long-chain fatty
acids, bovine serum albumin, and rabbit serum (Brouqui et
al., 2004; Parola et al., 2013; Parola and Raoult, 2001;
Raoult and Roux, 1999; Socolovschi et al., 2009).
Molecular methods using PCR based by amplification and  
sequencing of 16S rRNA gene and other genes  accepted for
the  characterization of specific groups of bacteria is the gold
standard  for the identification of vector-borne bacterial
agents in arthropods (Brouqui et al., 2004; Parola et al.,
2013; Parola and Raoult, 2001; Raoult and Roux, 1999;
Socolovschi et al., 2009). Identification strategies based on
PCR amplification followed by DNA sequencing of a
number of genes have been described for several bacteria
using genus-specific or species specific primers (Brouqui et
al., 2004; Parola et al., 2013; Raoult and Roux, 1999;
Socolovschi et al., 2009). Bacterial total DNA for PCR from
different arthropods such as lice, ticks, fleas, and flies can be
extracted by employing several methods like boiling
triturated arthropods in saline buffer or in PCR extraction
buffer, boiling hemolymph, phenol-chloroform extraction, or

91
recent extraction techniques by using a DNeasy Tissue kit
(QIAGEN, Hilden, Germany QIAamp Tissue Kit).
Molecular detection of bacteria in vectors has been
previously reviewed in detail (Brouqui et al., 2004; Parola et
al., 2013; Parola and Raoult, 2001; Raoult and Roux, 1999;
Socolovschi et al., 2009).

10. Diagnosis of vector-borne bacteria in humans

During the diagnosis of vector-borne bacterial diseases
epidemiological information such as pathogen distribution,
history of vector exposure, geographical origin, including
travel history and the clinical status of the patient should be
taken into considerations (Brouqui et al., 2004; Raoult and
Roux, 1999). Appropriate diagnosis of vector-borne bacterial
diseases requires a combination of compatible clinical and
laboratory findings, direct microscopic visualization or
immunodetection of infective organisms in blood or infected
tissue, microbial culture, serological testing, immunoblotting
and molecular diagnosis based on PCR (Brouqui et al., 2004;
Parola et al., 2013; Raoult and Roux, 1999).
Relapsing fever is most commonly diagnosed by standard
Giemsa staining of peripheral blood films and is still the gold

92
standard method for detection and to demonstrate the
presence of the spirochete (B. recurrentis and B. duttonii) in
the blood during the febrile period (Brouqui et al., 2004;
Cutler, 2010). Blood smears are stained according to Giemsa
or White. Mice or rats may also be inoculated
intraperitoneally, and they will demonstrate spirochetes in
their blood within a few days to 2 weeks (Brouqui et al.,
2004; Cutler, 2010). Immunological methods of diagnosis
relapsing fever are not satisfactory. Polymerase chain
reaction (PCR) is highly sensitive although not accessible to
many laboratories in endemic countries in east Africa
(Brouqui et al., 2004; Raoult and Roux, 1999).
Rickettsia spp. and Bartonella spp. diagnostics are based on
the detection of a specific antibody by indirect fluorescent
antibody testing (Brouqui et al., 2004; Parola et al., 2013;
Raoult and Roux, 1999). Cross-adsorption assay allowing
removal of the cross-reacting antibody is mandatory to avoid
cross reaction in closely related bacterial species (Brouqui et
al., 2004). Although sensitive and specific techniques such
as PCR and culture can be performed at reference
laboratories most cases worldwide are diagnosed by
serological analysis such as direct immunofuorescent assay
(DFA) (Raoult and Roux, 1999). The principal limitations

93
with serologic analysis include a usually negative result in
the acute phase when most patients first seek medical care,
poor sensitivity in cases treated early with doxycycline, and
an inability to distinguish between the various Rickettsia
species caused by cross-reactions (Brouqui et al., 2004;
Parola et al., 2013; Parola and Raoult, 2001; Raoult and
Roux, 1999).

11. Treatment of vector-borne bacterial diseases in

humans
Rickettsioses are best treated with doxycycline, 200 mg/day,
given for 1–7 days, depending on the severity of the disease
(Parola et al., 2013; Parola and Raoult, 2001; Raoult and
Roux, 1997; Raoult and Roux, 1999). Tick-borne relapsing
fever is treated by doxycycline, 200 mg given in a single oral
dose and also penicillin, erythromycin, or eftriaxone may
also be effective for treatment of severe forms of the disease
(Badiaga and Brouqui, 2012; Cutler et al., 2010a; Parola and
Raoult, 2001). Epidemic typhus is treated by administration
of tetracycline and chloramphenicol for 5 days (or for 2–4
days after defervescence) has been recommended for each
antibiotic. In outbreak situations, a single 200-mg oral dose

94
of doxycycline can be effective (Badiaga and Brouqui, 2012;
Raoult and Roux, 1999).
B. recurrentis has been successfully  treated with
chloramphenicol, penicillin, tetracycline,  and erythromycin 
the most  efficient of which is the  oral (a single dose of 0.5 g)
or iv administration of tetracycline and also erythromycin in a
single oral dose (0.5 g) is an equally  effective therapy in
pregnant women and children of, 8 years of age (Badiaga
and Brouqui, 2012; Cutler, 2010; Raoult and Roux, 1999).
Treatment of B. quintana in humans is a challenge and the
recommended regimen for patients with chronic bacteraemia
is a combination of gentamicin (3 mg/kg intravenously once
daily for 14 days) and doxycycline (200 mg orally once
daily) for 28 days; for patients with endocarditis, this
regimen is extended to 42 days (Badiaga and Brouqui, 2012;
Raoult and Roux, 1999).
Plague can be treated with gentamicin, oxytetracycline,
tetracycline, chloramphenicol and doxycycline (Eisen and
Gage, 2012; McElroy et al., 2010; Perry and Fetherston,
1997). However, streptomycin still remains the drug of
choice due to its bacteriolytic activity it should be
administered with care to prevent the development of
endotoxic shock (Eisen and Gage, 2012; McElroy et al.,

95
2010). All patients suspected of having bubonic plague
should be placed in isolation until 2 days after starting
antibiotic treatment to prevent the potential spread of the
disease should the patient develop secondary plague
pneumonia (Eisen and Gage, 2012; McElroy et al., 2010;
Perry and Fetherston, 1997).

12. Conclusion
The present review demonstrated that vector-borne zoonotic
bacterial diseases occur in many east African countries and
are more common than are generally known and are rarely
diagnosed (Cutler, 2010; Eisen et al., 2012; Mediannikov et
al., 2013b). Even though, these diseases pose severe public
health problems in residents as well as in international
travellers and cause huge economic losses (Cutler, 2010;
Mediannikov et al., 2013b) they are under reported and most
disease are neglected. Thus, this review is intended to
contribute to the better understanding and alert clinicians,
microbiologists, scientific and medical community working
in east African countries and in other countries around the
world about the importance of vector-borne zoonotic
bacterial diseases transmitted by ticks, lice, fleas and flies.
Increasing the knowledge and awareness on vector-borne

96
bacterial diseases and vector control in east African countries
is very crucial for disease prevention. Public education about
vector-borne bacterial diseases and arthropod control need to
receive great attention and widely disseminated to the public
via various means including television and radio
advertisements and need promotion on the websites of health
authorities. Research on arthropod-borne zoonotic bacteria in
humans, domestic and wild animals, reservoir hosts and
arthropods need to be intensified and also need to consider
the detection of pathogen RNA so as so to detect live disease
causative agents in arthropods. It is also equally important
for the future studies to address the vector competence of
several arthropod species in which bacterial DNA have been
detected in different countries in east Africa by
demonstrating their ability to acquire the pathogen from an
infected host and transmit it to nave susceptible animals
during the subsequent feeding under experimental or
controlled conditions.

97
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115
 Table 1. Common tick species and their vectorized zoonotic bacteria in east Africa
Tick species Country Feeding hosts Human Bacteria detected Reference
biting
Ar. Persicus Ethiopia, South Sudan Domestic birds yes B. anserina (Cutler et al., 2012)
Am. cohaerens ETH, KEY, SOM, S.Sud, UGA, ERT Cattle and yes R. africae, C. (Kumsa et al., 2014b;
other ruminants burneti Mutai et al., 2013)
Am. gemma ETH, KEY SOM Animals Yes R. africae, C. (Kumsa et al., 2014b;
burneti Mutai et al., 2013)
Am. lepidum East Africa Animals - R. africae (Morita et al., 2004; Mura
et al., 2008; Socolovschi
et al., 2007)
Am. hebraeum Zimbabwe,Mozambique Cattle and Yes R. africae (Mutai et al., 2013)
other ruminants
Am. variegatum East Africa Animals Yes R . africae, C. (Kumsa et al., 2014b;
burneti Lorusso et al., 2013;
Mutai et al., 2013)
Ha. leachi East Africa Animals yes SF
Hy. dromedarii ETH, KEY, SOM, S.Sud, UGA, ERT Camels and Yes R. aeschlimannii (Morita et al., 2004)
other ruminants
Hy. impeltatum ETH, KEY, SOM, S.Sud, TAN, Large domestic No report No report (Walker et al., 2003)
ERT,Chad Animals
Hy. m. rufipes East Africa Animals Yes R. aeschlimannii (Kumsa et al., 2014b;
Mura et al., 2008)
Hy. truncatum East Africa Domestic Yes R. aeschlimannii (Kumsa et al., 2014b;
animals Morita et al., 2004; Mutai
et al., 2013)
Or. moubata East Africa Domestic pigs Yes Borrelia duttoni (Cutler, 2010; Cutler et
and poultry al., 2010b)
Or. savignyi ETH, KEY, SOM, S.Sud, UGA, ERT Camels and Yes No report (Walker et al., 2003) 
cattle
Ot. megnini Kenya, Zimbabwe and Madagascar Domestic Yes No report (Walker et al., 2003) 
animals

116
Rh. annulatus ETH, KEY and S.Sudan Ruminants No report R. africae, R. (Mutai et al., 2013)
aeschlimannii
Rh. decoloratus East Africa Animals Yes R. africae, C. (Kumsa et al., 2014b)
burneti
Rh. geigyi UGA and S.Sudan Ruminants No report No report (Walker et al., 2003)
Rh. microplus Zambia, Malawi, Tanzania and Cattle and Yes R . africae (Yssouf et al., 2014)
Zimbabwe, Mozambique, other animals
Madagascar and Kenya
Rh. In all East Africa south of Ethiopia Animals Yes R . africae (Mutai et al., 2013;
appendiculatus and Somalia Yssouf et al., 2014)
Rh. e. evertsi East Africa Domestic Yes R . africae, C. (Kumsa et al., 2014b)
animals burneti
Rh. lunulatus East Africa Domestic No report Not documented (Walker, 2003a)
animals
Rh. muhsamae ETH, KEY, S.Sud, UGA and TAN Animals No report Not documented (Walker, 2003a)
Rh. praetextatus ERT, ETH, SOM, KEY, S.Sud, UGA Animals yes R. massiliae, C. (Kumsa et al., 2014b)
and TAN burnetii
Rh. pravus ERT, ETH, SOM, KEY, S.Sud, UGA Animals No report Not documented (Walker, 2003a)
and TAN
Rh. pulchellus ERT, ETH, SOM, KEY and TAN Ruminants Yes R . africae, C. (Kumsa et al., 2014b)
burneti
Rh. sanguineus East Africa Dog and other Yes R. conorii (Walker et al., 2003) 
animals
Rh. senegalensis S.Sudan and UGA Animals No report Not documented (Walker et al., 2003) 
Rh. simus Zambia, Malawi, Mozambique, Cattle and No report Not documented (Walker et al., 2003) 
Zimbabwe other ruminants
Rh. turanicus East Africa Animals No report Not documented (Walker et al., 2003) 
Rh. zambeziensis Tanzania , Zambia, Mozambique, Cattle and No report Not documented (Walker et al., 2003) 
Zimbabwe other ruminants

ETH=Ethiopia; KEY=Kenya; SOM=Somalia; S.Sud=South Sudan; UGA=Uganda; ER=Eritrea; TAN=Tanzania

117
Table 2. Lice species of humans and domestic animals and their associated zoonotic bacteria reported from east
African countries
Lice species Order Host Bacteria detected Country Reference
Pediculus humanus Anoplura Humans Rickettsia prowazekii, East Africa (Badiaga and Brouqui,
Borrelia recurrentis, 2012; Barker, 1994;
Bartonella quintana, Raoult and Roux,
Acinetobacter baumannii 1999)
Pediculus capitis Anoplura Humans Borrelia recurrentis, East Africa (Badiaga and Brouqui,
Bartonella quintana 2012; Barker, 1994;
Acinetobacter baumannii Raoult and Roux,
1999)
Pthirus pubis Anoplura Humans No report East Africa (Badiaga and Brouqui,
2012; Barker, 1994)
Bovicola (Damalina) bovis Mallophaga Cattle No report East Africa (Barker, 1994)
Linognathus vituli Anoplura Cattle Acinetobacter soli East Africa (Kumsa et al., 2012b)
Solenopotes capillatus Anoplura Cattle No report East Africa (Barker, 1994)
Haematopinus eurysternus Anoplura Cattle No report (Barker, 1994)
Haematopinus quadripertusus Anoplura Cattle No report East Africa (Barker, 1994)
Haematopinus tuberculatus Anoplura Cattle No report (Barker, 1994)
Bovicola (Damalina) ovis Mallophaga Sheep No report East Africa (Barker, 1994)
Linognathus ovillus Anoplura Sheep No report East Africa (Barker, 1994)
Linognathus pedalis Anoplura Sheep No report East Africa (Barker, 1994)
Linognathus africanus Anoplura Sheep No report (Barker, 1994)
Bovicola (Damalinia) caprae Mallophaga Goat No report East Africa (Barker, 1994)
Linognathus stenopsis Anoplura Goat No report (Barker, 1994)
Linognathus africanus Anoplura Goat No report East Africa (Barker, 1994)
Bovicola (Werneckiella) equi, Mallophaga equines No report East Africa (Barker, 1994)
Haematopinus asini. Anoplura equines No report East Africa (Barker, 1994)
Haematopinus suis Anoplura Pig No report East Africa (Barker, 1994)
Trichodectes canis Mallophaga Dog No report East Africa (Kumsa and
Mekonnen, 2011)
Heterodoxus spiniger Mallophaga Dog Acinetobacter pitti East Africa (Kumsa et al., 2012b)
Linognathus steosus Anoplura Dog No report East Africa (Barker, 1994)
Felicola subrostrata Mallophaga Cat No report East Africa (Barker, 1994)
 
118
Fig. 1. Map of Africa showing countries that belong to the

east Africa as defined by the United Nations

119
Fig. 2. Map of Africa showing very few Coxiella burnetii

infection prevalence and disease studies in humans and

animals in east African countries as compared to North,

west, middle and south countries in African.

120
3. TICKS

121
 
122
Ticks are obligate haematophagous arthropods that act as
reservoirs and/or vectors for a wide range of emerging and
re-emerging human and animal diseases worldwide. Ticks
rank second only after mosquitoes as vectors of human
infectious diseases (27). Approximately 10% of the currently
recognized tick species carry human and animal pathogens
(11,13). Ticks are incriminated to transmit several bacteria
like spotted fever group (SFG) rickettsiae, Borrelia spp.,
Coxiella burnetii, Ehrlichia spp., and Bartonella spp
(26,27,35).
In Ethiopia several species of ticks are common and widely
distributed throughout all regions (39). The genera
Amblyomma (8 spp.), subgenus Boophilus (2 spp.), genus
Haemaphysalis (4 spp.), Hyalomma (9 spp.), genus
Rhipicephalus (8 spp.) and genus Ixodes (1 sp.) are known to
exist in Ethiopia (22,33). Ticks are considered to have more
veterinary significance than medical importance in the
country. Most of the studies on the role of ixodid ticks as
vectors of human pathogens in Ethiopia were conducted
before the discovery of well-advanced molecular tools
during the 1950s and 60s (28,29).
Even though there have been a number of studies on the
prevalence, impact on the commercial values of skin and

123
hides of animals, distribution and epidemiology of ixodid
ticks in Ethiopia (16,22,33), only few previous studies were
made on bacteria in ixodid ticks in Ethiopia (19). For
instance, only very few previous studies were made on
Rickettsia species in Ethiopian ixodid ticks (23). Likewise,
the only previous study on Borrelia sp. in ixodid ticks from
Ethiopia is the recent report of 7.3% new Borrelia sp. in
Amblyomma cohaerens collected from cattle in southwest
Ethiopia (21). And also the earlier finding of C. burnetii in
5.3% Amblyomma variegatum and in 10.8% Hyalomma
truncatum ticks before half of a centurary is the only
previous report from Ethiopia (28). Thus, in the current
study we intended to address the occurrence and molecular
identity of vector-borne bacteria including Rickettsia spp.,
Borrelia spp., Bartonella spp. and Coxiella burnetii in
several species of ixodid ticks actively feeding on domestic
animals in nine Districts in Oromia Regional State in
Ethiopia using molecular tools. For this purpose DNA
extracted by using standard techniques from ticks was first
screened individually for human pathogenic bacteria by
genus or species-specific genes and then when it was
necessary genes were amplified by standard PCR followed
by sequencing and then BLAST and phylogenetic analysis.

124
Results of our study revealed the presence of several species
of zoonotic bacteria such as Rickettsia africae the agent of
African Tick Bite Fever (ATBF), Rickettsia aeschlimannii
the agent of Spotted Fever (SF), Rickettsia massiliae the
agent of Spotted Fever (SF), Coxiella burnetii the agent of Q
fever, and new Borrelia spp., related and similar to the agent
of tick-borne relapsing fever (TBRF) in different ixodid tick
species collected from domestic animals in nine districts in
Ethiopia.
In Ethiopia morphological identification of ticks is not easy
due to the great diversity and high prevalence of
Rhipicephalus sanguineus group ticks which are closely
related and very similar species (13). Thus, to address this
issue we tried to identify ixodid ticks of Ethiopia by
employing morphological (12,42,43), protein profiling by
MALDI-TOF MS method (14,24,46) and molecular tools
using 12S RNA gene (2). Hence, after creating a reliable
reference database of spectra obtained from the legs of tick
specimens followed by blind test against the created
reference database we correctly identified 11 ixodid tick
species by the MALDI-TOF-MS method.

125
 
126
3.1. Article 2
 

Spotted fever group rickettsiae in

ixodid ticks in Oromia, Ethiopia.

Kumsa, B., Socolovschi, C., Raoult, D., Parola, P.,

Aix Marseille Université, URMITE, UM63, CNRS 7278,

IRD 198,
Inserm 1095, 13005 Marseille, France.

Ticks and Tick-borne diseases6 (2015) 8–15

127
 
136 
 Ticks and Tick-borne Diseases 6 (2015) 8–15

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

journal homepage: www.elsevier.com/locate/ttbdis

Original article

Spotted fever group rickettsiae in ixodid ticks in Oromia, Ethiopia

Bersissa Kumsa, Cristina Socolovschi, Didier Raoult, Philippe Parola ∗
Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, 13005 Marseille, France

a r t i c l e i n f o a b s t r a c t

Article history: In Ethiopia, information on the transmission of human zoonotic pathogens through ixodid ticks remains
Received 16 May 2014 scarce. To address the occurrence and molecular identity of spotted fever group rickettsiae using molec-
Received in revised form 19 August 2014 ular tools, a total of 767 ixodid ticks belonging to thirteen different species were collected from domestic
Accepted 22 August 2014
animals from September 2011 to March 2014. Rickettsia africae DNA was detected in 30.2% (16/53) Ambly-
Available online 26 September 2014
ommma variegatum, 28.6% (12/42) Am. gemma, 0.8% (1/119) Am. cohaerens, 18.2% (4/22) Amblyomma
larvae, 6.7% (2/60) Amblyomma nymphs, 0.7% (1/139) Rhipicephalus (Boophilus) decoloratus and 25% (1/4)
Keywords:
nymphs of Rh. (Bo.) decoloratus. A markedly low prevalence of R. africae was recorded in both Am. cohaerens
Ixodid ticks
Domestic animals
and Rh. (Bo.) decoloratus (p < 0.0001) compared with that in Am. variegatum and Am. gemma. The preva-
Spotted fever rickettsiae lence of R. africae was markedly low in the western districts (Gachi and Abdela) (p < 0.0001); however, the
Oromia, Ethiopia prevalence of R. africae was relatively high in the central (Ada’a, Wolmara and Arsi) and eastern (Arero,
Moyale and Yabelo) districts, where Am. variegatum and Am. gemma were predominantly associated with
R. africae, respectively. R. aeschlimannii DNA was detected in 45.4% (5/11) Hyalomma marginatum rufipes
and 2.2% (1/46) Hy. truncatum. Moreover, the first report of R. massiliae DNA in 1.9% (1/52) Rhipicephalus
praetextatus ticks in Ethiopia is presented herein. Altogether, these results suggest that the transmission
of spotted fever group rickettsiae through ixodid ticks is a potential risk for human health in different
parts of Ethiopia. Clinicians in this country should consider these pathogens as a potential cause of febrile
illness in patients.
© 2014 Elsevier GmbH. All rights reserved.

Introduction
Ticks are obligate hematophagous arthropods that act as reser-
voirs and vectors for a wide range of human and animal pathogens
worldwide. Approximately 10% of the currently recognized tick
species carry human and animal pathogens. Currently, ticks and
mosquitoes are the major vectors for human and animal disease
agents (Jongejan and Uilenberg, 2004; Parola and Raoult, 2001).
Recent studies have indicated an increase in the spectrum of tick-
borne pathogens affecting humans and animals (Dantas-Torres
et al., 2012; de la Fuente and Estrada-Pena, 2012; Nicholson et al.,
2010).
Ticks are common and widely distributed throughout Ethiopia
(Mekonnen et al., 2001). Several species of ixodid ticks have been
identified in Ethiopia and are considered to have more veterinary
significance than medical importance (Kumsa et al., 2012). Most of
∗ Corresponding author at: URMITE, UMR CNRS 7278, IRD 198, INSERM U1095,
Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France.
Tel.: +33 04 91 32 43 75; fax: +33 04 91 38 77 72.
E-mail address: philippe.parola@univ-amu.fr (P. Parola).
the studies on the role of ixodid ticks as vectors of human pathogens
in Ethiopia were conducted before the discovery of well-advanced
molecular tools during the 1950s and 60s (Burgdorfer et al., 1973;
Philip et al., 1966). Indeed, the isolation of Rickettsia prowazekii, the
causative agent of epidemic typhus, from Amblyomma ticks feeding
on Ethiopian cattle remains a mystery and had never been con-
firmed in any other studies conducted in Ethiopia (Burgdorfer et al.,
1973).
Spotted fever group (SFG) rickettsiae are small, obligate intra-
cellular, short rod, Gram-negative bacteria belonging to the genus
Rickettsia, the family Rickettsiaceae and the order Rickettsiales
(Parola et al., 2013). The genus Rickettsia comprises 3 main
biogroups: the ‘spotted fever group’ (SFG), primarily transmitted
through ixodid ticks, except R. felis and R. akari, which are vec-
tored through fleas and mites, respectively; the ‘typhus group’ (TG),
transmitted through fleas and lice; and the ‘scrub typhus group’
(STG), primarily vectored through chiggers (Parola, 2011; Parola
et al., 2013). Currently, the genus Rickettsia comprises 31 species
that cause diseases in vertebrate hosts, including humans, domes-
tic animals, birds, and wildlife. Some ticks have also been implicated
as SFG rickettsiae reservoirs, as these insects maintain rickettsiae
both transstadially and transovarially.

http://dx.doi.org/10.1016/j.ttbdis.2014.08.001
1877-959X/© 2014 Elsevier GmbH. All rights reserved.
 B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15
In humans, spotted fever rickettsia induces symptoms that
generally include fever, headache, myalgia, rash, local lym-
phadenopathy, and an eschar at the site of the tick bite, which
might be useful in diagnosis (Parola et al., 2013). Rickettsia africae,
the causative agent of ATBF, typically causes symptoms, includ-
ing inoculation eschars, fever, regional lymphadenopathies and the
frequent lack of cutaneous rash or pale vesicular eruptions, and
the distribution of this pathogen is consistent with the geographi-
cal distribution of Amblyomma ticks (Parola et al., 2013). Rickettsia
aeschlimannii is an emerging pathogen recently detected in human
patients in Morocco, South Africa, Algeria, Tunisia and Greece,
causing clinical symptoms resembling Mediterranean spotted fever
caused by R. conorii (fever, generalized maculopapular rashes and
an escar at the tick-bite site) (Parola et al., 2013). Rickettsia massil-
iae is a pathogenic SFG rickettsia associated with clinical symptoms,
such as fever, eschar, night sweats, headache, maculopapular rash
and necrotic skin lesions, in human patients in America, Europe and
Africa (Parola et al., 2013).
Previous studies on rickettsiae in Ethiopian ixodid ticks have
documented the presence of Rickettsia spp. in Amblyomma var-
iegatum, Amblyomma cohaerens and Rhipicephalus spp. in central
Ethiopia (Philip et al., 1966) and Amblyomma spp. (Amblyomma
gemma, Am. variegatum and Am. cohaerens) in the central and
eastern regions of Ethiopia (Burgdorfer et al., 1973). R. aeschli-
mannii, the agent of SFG rickettsiosis, had been detected in
Hyalomma marginatum rufipes and R. africae, the agent of African
tick bite fever (ATBF), has been detected in Amblyomma lep-
idum and Am. variegatum ticks from eastern Ethiopia (Mura et al.,
2008), and also R. africae had been detected in pools of Ambly-
omma and Rhipicephalus ticks (Pader et al., 2012) (summarized in
Fig. 1).
In Ethiopia, where the environment is suitable for ticks, 84% of
the population is involved in agriculture and farmers and their fam-
ily members interact with ectoparasite-infested animals on daily
life activities. Moreover, the confirmatory diagnosis of fever and
other diseases with unknown etiologies is not commonly practiced,
reflecting the lack of health centers in many regions of the coun-
try. Therefore, information concerning ectoparasite-borne bacteria
is extremely important. To update the knowledge on tick-borne
rickettsiae in Ethiopia, the aim of the present study was to address
the occurrence and molecular identity of Rickettsia species in ixo-
did ticks collected from domestic animals in nine districts in Oromia
Regional State in Ethiopia.
Materials and methods
Study areas and animals
In this study, tick samples were collected from cattle, sheep,
dogs and cats in the following districts in Oromia Regional
State in Ethiopia: Arsi (7◦ 56� 2.36�� N, 39◦ 39� 6.54�� E), Wol-
mara (9◦ 6� 17.87�� N, 38◦ 28� 8.50�� E), Kimbibit (9◦ 24� 39.95�� N,
39◦ 21� 14.75�� E), Ada’a (9◦ 31� 60.00�� N, 38◦ 18� 0.00�� E), Bedele
(8◦ 27� 1.76�� N, 36◦ 21� 5.08�� E), Gachi (9◦ 14� 2.57�� N, 35◦ 54� 48.46�� E),
Arero (4◦ 43� 33.28�� N, 38◦ 45� 46.78�� E), Moyale (3◦ 31� 60.00�� N,
39◦ 3� 0.00�� E) and Yabelo (4◦ 53� 41.91�� N, 38◦ 6� 0.59�� E). The
districts are located in six zones in the central, southwestern
and southeast regions of the country, with various climates
and agroecology. The livestock in the study areas are tradi-
tionally managed under extensive production systems (CSA,
2008).
All the study animals were selected irrespective of sex and age.
The animals were categorized into two age groups, young (up to
one year) and adult (older than one year), according to a previous
publication (Kumsa et al., 2012).
9
Collection and identification of ticks
Ticks were collected from September through November of
2011. A thorough visual examination of the body surfaces of each
study animal was conducted to establish the presence or absence
of ticks. All observed ticks attached to the skin of each animal were
carefully removed using forceps or by hand to avoid any damage
to the body, and the specimen were individually placed into small,
pre-labeled plastic tubes containing 70% ethanol for subsequent
identification as previously described (Kumsa et al., 2012, 2014a).
All ticks from the same animal were placed into the same vial and
transported to the Laboratory of the World Health Organization
Collaborative Center for Rickettsial Diseases and Arthropod-borne
Bacterial Diseases in Marseille, France.
The morphological identification of ticks and molecular studies
were performed from January 2012 through March 2014. All adult
ticks were identified at the species level, and the larvae and nymphs
were microscopically identified at the genus level using previously
described morphological identification keys (Hoogstraal, 1956;
Walker et al., 2000, 2003). The sex and stage of each tick was deter-
mined, and photographs of the dorsal and ventral body parts of
each tick specimen were captured. The tick genera are abbreviated
as previously described (Dantas-Torres, 2008).
DNA extraction from ticks
Prior to DNA extraction, each tick specimen was rinsed twice in
sterile water for 15 min, dried on sterile filter paper, and longitu-
dinally dissected into two equal halves. One half of each specimen
was retained as reserve sample to avoid the risk of losing samples
for any reason during or after DNA extraction (Kumsa et al., 2014a,
2014b). Genomic DNA was individually extracted from a total of
767 tick specimens using the QIAamp DNA tissue extraction kit
(Qiagen, Hilden, Germany) according to the manufacturer’s instruc-
tions. In engorged tick DNA was extracted from small portion of
the anterior part of one half of the ticks so as to minimize the PCR
inhibitory effect of large amount of blood in the abdomen. The DNA
from each tick specimen was eluted in 100 �l of Tris–EDTA (TE)
buffer and stored at −20 ◦ C under sterile conditions to preclude
contamination until the sample was used for PCR. To avoid cross-
contamination among the samples during DNA extraction, the
DNA extracting EZI Advanced XL Robot (Qiagen, Hilden, Germany)
was thoroughly disinfected after each extraction according to the
manufacturer’s recommendations. The second half of each tick
specimen was stored at −80 ◦ C as a backup sample.
Molecular detection of Rickettsia species
As a first step, all DNA samples were individually tested
using a genus-specific qPCR system targeting the gltA gene
(RKND03 system (Rolain et al., 2002): RKND03F-5� -GTG-AAT-
GAA-AGA-TTA-CAC-TAT-TTA-T-3� , RKND03R- 5� -GTA-TCT-TAG-
CAA-TCA-TTC-TAA-TAG-C-3� and RKND03P- 6-FAM-CTA-TTA-
TGC-TTG-CGG-CTG-TCG-GTT-C-TAMRA) in SFG rickettsiae and the
Rpr274P gene in typhus group rickettsiae as previously described
(Mediannikov et al., 2010a; Socolovschi et al., 2012). Sterile water
was used as a negative control, and DNA from R. montanensis and
R. typhi were used as positive controls for SFG and typhus group
Rickettsia, respectively. All DNA samples positive for the gltA gene
(RKND03 system) were further confirmed using species-specific
genes for SFG Rickettsia mentioned below.
The Amblyomma spp. (n = 37) and Rhipicephalus (Boophilus)
decoloratus (n = 2) DNA samples positive for SFG Rickettsia were fur-
ther tested using a previously described R. africae species-specific
qPCR targeting the ITS gene (Mediannikov et al., 2012a). Sterile
10 B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15
Fig. 1. Map of Ethiopia showing the areas in which SFG rickettsiae has previously been reported.
water was used as a negative control, and DNA from R. africae was
used as positive control.
Hyalomma spp. DNA samples (n = 6) positive for SFG Rickettsia
were further tested using a previously described species-specific
qPCR targeting the Sca1 gene (a cell-surface antigen) of R. aeschli-
mannii with the same primers and probes used before (Socolovschi
et al., 2012). Sterile water was used as a negative control, and DNA
from R. aeschlimannii was used as positive control.
Rhipicephalus sp. DNA sample (n = 1) positive for SFG Rickettsia
was further tested using a previously described species-specific
qPCR targeting hypothetical proteins of R. massiliae with the same
primers and probes used before (Socolovschi et al., 2012). Sterile
water was used as a negative control, and DNA from R. massiliae
was used as positive control.
Ethical statement
Ethical approval for the collection of ticks from domestic
animals was obtained from the animal research ethics board
(Agreement # 14/160/550/2011) of the College of Veterinary
Medicine and Agriculture at Addis Ababa University. All neces-
sary oral permits were obtained for the field studies, including
permission from each animal owner and the administration and
agricultural office of each district. The collection of ticks from ani-
mals was not harmful or contrary to the welfare of the animals.
Data analysis
Microsoft Excel was used for data management. Data were strat-
ified by districts for analysis and then descriptive statistics such as
percentages and means, were employed to estimate and compare
the prevalence of Rickettsia DNA among different tick species in dif-
ferent districts. Statistical analysis was performed using EpiInfoTM 7
(CDC, 2008). The Mantel–Haenszel (MH) test in EpiInfoTM 7 was
used to determine the relationships between the prevalence of
Rickettsia DNA in different tick species and genera and districts of
collection. A p-value of <0.05 was considered significant.
Results
Ticks were collected from a total of 244 animals (206 cattle, 29
sheep, seven dogs and two cats) in nine districts: Abdela (37 cattle
and fifteen sheep), Gachi (29 cattle and three sheep), Ada’a (nine-
teen cattle, seven dogs and two cats), Wolmara (seventeen cattle
and one sheep), Kimbibit (23 cattle and ten sheep), Arsi (31 cat-
tle), Arero (23 cattle), Moyale (eighteen cattle) and Yabelo (nine
cattle). Overall, 329 male ticks, 314 female (260 non-engorged
and 54 engorged) ticks, 98 nymphs and 26 larvae of ticks were
collected. Greater proportion of the ticks was collected from cat-
tle 89.9% (689/767) while lower proportion was collected from
sheep 6.4% (49/767), from dogs 3% (23/767) and from cats 0.8%
(6/767). From each infested animal from one up to ten ticks were
collected. Further information on tick collection is presented on
Table 1.
A total of 767 ixodid ticks comprising thirteen different species
(Table 2) were screened for both spotted fever and typhus group
rickettsiae DNA using molecular methods. The overall prevalence
of SFG rickettsiae was 6% (46/767) in all ticks collected from nine
Ethiopian districts. In the study 6.5% (45/689) of the ticks from cattle
and 16.7% (1/6) tick from one cat were positive for spotted fever
group rickettsiae, however; spotted fever group rickettsiae was not
detected in ticks taken from sheep (0/49) and dogs (0/23).
 B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15 11

Table 1
Summary of species of ticks collected from different domestic animal in 9 districts in Oromia, Ethiopia.

Tick spp. District Animal spp. No. of ticks No. of each tick No. of
(number) collected spp. collected engorged ticks
(m = male, from one
f = female) animal

Amblyomma cohaerens (n = 119) Abdela Cattle (19) 39 (23m, 16f) 1–3 4 female
Sheep (8) 10 (6m, 4f) 1–2
Gachi Cattle (24) 49 (28m, 21f) 1–3 4 female
Sheep (1) 2 (1m, 1f) 2
Arsi Cattle (2) 2 (2m) 1
Arero Cattle (9) 12 (8m, 4f) 1–2 1 female
Moyale Cattle (2) 3 (2m, 1f) 1–2
Yabelo Cattle (1) 2 (1m, 1f) 2
Amblyomma gemma (n = 28) Ada’a Cattle (1) 1 (1m) 1
Arero Cattle (8) 13 (7m, 6f) 1–2 2 female
Moyale Cattle (11) 20 (10m, 10f) 1–2 2 female
Yabelo Cattle (5) 8 (4m, 4f) 1–2 1 female
Amblyomma lepidum (n = 2) Arero Cattle (1) 1 (1f) 1
Moyale Cattle (1) 1 (1f) 1
Amblyomma variegatum (n = 119) Gachi Cattle (3) 3 (3m) 1
Sheep (2) 2 (2m) 1
Ada’a Cattle (9) 13 (10m, 3f) 1–2
Wolmara Cattle (6) 7 (6m, 1f) 1–2 1 female
Arsi Cattle (19) 25 (20m, 5f) 1–2 2 female
Arero Cattle (2) 2 (2m) 1
Yabelo Cattle (1) 1 (1m) 1
Amblyomma spp. larva (n = 22) Abdela Cattle (8) 8 1
Sheep (2) 2 1
Gachi Cattle (5) 5 1
Ada’a Cattle (4) 4 1
Wolmara Cattle (2) 2 1
Yabelo Cattle (1) 1 1
Amblyomma spp. nymph (n = 60) Abdela Cattle (13) 13 1
Sheep (12) 13 1–2
Gachi Cattle (11) 11 1
Sheep (2) 2 1
Ada’a Cattle (6) 6 1
Cat (1) 1 1
Wolmara Cattle (1) 1 1
Arsi Cattle (10) 10 1
Arero Cattle (1) 1 1
Moyale Cattle (1) 1 1
Yabelo Cattle (1) 1 1
Rhpicephalus (Boophilus) decoloratus (n = 139) Abdela Cattle (14) 14 (14f) 1 3 female
Gachi Cattle (12) 14 (2m, 12f) 1–2 1 female
Sheep (1) 1 (1f) 1
Ada’a Cattle (8) 11 (3m, 8f) 1–2
Wolmara Cattle (15) 31 (16m, 15f) 1–3 2 female
Kimbibit Cattle (15) 20 (7m, 13f) 1–2 1 female
Sheep (1) 3 (3f) 3 2 female
Arsi Cattle (24) 31 (6m, 25f) 1–2 8 female
Arero Cattle (10) 10 (10f) 1
Moyale Cattle (3) 3 (3f) 1
Yabelo Cattle (1) 1 (1f) 1
Rhipicephalus (Boophilus)decoloratus larva (n = 4) Ada’a Cattle (2) 2 1
Kimbibit Cattle (2) 2 1
Rhipicephalus(Boophilus)decoloratus nymph (n = 31) Abdela Cattle (3) 3 1
Gachi Cattle (6) 6 1
Ada’a Cattle (4) 4 1
Wolmara Cattle (11) 11 1
Kimbibit Cattle (6) 6 1
Arero Cattle (1) 1 1
Rhipicephalus praetextatus (n = 52) Ada’a Cattle (7) 9 (4m, 5f) 1–2
Kimbibit Cattle (16) 23 (12m, 11f)) 1–3
Sheep (9) 12 (5m, 7f) 1–2
Arsi Cattle (6) 8 (5m, 3f) 1–2
Rhipicephalus eversti evetsi (n = 37) Abdela Cattle (1) 1(1m) 1
Gachi Cattle (3) 3 (2m, 1f) 1
Sheep (1) 1 (1f) 1
Ada’a Cattle (5) 8 (5m, 3f) 1–2
Wolmara Cattle (2) 4 (2m, 2f) 2
Sheep (1) 1 (1f) 1
Kimbibit Cattle (4) 7 (4m, 3f) 1–2
Arsi Cattle (6) 10 (6m, 4f) 1–2
Arero Cattle (1) 1 (1f) 1
Moyale Cattle (1) 1 (1f) 1 1 female
12 B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

Table 1 (Continued)

Tick spp. District Animal spp. No. of ticks No. of each tick No. of
(number) collected spp. collected engorged ticks
(m = male, from one
f = female) animal

Rhipicephalus pulchullus (n = 118) Ada’a Cattle (2) 2 (1m, 1f) 1

Dog (1) 2 (1m, 1f)) 2
Arero Cattle (22) 52 (30m, 22f) 1–4 4 female
Moyale Cattle (18) 38 (19m, 19f) 1–4 6 female
Yabelo Cattle (8) 24 (14m, 10f) 1–5 3 female
Rhipicephalus sanguienus (n = 14) Ada’a Dog (7) 14 (9m, 5f) 1–6
Rhipicephalus spp. nymph (n = 9) Ada’a Cattle (1) 1 1
Dog (3) 4 1–2
Arero Cattle (2) 2 1
Moyale Cattle (2) 2 1
Hyalomma marignatum rufipes (n = 11) Ada’a Cattle (1) 1 (1m) 1
Wolmara Cattle (1) 2 (1m, 1f)) 2
Kimbibit Cattle (1) 1 (1m) 1
Arero Cattle (2) 2 (2f)) 1
Moyale Cattle (4) 5 (4m, 1f)) 1–2
Hyalomma truncatum (n = 46) Ada’a Cattle (13) 23 (13m, 10f) 1–2 1 female
Wolmara Cattle (3) 6 (3m, 3f) 2
Kimbibit Cattle (1) 2 (1m, 1f) 2
Arsi Cattle (10) 15 (11m, 4f) 1–2
Haemaphysalis leachi (n = 6) Ada’a Dog (1) 1 (1m) 1
Cat (2) 5 (1m, 4f) 1–4 2 female
Haemaphysalis spinulosa (n = 2) Ada’a Dog (1) 2 (2f) 2
Total 767 ticks 9 districts 244 animals 329m, 314f, 26 1–10 ticks 54 females
larvae, 98
nymphs

R. africae DNA was detected in 30.2% (16/53) Am. variegatum,
28.6% (12/42) Am. gemma, 0.8% (1/119) Am. cohaerens, 18.2% (4/22)
Amblyomma larvae, 6.7% (4/60) Amblyomma nymphs, 0.7% (1/139)
Rh. (Bo.) decoloratus and 25% (1/4) nymphs of Rh. (Bo.) decoloratus
(Table 2). The overall prevalence of R. africae was significantly
higher in both Am. variegatum and Am. gemma than in the other tick
species (Am. cohaerens and Rh (Bo.) decoloratus) (Mantel–Haenszel
(MH), p < 0.0001). In the southwestern districts (Gachi and Abdela),
the overall prevalence of R. africae was markedly low compared
with the central and southeastern districts (MH, p < 0.0001). In the
central districts (Ada’a, Wolmara and Arsi), Am. variegatum was
predominantly associated with R. africae, whereas Am. gemma was
predominantly associated with this species in the southeastern dis-
tricts (Arero, Moyale and Yabelo) (Fig. 2). This study is the first to
report R. africae in Am. gemma, Rh (Bo). decoloratus, in larval and
nymphal ticks in Ethiopia. Statistically significant variation was not
found (MH, p < 0.07) in the prevalence of R. africae between 3/54
(5.5%) engorged and 4/260 (1.5%) non engorged female ticks.
R. aeschlimannii DNA was detected in 45.4% (5/11) Hy. m. rufipes
and 2.2% (1/46) Hyalomma truncatum (Table 2). The prevalence of
R. aeschlimannii was significantly higher in Hy. m. rufipes than in
Hy. truncatum ticks (MH, p = 0.001).
R. massiliae DNA was detected in 1.9% (1/52) Rhipicephalus prae-
textatus ticks (Table 2).
Spotted fever group rickettsia DNA was not detected in Haema-
physalis (Hae. leachi (n = 6) and Hae. spinulosa (n = 2) ticks collected
from domestic animals. Similarly, SFG rickettsiae were not detected
in Am. lepidum, Rhipicephalus evertsi evertsi, Rhipicephalus pulchellus
and Rhipicephalus sanguineus ticks (Table 2).
We did not detect any typhus group rickettsia DNA in the 767
ixodid ticks collected from domestic animals in Oromia.
Discussion
The overall prevalence of pathogenic SFG rickettsiae was
detected in 6% (46/767) of ixodid ticks belonging to four genera
(Amblyomma, Rhipicephalus and Hyalomma) collected from ani-
mals in nine districts, highlighting the importance of hard ticks for
human health in Ethiopia. Our molecular strategy was based on
recent scientific studies, and the results were confirmed through
the amplification of two different target genes for each positive
result using positive and negative controls as previously described
(Mediannikov et al., 2012a; Socolovschi et al., 2012).
The absence of spotted fever group rickettsiae in ticks collected
from sheep and dogs most probably reflect the very lower percent-
age of sheep 11.9% (29/244) and dogs 2.9% (7/244) as compared
to the very higher percentage of ticks from cattle as well as the
greater number of cattle 84.4%(206/244) studied. Future studies
with representative number of domestic animals and their ticks are
necessary to address the comparative importance of animal species
and their ticks in Ethiopia.
The detection of R. africae, the agent of African tick bite fever,
in Am. variegatum confirms the results of a previous report associ-
ating R. africae with this Ethiopian tick species (Mura et al., 2008).
Similarly, this spotted fever group rickettsia, erroneously consid-
ered as R. conorii, the agent of Mediterranean spotted fever, was
reported in Am. variegatum ticks from Ethiopia approximately 41
years ago (Burgdorfer et al., 1973; Philip et al., 1966). This erro-
neous consideration of spotted fever group rickettsia as R. conorii,
vectored by Rhipicephalus spp., at that time in Ethiopia was due
to the fact that new techniques such as species sensitive and spe-
cific serology, shell vial assay and molecular methods that enabled
the isolation, identification and characterization of most of these
bacteria including R. africae, vectored principally by Amblyomma
spp., were developed only during the last 20 years after the 1990
(Raoult et al., 2001). In consistent with our observation R. africae
has been detected in Am. variegatum ticks collected from cattle in
several African countries including in (7/26) in Kenya (Mutai et al.,
2013), in (22/39) Uganda and 22/141 Nigeria (Lorusso et al., 2013),
in 4–8% in Nigeria (Reye et al., 2012), in Senegal (Mediannikov et al.,
2010b), in 11/12 in Sudan (Morita et al., 2004) and in 6/6 Mali, 6/6
Niger and 1/13 Burundi (Parola et al., 2001). Similarly, in support of
our finding R. africae was detected in A. gemma ticks collected from
cattle in Kenya (Mutai et al., 2013). Results of our study suggest that
A. gemma is potentially an important vector for R. africae in addition
to the already known Am. variegatum in Ethiopia. The identification
of R. africae in Am. gemma and Rh. (Bo.) decoloratus in the present
study expands the current knowledge concerning tick species that
Table 2
Prevalence of SFG rickettsiae in ixodid ticks using species-specific gene qPCR in 9 districts in Oromia, Ethiopia.

Zones IluAba Bora East Showa West Showa North Showa Arsi Borana Total

District Abdela Gachi Ada’a Wolmara Kimbibit Arsi Arero Moyale Yabelo

1. Prevalence of R. africae in Amblyomma spp. and Rh (Bo). decoloratus

Am. cohaerens 0/49 1/51 (2%) – – – 0/2 0/12 0/3 0/2 (0%) 1/119 (0.8%)
Am. gemma – – 1/1 (100%) – – – 6/13 (46.1%) 4/20 (20%) 1/8 (12.5%) 12/42 (28.6%)
Am. lepdium – – – – – – 0/1 0/1 – 0/2
Am. variegatum – 0/5 4/13 (30.8%) 5/7 (71.4%) – 7/25 (28%) 0/2 – 0/1 16/53 (30.2%)
Amblyomma larva 0/10 1/5 (20%) 2/4 (50%) ½ (50%) – – – – 0/1 4/22 (18.2.%)
Amblyomma nymph 0/26 1/13 (7.7%) 1/7 (14.3%) 0/1 – 1/10 (10%) 0/1 0/1 1/1 (100%) 4/60 (6.7%)
Overall Amblyomma spp. 0/85 3/74 (4%) 8/25 (32%) 6/10 (60%) – 8/37 (21.6%) 6/29 (20.7%) 4/25 (16%) 2/13(15.4%) 37/298 (12.4%)
Rh (Bo). decoloratus 0/14 0/15 0/11 0/31 0/23 1/31 (3.2%) 0/10 0/3 0/1 1/139 (0.7%)
Rh (Bo). decoloratus larva – – 1/2 (50%) – 0/2 – – – – ¼ (25%)
Rh (Bo). decoloratus nymph 0/3 0/6 0/4 0/11 0/6 – 0/1 – – 0/31
Overall Rh (Bo). decoloratus spp. 0/17 0/21 1/17 (3.2%) 0/42 0/31 1/31 (3.2%) 0/11 0/3 0/1 2/174 (1.1%)
Overall tick spp. 0/102 3/95 (3.1%) 9/42 (26.4%) 6/52 (11.5%) 0/31 9/68 (13.2%) 6/40 (15%) 4/28 (14.3%) 2/14 (14.3%) 39/472 (8.3%)
2. Prevalence of R. massiliae in Rhipicephalus spp.
Rh. praetextaatus – – 1/9 (11.1%) – 0/35 0/8 – – – 1/52 (1.9%)
Rh. e.evertsi 0/1 0/4 0/8 0/5 0/7 0/10 0/1 0/1 – 0/37
Rh. pulchellus – – 0/4 – – – 0/52 0/38 0/24 0/118
Rh. sanguineus – – 0/14 – – – – – – 0/14
Rhipicephalus spp. nymph – – 0/5 – – – 0/2 0/2 – 0/9
Overall Rhipicephalus spp. 0/1 0/4 1/40 (2.5%) 0/5 0/42 0/18 0/55 0/41 0/24 1/230 (0.4%)
3. Prevalence of R. aeschlimannii in Hyalomma spp.
Hy. m. rufipes – – 1/1 (100%) 0/2 1/1 (100%) – ½ (50%) 2/5 (40%) – 5/11 (45.4%)
B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

Hy. truncatum – – 0/23 0/6 0/2 1/15 (6.7%) – – – 1/46 (2.2%)

Overall Hyalomma spp. – – 1/24 (4.2%) 0/8 1/3 (33.3%) 1/15 (6.7%) ½ (50%) 2/5 (40%) – 6/57 (10.5%)
4. Prevalence of SFG rickettsia in Haemaphysalis spp.
Hae. leachi – – 0/6 – – – – – – 0/6
Hae. spinulosa – – 0/2 – – – – – – 0/2
Overall Haemaphysalis spp. 0/8 – – – – – – 0/8
Overall prevalence of SFG rickettsiae in ticks 0/103 3% (3/99) 9.6% (11/114) 9.2% (6/65) 1.3% (1/76) 9.9% (10/101) 7.2% (7/97) 8.1% (6/74) 5.3% (2/38) 6% (46/767)
13
14 B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15
Fig. 2. Map of Ethiopia showing the geographical distribution of SFG rickettsiae and their host ticks in the present study.
host R. africae in Ethiopia. The absence of significant variations in
the prevalence of R. africae between engorged and none engorged
female ticks most probably reflects the avoiding of lager portion of
abdomen of engorged ticks containing large blood.
We observed a strong geographic correlation between the high
prevalence of R. africae (in Am. variegatum in the central districts
and Am. gemma in the southeastern districts) and the distribution
of different Amblyomma species across Oromia, consistent with the
previously established geographical distribution of Am. variegatum
as the predominant Amblyomma spp. in central Ethiopia (Kumsa
et al., 2012; Mekonnen et al., 2001) and Am. gemma as the predom-
inant Amblyomma spp. in the southeastern and eastern regions of
Oromia, Ethiopia (Regassa, 2001). Am. cohaerens is the predomi-
nant tick species in western Ethiopia, where the climate is humid
and wet for most of the year, whereas Am. variegatum is the pre-
dominant tick species in areas with high rainfall in central and
other regions of Ethiopia (Mekonnen et al., 2001), and Am. gemma
is restricted to the semi-arid regions east of Rift Valley in Ethiopia
(Regassa, 2001). The high prevalence of R. africae in Am. gemma has
also been reported in Kenya (Mutai et al., 2013).
The markedly low prevalence of 1.9% (3/159) R. africae in west-
ern Oromia (Gachi and Abdela districts), where Am. cohaerens is the
predominant Amblyomma spp. (Kumsa et al., 2012), compared with
that in the central (Ada’a, Wolmara and Arsi) (21.6%, 22/102) and
southeastern (Arero, Moyale and Yabelo) (17.9%, 12/67) districts is
consistent with previous reports of low levels of SFG rickettsiae in
Am. cohaerens compared with Am. variegatum and Am. gemma ticks
in Ethiopia (Burgdorfer et al., 1973; Philip et al., 1966). In contrast,
the absence of R. africae DNA in 109 Am. cohaerens has been
recently reported in southwestern Ethiopia (Mediannikov et al.,
2013). This inconsistency likely reflects differences in the number
of Amblyomma ticks tested and the geographical locations exam-
ined in the two studies. In the present study, we examined a total
of 298 Amblyomma ticks comprising four different species in nine
districts in Oromia; however, in the previous study (Mediannikov
et al., 2013), only 109 Amblyomma ticks representing Am. cohaerens
in a single district in southwest Ethiopia were studied.
The low prevalence of R. africae in 1.1% Rh. (Bo.) decoloratus
compared with the prevalence of this species in Amblyomma ticks
(12.4%) is consistent with the argument that the rate of R. africae
infection in Rhipicephalus ticks is typically low, reflecting con-
comitant co-feeding with Amlyomma ticks (Parola et al., 2013). In
addition, Rh. (Bo.) decoloratus is infected only when cohabiting with
Amblyomma ticks, regarded as the primary reservoir of R. africae
(Macaluso et al., 2003; Mediannikov et al., 2013). R. africae has also
been reported in Rh (Bo.) decoloratus ticks in Liberia (Mediannikov
et al., 2012b) and Nigeria (Ogo et al., 2012).
The prevalence of R. aeschlimannii in 45.4% of Hy. m. rufipes
ticks confirms a previous report in eastern Ethiopia (Mura et al.,
2008) and previous studies in other countries, reporting a preva-
lence of 44.8% in Senegal (Mediannikov et al., 2010a), 60% in Corsica
(Matsumoto et al., 2004), 33.3% in Niger and 15% in Mali (Parola
et al., 2001) and Camargue in southern France (Socolovschi et al.,
2012). R. aeschlimannii has been detected in Hy. truncatum in Sene-
gal (Mediannikov et al., 2010a), Sudan (Morita et al., 2004) and
Kenya (Mutai et al., 2013). The significantly lower prevalence of
R. aeschlimannii in Hy. truncatum suggests that Hy. m. rufipes is
the principal vector of this bacterium, as previously suggested
(Demoncheaux et al., 2012; Parola et al., 2013).
Interestingly, this study is the first to report R. massiliae in 1.9%
(1/52) Rh. praetextatus in Ethiopia. Consistent with this observation,
R. massiliae has been previously detected in several Rhipicephalus
spp. in other African countries, including 8.1% (2/37) in Rh. muh-
samae in Mali (Parola et al., 2001), 8.2% (5/61) in Rh. senegalensis in
Guinea (Mediannikov et al., 2012b) and in 22.4% (11/49) in Rh. guil-
honi in Senegal (Mediannikov et al., 2010a). Rh. praetextatus is one of
 B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15
the predominant tick species observed on ruminants in the central,
north and eastern regions of Ethiopia (Mekonnen et al., 2001).
Although published reports are not available on human hard
tick infestations in Ethiopia, Am. variegatum, Am. cohaerens and Rh.
praetextatus have been reported to feed on humans (Dantas-Torres
et al., 2012; Jongejan and Uilenberg, 2004; Parola and Raoult, 2001).
The significance of the present study is further strengthened
through recent reports of SFG rickettsiae in humans in Ethiopia,
including R. africae in a French man who stayed for 2 months in
western Ethiopia (Stephany et al., 2009) and 2.9% (3/102) SFG Rick-
ettsia DNA in dried thin blood smears prepared to test for malaria
in children with febrile illnesses at Soddo Christian Hospital in
Wolaitta Soddo in southern Ethiopia (Aarsland et al., 2012).
The absence of any typhus group rickettsiae in ixodid ticks
in this study is consistent with previous studies that did not
detect this bacterium in hard ticks in Ethiopia (Mediannikov et al.,
2012a; Mura et al., 2008; Pader et al., 2012), suggesting that other
hematophagous arthropods might play a role in the transmission
of TG rickettsiae.
In conclusion, the findings presented herein provide additional
clear information on the geographic distribution and SFG rick-
ettsia species detected in different ixodid ticks in various regions of
Ethiopia. These results suggest that physicians managing patients
with fever of unknown etiology in Ethiopia and those who care for
travelers from Ethiopia should consider the presence of several SFG
rickettsiae as potential causative agents. In addition, future studies
are needed to address the isolation and culture of SFG rickettsiae
from ticks collected from animals and vegetation (questing ticks),
and in blood samples from human, domestic and other reservoir
animals and determine the public health significance of this bac-
terium in Ethiopia.
Conflict of interest
The authors declare that no competing interests exist.
Acknowledgments
The authors would like to thank the domestic animal owners
in the different districts of Oromia for allowing the collection of
ticks from their animals. We would also like to thank the laboratory
technicians from URMITE, Marseille, particularly Veronique Brice
and Annick Bernard, for technical support.
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3.2. Article 3

Occurrence and genotyping

of Coxiella burnetii in ixodid ticks
in Oromia, Ethiopia

Bersissa Kumsa, Cristina Socolovschi, Lionel

Almeras, Didier Raoult, Philippe Parola *

Aix Marseille Université, URMITE, UM63, CNRS

7278, IRD 198, Inserm 1095, 13005 Marseille,
France.

PLOS Neglected Tropical Diseases (Under Review)

137 
 
138 
Manuscript
Click here to download Manuscript: C. burnetii in ixodid ticks in Oromia for PLOS Neglected Tropical Diseases__final.docx

1 Occurrence and genotyping of Coxiella burnetii in ixodid ticks in Oromia, Ethiopia

3 Bersissa Kumsa, Cristina Socolovschi, Lionel Almeras, Didier Raoult, Philippe Parola *

5 Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales

6 Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO

7 collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de

8 Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France.

10 *Address for correspondence: Pr. Philippe Parola. Unité de Recherche en Maladies

11 Infectieuses et Tropicales Emergentes (URMITE), Faculté de Médecine, 27 bd Jean Moulin,

12 13385 Marseille cedex 5, France. Phone: + 33 (0) 4 91 32 43 75. Fax: + 33 (0)4 91 38 77 72.

13 E-mail address: philippe.parola@univ-amu.fr

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20 Proposed Journal for submission: PLOS Neglected Tropical Diseases

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26 Abstract

27 Background: Although ticks are widely distributed in all parts of Ethiopia, there is a paucity

28 of information on Coxiella burnetii in hard ticks from this country. The present study was

29 conducted from September 2011 to March 2014 to address the occurrence and genotypes of

30 C. burnetii using molecular methods in a total of 842 ticks collected from domestic animals.

31 Methodology/Principal findings: Ticks collected from domestic animals in Ethiopia were

32 tested for Coxiella burnetii by quantitative (q) real time PCR targeting two different genes

33 followed by multi-spacer sequence typing (MST) to determine the genotypes. An overall

34 prevalence of 6.4% (54/842) of C. burnetii was recorded. Coxiella burnetii was detected in

35 28.6% (14/49) of Amblyomma gemma, 25% (31/124) of Rhipicephalus pulchellus, 7.1%

36 (1/14) of Hyalomma marginatum rufipes, 3.2 % (2/62) of Amblyomma variegatum, 3.1%

37 (4/128) of Amblyomma cohaerens, 1.6% (1/63) of Rhipicephalus praetextatus and 0.6%

38 (1/153) of Rhipicephalus (Boophilus) decoloratus. Significantly higher overall frequencies of

39 C. burnetii DNA were observed in Am. gemma and Rh. pulchellus than in other tick species

40 (Mantel-Haenszel (MH), p<0.0001). The overall frequency of C. burnetii was significantly

41 higher (MH, p<0.0001) in ticks from southeastern districts (Arero, Moyale and Yabelo) than

42 from other districts. This study demonstrated the presence of C. burnetii genotype MST 18 in

43 ticks in southeastern districts, and genotype MST 20 was found in ticks in central districts.

44 Conclusions/Significance: This study highlights the importance of ticks in the epidemiology

45 of C. burnetii in Ethiopia. Clinicians in Ethiopia and travelers returning from Ethiopia with

46 fevers of unknown etiology should consider C. burnetii as one of the possible causes of

47 febrile illness in patients.

48

49 Keywords: Ixodid ticks, domestic animals; Coxiella burnetii; Oromia, Ethiopia

50

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51 Author Summary

52 In Ethiopia, there is lack of information on Coxiella burnetii in hard ticks. Thus, the present

53 study was conducted to determine the occurrence and genotypes of Coxiella burnetii in ticks

54 collected from domestic animals in Ethiopia using molecular tools. Our study recorded an

55 overall frequency of 6.4% (54/842) C. burnetii in ticks. Coxiella burnetii was detected in

56 seven species of ticks: Amblyomma gemma, Rhipicephalus pulchellus, Hyalomma

57 marginatum rufipes, Amblyomma variegatum, Amblyomma cohaerens, Rhipicephalus

58 praetextatus and Rhipicephalus (Boophilus) decoloratus. Significantly higher overall

59 frequencies of C. burnetii DNA were observed in Am. gemma and Rh. pulchellus than in the

60 other tick species (Mantel-Haenszel (MH), p<0.0001). The overall frequency of C. burnetii

61 was significantly higher (MH, p<0.0001) in ticks from southeastern districts (Arero, Moyale

62 and Yabelo) than in other districts in Oromia. Our study showed the circulation of C. burnetii

63 genotype MST 18 in ticks in southeastern districts, and genotype MST 20 was found in ticks

64 in central districts. This study highlights the possible importance of ticks in the epidemiology

65 of C. burnetii in Ethiopia.

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 76 Introduction

77 Ticks are one of the major causes of health problems in both humans and animals [1]. Ticks

78 are vectors and reservoirs for several emerging and reemerging infectious pathogens of

79 medical and veterinary importance [2,3]. In Ethiopia, ticks are widely distributed in all agro-

80 ecological zones [4]. Most previously published data on ticks in Ethiopia focus on species

81 distribution, and thus only a very few molecular studies have been conducted on the

82 occurrence of pathogens in ticks in the country.

83 Coxiella burnetii, the causative agent of Q fever, is a small (3-5 µm) polymorphic obligate

84 intracellular Gram-negative bacteria belonging to the genus Coxiella, family Coxiellaceae,

85 order Legionellales, class Gammaproteobacteria and phylum Proteobacteria [5,6]. C. burnetii

86 mainly affects macrophages. It is able to build highly infective spore-like forms that are

87 resistant to environmental influences and therefore can stay infective for several months.

88 Thus, the bacterium is highly resistant to environmental stresses, such as high temperature,

89 osmotic pressure, and ultraviolet light. It can also survive standard disinfectants and is

90 resistant to many other environmental factors [5]. Due to its cosmopolitan distribution, high

91 resistance to environmental stresses, aerosol route of infection, non-specific nature of its

92 symptoms and clinical signs and low infectious dose (1-10 bacteria), C. burnetii is considered

93 a potential bioterrorist agent [6].

94 In humans, C. burnetii causes non-pathognomonic clinical signs ranging from asymptomatic

95 or mildly symptomatic to fatal disease. Acute Q fever can be asymptomatic, or it can

96 manifest as atypical pneumonia, granulomatous hepatitis or appear as a self-limited flu-like

97 syndrome [7]. Syndromes, such as endocarditis, osteomyelitis, septic arthritis, infected aortic

98 aneurysms, chronic hepatitis or chronic pneumonia, have been described as the chronic type

99 of Q fever. In pregnant woman, infection may lead to premature delivery or abortion [5,8].

100 Farmers, veterinarians, abattoir and dairy workers and laboratory technicians are populations

4
101 that are at high risk of contracting infection as an occupational zoonosis [9]. Although

102 inhalation of C. burnetii is considered the main means of infection in humans, consumption

103 of raw milk and milk-products, transplacental infection, intradermal inoculation, blood

104 transfusion, and contact with urine, feces and semen of infected animals are also regarded as

105 possible routes of infection. Excretion of C. burnetii by domestic ruminants is implicated as a

106 major cause of environmental contamination and the source for human infection [5,7].

107 In animals, C. burnetii is known to cause infection in a wide range of species, including

108 ruminants, companion animals, birds and reptiles [5,7]. However, domestic ruminants are

109 implicated as the common source of infection for humans. As is the case of humans,

110 infections in animals are generally asymptomatic [9]. In small ruminants, C. burnetii causes

111 reproductive disorders, such as abortions, premature delivery, delivery of weak offspring,

112 stillbirth in the final stage of gestation, postpartum metritis and infertility [9,10]. In cattle, C.

113 burnetii generally causes less obvious clinical signs, but recent studies have shown that

114 abortion and irregular repeat breeding, metritis and infertility are important risk indicators. In

115 ruminants, C. burnetii is shed via birth fluids and the placenta during normal parturition or

116 abortion and in vaginal mucus, milk, feces, urine and semen. Inhalation or ingestion of C.

117 burnetii with contaminated feed and water from the environment is the major source of

118 infection in animals [9,10].

119 Hard and soft ticks are one of the most important arthropods known to be naturally infected

120 with C. burnetii [5,6,8]. So far, C. burnetii has been reported in more than 40 different tick

121 species in several countries [5]. Ticks acquire C. burnetii during a blood feeding on infected

122 animals and can transmit the bacterium to other mammals during the next blood meal or by

123 aerogenic spread of dried tick fecal excretions, thus playing a role in the maintenance of C.

124 burnetii in the environment [11]. In infected ticks, C. burnetii multiplies in the middle gut

125 cells resulting in high titers of viable organisms that are expelled with feces. Ticks are

5
126 considered important reservoirs and potential vectors due to both transstadial and transovarial

127 transmission of C. burnetii to their offspring in some tick species [5,6]. Furthermore, some

128 previous authors have postulated that there is an increase in the virulence of C. burnetii after

129 passage through ticks. Ticks play an important role in the epidemiology of Q fever by

130 contaminating the environment by excreting C. burnetii via their feces, saliva and coxal

131 fluids. However, no evidence is available reporting the transmission of C. burnetii to humans

132 by blood-feeding ticks [6]. Recently, C. burnetii DNA has been detected in 8.3% of

133 Ornithodoros sonrai in Senegal [11], in fleas in Cyprus [12] and Egypt [13], and in biting flies

134 in the USA [14].

135 In sub-Saharan African countries, including Ethiopia, the importance of C. burnetii as a cause

136 of febrile illness in humans is poorly understood due to the lack of capacity of developed

137 laboratories [15]. Included in the few studies from African countries are reports of antibodies

138 against C. burnetii in the sera of 1%-24% of humans in 7 West African countries [16], 30.9%

139 of humans, 28.3% of cattle, 18.2% of sheep and 32% of goats in Kenya [17], 24.2% of goats

140 and 18.5% of sheep in Gambia [18] and C. burnetii DNA in the blood of 7.8% of cattle and

141 7.5% of goats in Tanzania [19] and 0.3% of febrile patients in Algeria and 0.4% in Senegal

142 [20] has been reported.

143 The only previous study performed nearly half a century ago in Ethiopia [21] reported the

144 detection of C. burnetii in 5.3% of Amblyomma variegatum and 10.8% of Hyalomma

145 truncatum ticks. More recently, an epidemiological study highlighted the seroprevalence of

146 C. burnetii in 31.6% of cattle, 90% of camels and 54.2% of goats in eastern Ethiopia [22]. To

147 obtain better information about the involvement of tick species as reservoirs or vectors of Q

148 fever, in the present study, we aimed to determine the occurrence and genotype of C. burnetii

149 in different ticks species collected from domestic animals in different agro-ecological zones

150 and animal management systems in 9 districts in the Oromia Regional State of Ethiopia.

6
151 Materials and methods

152 Study areas and animals

153 Ixodid ticks were collected from cattle, sheep, dogs and cats in the Arsi, Wolmara, Kimbibit

154 Ada’a, Abdela, Gachi, Arero, Moyale and Yabelo districts in Oromia as described in a

155 previous publication [23]. These districts are located in 6 zones of the central, southwestern

156 and southeast parts of Ethiopia with various climates, agro-ecological and animal

157 management systems (Fig 1). Livestock comprising cattle, goats, sheep and camels in Arero,

158 Moyale and Yabelo districts (Borana zone) are kept under a pastoral type of animal

159 management. This system is characterized by very large animal populations, and livestock are

160 the main source of human food from milk and milk-byproducts and the main source of

161 income for farmers, whereas in all other study districts, animals, including cattle, sheep, goats

162 and equines, are kept under a crop-animal mixed type of management with extensive

163 production systems [22,24]. Animals were selected irrespective of their sex and age. Animals

164 were categorized into two age groups, young (up to one year) and adult (older than one year),

165 accordingly to previous publications [23].

166 Collection and identification of ticks

167 Ticks were collected from September through November of 2011. All the body surfaces of

168 each study animal were thoroughly examined visually to establish the presence or absence of

169 ticks. Ticks attached to the skin of each animal were carefully removed manually using

170 forceps or by hand to avoid any damage to the body and placed into separate, pre-labeled,

171 small, plastic tubes containing 70% ethanol for subsequent identification as previously

172 described. Ticks from the same animal were put in the same vial and transported to the

173 laboratory (URMITE, Marseille, France). Ticks were morphologically identified under a

174 microscope using identification keys described previously [25–27]. Species determination

175 was possible for adult specimens, whereas identification to the genus level was performed for

7
176 larvae and nymphs. For each tick specimen, the sex and developmental stage was determined,

177 and a photograph of the dorsal and ventral body parts was captured. Tick genera are

178 abbreviated as has been described previously [28].

179 DNA extraction from ticks

180 Prior to DNA extraction, each tick specimen was rinsed twice in sterile water for 15 minutes

181 and then dried on sterile filter paper. Each specimen was longitudinally cut into two equal

182 halves [29,30]. One half of each specimen was kept as reserve sample to avoid the risk of

183 losing samples for any reason during or after DNA extraction [23]. Genomic DNA was

184 individually extracted from a total of 842 tick specimens using the QIAamp DNA tissue

185 extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The

186 DNA from each tick specimen was eluted in 100 µl of Tris EDTA (TE) buffer and stored at -

187 20°C under sterile conditions to preclude contamination until the sample was used for PCR.

188 To avoid cross-contamination among samples during DNA extraction, all parts of the DNA

189 extracting EZI Advanced XL Robot were disinfected after each batch of extraction as per

190 recommendations of the manufacturers. The second half of each tick specimen was stored at -

191 80°C as a backup sample.

192 Molecular detection of C. burnetii by quantitative PCR

193 DNA samples were individually tested using C. burnetii-specific qPCR with primers and

194 probes designed for the amplification of the COX spacer sequence as previously described

195 [11]. DNA samples that were positive for the COX spacer sequence were further confirmed

196 by the more sensitive C. burnetii-species specific IS30A spacer as has been performed

197 previously using the same primers and probes [11,31]. Sterile water was used as a negative

198 control, whereas DNA from C. burnetii was used as a positive control. A sample was

199 considered positive when the PCRs were positive for the two different above-mentioned

200 specific genes.

8
201 Genotyping of C. burnetii detected in ticks

202 Multispacer sequence typing (MST) was used to determine the genotypes of C. burnetii in

203 randomly selected representative tick DNA samples that were positive for C. burnetii by

204 qPCR. Seven of the 10 spacer regions in the C. burnetii genome that exhibited higher

205 variation for differentiating the genotypes (Cox2, Cox5, Cox18, Cox37, Cox56, Cox57 and

206 Cox61 manufactured by Eurogentec, Seraing, Belgium) were selected for standard PCR using

207 similar PCR conditions and sequences of primers as described previously [11,31,32]. The

208 genotypes identified by MST were compared to genotypes included in the MST database

209 containing C. burnetii genotypes from countries in Europe and other parts of the world

210 (http://ifr48.timone.univ-mrs.fr/MST_Coxiella/mst/). Detection of amplified products,

211 cleaning of excess primers and nucleotides from DNA, sequencing, assembling and edition of

212 sequences were all performed as described previously [29,30].

213 Ethical statement

214 Ethical approval for the collection of ticks from domestic animals was obtained from the

215 animal research ethics board (Agreement # 14/160/550/2011) of the College of Veterinary

216 Medicine and Agriculture of Addis Ababa University. All necessary permits were obtained

217 from the administration and agricultural office of each district and from each animal owner.

218 Data analysis

219 Microsoft Excel was used for data management. Descriptive statistics, such as percentages

220 and means, were employed to summarize the proportions of ticks positive for C. burnetii

221 DNA. Statistical analyses were performed with EpiInfoTM7. Associations between the

222 prevalence of C. burnetii DNA among different tick species and genera and districts of

223 collection were determined using the Mantel-Haenszel (MH) test with the statistical software

224 EpiInfoTM7. All differences were considered significant at p-values < 0.05.

225

9
226 Results

227 Ticks in the present study were collected from a total of 245 animals (207 cattle, 29 sheep,

228 seven dogs and two cats) in nine districts: Abdela (37 cattle and fifteen sheep), Gachi (29

229 cattle and three sheep), Ada’a (20 cattle, seven dogs and two cats), Wolmara (seventeen cattle

230 and one sheep), Kimbibit (23 cattle and ten sheep), Arsi (31 cattle), Arero (23 cattle), Moyale

231 (eighteen cattle) and Yabelo (nine cattle). The 842 ticks collected consisted of 373 males, 343

232 females, 100 nymphs and 26 larvae. Overall, tick species collected comprised Am. cohaerens

233 (128: 76 m, 52 f), Am. gemma (49: 27 m, 22 f), Am. lepidum (2 f), Am. variegatum (62: 51 m,

234 11 f), Amblyomma larvae (22), Amblyomma nymphs (60), Rh. (Bo.) decoloratus (153: 37 m,

235 116 f), Rh. (Bo.) decoloratus larvae (4), Rh. (Bo.) decoloratus nymphs (31), Rh. praetextatus

236 (63: 34 m, 29 f), Rh. pulchellus (126: 69 m, 57 f), Hy. m. rufipes (14: 10 m, 4 f),

237 Rhipicephalus spp. nymphs (9), Hy. truncatum (53: 33 m, 20 f), Rh. sanguineus (14: 9 m, 5

238 f), Hae. leachci (6: 2 m,4 f) and Hae. spinulosa (2 f). The majority of the ticks were collected

239 from cattle 90.5% (762/842) and a small proportion was collected from sheep 6% (51/842),

240 from dogs 2.7% (23/842) and from cats 0.7% (6/842).

241 The present study recorded an overall frequency of 6.4% (54/842) of C. burnetii DNA in 7

242 species of ixodid ticks out of the total of 13 different species tested using molecular tools

243 (Table 1).

244 C. burnetii DNA was detected in 28.6% (14/49) of Am. gemma, 25% (31/124) of Rh.

245 pulchellus, 7.1 % (1/14) of Hy. m. rufipes, 3.2 % (2/62) of Am. variegatum, 3.1% (4/128) of

246 Am. cohaerens, 1.6% (1/63) of Rh. praetextatus and 0.6% (1/153) of Rh. (Bo.) decoloratus

247 (Table 1). Photographs of ticks positive for C. burnetii is depicted on Fig 2. The overall

248 prevalence of C. burnetii DNA was significantly higher in both Am. gemma and Rh.

249 pulchellus than in the other positive tick species (Mantel-Haenszel (MH), p<0.0001). This

250 study is the first to report finding C. burnetii DNA in Am. gemma, Rh. pulchellus Rh (Bo).

10
251 decoloratus, Hy. m. rufipes and Rh. praetextatus ticks from Ethiopia. Overall, C. burnetii

252 DNA was most frequently found in the Rhipicephalus (7.4%; 33/444) and Amblyomma

253 (6.2%; 20/323) genera than in the Hyalomma (1.5%; 1/67) tick genus. However, C. burnetii

254 was not detected in Haemaphysalis genus (0/8) ticks (Table 1).

255 The overall proportion of positive C. burnetii DNA in ticks was 1.2% (1/80) in Kimbibit,

256 3.3% (4/121) in Arsi, 20% (21/105) in Arero, 27.8% (22/79) in Moyale and 15% (6/40) in

257 Yabelo districts (Table 1). Conversely, C. burnetii DNA was not detected in any ticks from

258 the Abdela (0/107), Gachi (0/107), Ada’a (0/128) and Wolmara (0/75) districts in western

259 and central Oromia (Table 1 and Fig 1). The overall frequency of positive C. burnetii DNA

260 was significantly higher in ticks collected from the Arero, Moyale and Yabelo (Borana zone)

261 southeastern districts than from the Kimbibit and Arsi central districts (Oromia regional state)

262 (MH, p<0.0001) (Fig 1). In this study, 7.1% (54/762) of the ticks from cattle were positive

263 for C. burnetii DNA; however, C. burnetii DNA was not detected in ticks taken from sheep

264 (0/51), dogs (0/23) and (0/6) cats (Table 1).

265 Multispacer sequence typing (MST) of C. burnetii from positive tick samples showed the

266 presence of two different genotypes: the first genotype, MST 18, was found in ticks from the

267 Borana zone (Arero, Moyale and Yabelo districts), whereas the second genotype, MST 20,

268 was detected in ticks from the central districts (Kimbibit and Arsi) (Fig 1). In southeastern

269 districts, genotype MST 18 was detected in Am. cohaerens in the Arero and Moyale districts,

270 in Hy. m. rufipes in the Moyale district, and in Rh. pulchellus and Am. gemma in the Arero,

271 Moyale and Yabelo districts. In the central districts, genotype MST 20 was detected in Rh.

272 praetextatus in the Kimbibit district and in Am. cohaerens, Am. variegatum and Rh. (Bo.)

273 decoloratus in the Arsi district.

274

275

11
276 Discussion

277 The overall frequency of C. burnetii (6.4%; 54/842) in 7 species of ixodid ticks collected

278 from cattle may highlight the possible importance of ticks in the epidemiology of C. burnetii

279 in Ethiopia. A reliable molecular strategy was used that has been validated in recent studies,

280 and our findings were confirmed by positive and negative controls and by amplification of

281 several target genes as previously described [11].

282 The detection of C. burnetii in Am. variegatum for the second time in Ethiopia confirms the

283 earlier report made some 48 years ago in central Ethiopia [21]. In line with our findings, C.

284 burnetii has been recently detected in 19.6% of Am. variegatum in Nigeria [33], in up to

285 37.6% of A. variegatum in two regions of Senegal [11], in 2.5% of pools of Am. variegatum

286 from cattle and in 20% of pools from dogs in Kenya [17]. These studies are the only reports

287 available in ticks from Africa.

288 Our observation of C. burnetii in Am. gemma, Rh. (Bo.) decoloratus, Rh. pulchellus, Hy. m.

289 rufipes, Am. cohaerens and Rh. praetextatus for the first time in Ethiopia is additional

290 knowledge about the species of ticks that host C. burnetii in this country. Consistent with our

291 observation, C. burnetii has been reported in 30% of Rh. (Bo.) decoloratus and in 1.7 to

292 20.9% of Hy. m. rufipes in Senegal [11] and in 20% of pools of Rh. (Bo.) decoloratus in

293 Kenya [17]. Similarly, C. burnetii has recently been detected in different species of ixodid

294 ticks in France [31,34], Italy [35], Spain [36], Germany [37], Australia [38], Argentina [39],

295 Slovakia and Hungary [40], Japan [41] and in 8 species of ticks in China [42].

296 The finding of significantly higher C. burnetii in both Am. gemma and Rh. pulchellus

297 compared with all the other tick spp. most probably indicates the greater importance of these

298 two spp. in the epidemiology of this zoonosis than the other ticks. When considering the

299 established fact that these two tick species are the most predominant species in southeastern

300 Oromia in the Borana zone [4] where animals are kept under a pastoral type of management

12
301 and there is much closer contact between animals and pastoralists due to common

302 consumption of raw milk and meat, it is implied that there is a high likelihood of transmission

303 of C. burnetii to humans in the study districts of this zone. It is hypothesized that ticks

304 become infected during blood feeding on infected animals and then act as reservoirs of C.

305 burnetii and play an important role in maintaining the bacteria in the environment, which

306 may cause infection in wild vertebrates, domestic animals and humans [5,10].

307 However, contrary to our present finding, C. burnetii was not detected in ticks studied in

308 Hungary [43], in ticks in Germany [37] or in ticks in Sweden [44]. This variation is most

309 probably attributed to differences in animal management factors, for example that domestic

310 animals are housed within farms throughout their lifespan and are not allowed to graze on

311 pastures in some European countries and the systematic use of acaricides, such as

312 deltamethrin, that reduce tick populations and alter the ability of ticks to carry C. burnetii.

313 This is different from Ethiopia where animals graze in communal pastures throughout the

314 year that serve as an infectious source for blood feeding ticks, and there is no regular use of

315 acaricides as has been shown before [36]. In support of this fact, a recent serological study

316 reported antibodies against C. burnetii in 54.2% of goats, 31.6% of cattle and 90% of camels

317 in eastern parts of Ethiopia [22].

318 In our study, a statistically significantly higher (MH, p<0.0001) prevalence of positive C.

319 burnetii DNA in ticks was observed in southeastern districts from the Borana zone (Arero,

320 Moyale and Yabelo) than in the central districts (Kimbibit and Arsi). This difference is most

321 probably attributable to factors such as the presence of larger population of cattle, goats,

322 sheep and camel that are usually managed by a pastoral type of management characterized by

323 unrestricted movement of animals in search of water and grazing pasture in districts in the

324 Borana zone. This is in contrast with the central districts that practice a crop-livestock mixed

325 type of farming with restricted movement of ruminants and equines and the absence of

13
326 camels. In support of the finding of the highest frequency of C. burnetii in camel-rearing

327 districts in the Borana zone of the present study, being a camel breeder was reported as a very

328 important risk factor for human Q-fever seropositivity in Chad [45]. Despite the greater

329 number of ticks tested in Abdela (0/107), Gachi (0/107), Ada’a (0/128) and Wolmara (0/75)

330 from the western and central districts of Oromia, C. burnetii was not detected (Table 1). In

331 agreement with our report, a significantly higher prevalence of C. burnetii was recorded in

332 ticks from areas where higher populations of ruminants are kept in Senegal [11] and Kenya

333 [17]. Also in support of our observations, the presence of ticks on animals was reported as the

334 most important risk factor for the occurrence of Q fever and abortion in northern Cyprus [46].

335 Furthermore, several previous studies linked higher populations of ruminants with outbreaks

336 of Q-fever in humans in several countries around the world [47,48].

337 For the first time in Ethiopia, this study provides new information on the presence of two

338 different genotypes of C. burnetii in ticks, MST 18 in ticks from animals in the Borana zone

339 and MST 20 in ticks from animals in the central districts. This finding is in line with previous

340 reports of several genotypes of C. burnetii in ticks [11]. Genotype MST18 was previously

341 identified from human and animal (sheep and goats) clinical samples in Hungary [49],

342 France, Italy, Romania, Greece, Slovakia, Germany and Spain [32,50]. Similarly, genotype

343 MST 20 has been identified in samples from cattle, goat and cow’s milk in Hungary [49],

344 from cow milk and milk products from several countries in Europe, including France, the

345 Netherlands, Portugal, Spain, Switzerland and the United Kingdom, in human clinical

346 samples from France, in a cow’s placenta from Germany and in rodents and in cow’s milk,

347 soil, and goat placental material from the United States [32] and other parts of the world

348 (Qatar and Saudi Arabia), suggesting the worldwide occurrence of this genotype, which

349 supports our finding. Previous studies have shown that genotype MST 20 is usually

350 associated with cattle and their products [49,50]. C. burnetii genotyping is important for

14
351 identifying the source of infection for humans and animals. It is important to note that

352 additional genotypes not detected by our sampling may be circulating in other parts of the

353 country. In the only previous report of C. burnetii genotypes from Africa, MST 6, 35, and 36

354 were detected in ticks in Senegal, and genotype MST 19 was recorded in human patients with

355 endocarditis [11].

356 In conclusion, the findings of this study provide additional information on the geographic

357 distribution and genotypes of C. burnetii in different ticks in Ethiopia. The overall frequency

358 of C. burnetii in ticks from the Borana zone is higher than all other zones. Medical and

359 veterinary professionals in Ethiopia should consider C. burnetii as an emerging pathogen in

360 the country. Ethiopian physicians managing patients with fevers of unknown etiology and

361 travelers with unexplained fevers returning from Ethiopia should consider C. burnetii as one

362 of the potential causative agents. Future studies are necessary to address the isolation, culture,

363 and genotypes of C. burnetii in arthropods, human blood, domestic and other reservoir host

364 animals and the vector competence of these tick species for C. burnetii and its public health

365 and economic significance in Ethiopia.

366 Acknowledgments

367 The authors greatly acknowledge domestic animal owners in the different districts of Oromia

368 for their kindness in allowing us to collect ticks from their animals.

369

15
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522 47. De Lange MM, Schimmer B, Vellema P, Hautvast JL, Schneeberger PM, van
523 Duijnhoven YT (2014) Coxiella burnetii seroprevalence and risk factors in
524 sheep farmers and farm residents in The Netherlands. Epidemiol Infect 142:
525 1231-1244. S0950268813001726 [pii];10.1017/S0950268813001726 [doi].

526 48. Schimmer B, Schotten N, van EE, Hautvast JL, Schneeberger PM, van Duijnhoven
527 YT (2014) Coxiella burnetii seroprevalence and risk for humans on dairy
528 cattle farms, the Netherlands, 2010-2011. Emerg Infect Dis 20: 417-425.
529 10.3201/eid2003.131111 [doi].

530 49. Sulyok KM, Kreizinger Z, Hornstra HM, Pearson T, Szigeti A, Dan A, Balla E, Keim
531 PS, Gyuranecz M (2014) Genotyping of Coxiella burnetii from domestic
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532 ruminants and human in Hungary: indication of various genotypes. BMC Vet
533 Res 10: 107. 1746-6148-10-107 [pii];10.1186/1746-6148-10-107 [doi].

534 50. Astobiza I, Tilburg JJ, Pinero A, Hurtado A, Garcia-Perez AL, Nabuurs-Franssen
535 MH, Klaassen CH (2012) Genotyping of Coxiella burnetii from domestic
536 ruminants in northern Spain. BMC Vet Res 8: 241. 1746-6148-8-241
537 [pii];10.1186/1746-6148-8-241 [doi].
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581 Figure Legends
582

583 Fig 1. Map of Oromia showing the overall prevalence of C. burnetii in ticks and MST in each

584 study district.

585

586 Fig 2. Picture of ticks positive for C. burnetii in 9 districts in Oromia, Ethiopia.

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 626 Table 1. Prevalence of C. burnetii in ixodid ticks using species specific gene qPCR in 9
627 districts in Oromia, Ethiopia.
628
Zones IluAba Bora East Showa West North Arsi Borana
Showa
Showa

District Abdela Gachi Ada’a Wolmara Kimbibit Arsi Arero Moyale Yabelo Total

Am. cohaerens 0/52 0/57 - - - ½(50%) 1/12(8.3%) 2/3(66.7%) 0/2 4/128 (3.1%)
Cattle (n=58) 0/41 0/55 - - - ½(50%) 1/12(8.3%) 2/3(66.7%) 0/2 4/115 (3.5%)
Sheep(n=9) 0/11 0/2 - - - - - - - 0/13
Am. gemma - - 0/1 - - - 5/15(33.3% 7/23(30.4% 2/10(20%) 14/49(28.6%)
) )
Cattle (n=25) - - 0/1 - - - 5/15(33.3% 7/23(30.4% 2/10(20%) 14/49(28.6%)
) )
Am. lepidum - - - - - - 0/1 0/1 - 0/2
Cattle (n=2) - - - - - - 0/1 0/1 - 0/2
Am. variegatum - 0/5 0/14 0/9 - 2/31 (6.4%) 0/2 - 0/1 2/62 (3.2%)
Cattle (n=40) - 0/3 0/14 0/7 2/31 (6.4%) 0/2 - 0/1 2/60 (3.3%)
Sheep(n=2) 0/2 - - - - - - - 0/2
Amblyomma 0/10 0/5 0/4 0/2 - - - - 0/1 0/22
larva
Cattle (n=20) 0/8 0/5 0/4 0/2 - - - - 0/1 0/20

Sheep(n=2) 0/2 - - - - - - - - 0/2

Amblyomma 0/26 0/13 0/7 0/1 - 0/10 0/1 0/1 0/1 0/60
nymph
Cattle (n=44) 0/13 0/11 0/6 0/1 - 0/10 0/1 0/1 0/1 0/44

Sheep(n=14) 0/13 0/2 - - - - - - - 0/15

Cat(n=1) - - 0/1 - - - - - - 0/1

Overall 0/88 0/80 0/26 0/12 - 3/43 (7%) 6/31 9/28 2/15(13.3%) 20/323(6.2%)
Amblyomma (19.3%) (32.1%)
spp.
Rh (Bo). 0/14 0/17 0/11 0/37 0/23 1/37 (2.7%) 0/10 0/3 0/1 1/153 (0.6%)
decoloratus
Cattle (n=104) 0/14 0/16 0/11 0/37 0/20 1/37 (2.7%) 0/10 0/3 0/1 1/149 (0.7%)

Sheep(n=2) - 0/1 - - 0/3 - - - - 0/4

Rh (Bo). - - 0/2 - 0/2 - - - - 0/4

decoloratus
larva
Cattle (n=2) - - 0/2 - 0/2 - - - - 0/4

Rh (Bo). 0/3 0/6 0/4 0/11 0/6 - 0/1 - - 0/31

decoloratus
nymph
Cattle (n=31) 0/3 0/6 0/4 0/11 0/6 - 0/1 - - 0/31

Rh. praetextatus - - 0/12 - 1/38(2.6 0/13 - - - 1/63(1.6%)

%)
Cattle (n=30) - - 0/12 - 1/26(3.8 0/13 - - - 1/51(2%)
%)
Sheep(n=9) - - - - 0/12 - - - - 0/12

Rh. e. evertsi 0/2 0/4 0/11 0/6 0/7 0/12 0/1 0/1 - 0/44

Cattle (n=23) 0/2 0/3 0/11 0/4 0/7 0/12 0/1 0/1 - 0/41

22
 Sheep(n=2) 0/1 0/2 - - - - - 0/3

Rh. pulchellus - - 0/4 - - - 15/58(25.9 12/40(30%) 4/24(16.7%) 31/126(24.6%

%) )
Cattle (n=50) - - 0/2 - - - 15/58(25.9 12/40(30%) 4/24(16.7%) 31/124(25%)
%)
Dog (n=1) - - 0/2 - - - - - - 0/2

Rh. sanguineus - - 0/14 - - - - - - 0/14

Dog (7) - - 0/14 - - - - - - 0/14

Rhipicephalus - - 0/5 - - - 0/2 0/2 - 0/9

spp. nymph
Cattle (n=5) - - 0/1 - - - 0/2 0/2 - 0/5

Dog (3) - - 0/4 - - - - - - 0/4

Overall 0/19 0/27 0/63 0/54 1/76(1.3 1/62(1.6%) 15/72(20.8 12/46(26.1 4/25(16%) 33/444
Rhipicephalus %) %) %) (7.4%)
spp.
Hy. m. rufipes - - 0/2 0/3 0/2 - 0/2 1/5 (20%) - 1/14 (7.1%)

Cattle (n=5) - - 0/2 0/3 0/2 - 0/2 1/5 (20%) - 1/14 (7.1%)

Hy. truncatum - - 0/29 0/6 0/2 0/16 - - - 0/53

Cattle (n=27) - - 0/29 0/6 0/2 0/16 - - - 0/53

Overall - - 0/31 0/9 0/4 0/16 0/2 1/5 (20%) - 1/67 (1.5%)
Hyalomma spp.
Hae. leachci - - 0/6 - - - - - - 0/6
Dog (1) - - 0/1 - - - - - - 0/1
Cat (2) - - 0/5 - - - - - - 0/5
Hae. spinulosa - - 0/2 - - - - - - 0/2
Dog (1) - - 0/2 - - - - - - 0/2
Overall 0/8 - - - - - - 0/8
Haemaphysalis
spp.
Overall 0/107 0/107 0/128 0/75 1/80(1.2 4/121(3.3 21/105(20 22/79(27.8 6/40(15%) 54/842(6.4%)
prevalence in %) %) %) %)
ticks
629

23
Figure
Click here to download high resolution image
Figure
Click here to download high resolution image
Figure
Click here to download high resolution image
3.3. Article 4
 

New Borrelia species detected in

ixodid ticks in Oromia, Ethiopia

Bersissa Kumsa1, Cristina Socolovschi1, Didier

Raoult1, Philippe Parola1

1
Aix Marseille Université, URMITE, UM63,
CNRS 7278, IRD 198, Inserm 1095, 13005
Marseille, France.

Ticks and Tick-Borne diseases (Under Review)

165 
 
167 
Manuscript

1 New Borrelia species detected in ixodid ticks in Oromia, Ethiopia

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2 2
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5 3 Bersissa Kumsa, Cristina Socolovschi, Didier Raoult, Philippe Parola *
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7 4 Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, 13005
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10 5 Marseille, France.
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24 11 *Address for correspondence:
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27 12 URMITE, UMR CNRS 7278, IRD 198, INSERM U1095
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29 13 Faculté de Médecine
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32 14 27 Bd Jean Moulin
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34 15 13385 Marseille cedex 5 France
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16 Tel.: +33 (0)4 91 32 43 75
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39 17 Fax: + 33 (0)4 91 38 77 72
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41 18 E-mail: philippe.parola@univ-amu.fr
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 26 Abstract
1
2 27 Little is known about Borrelia species transmitted by hard ticks in Ethiopia. The present
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5 28 study was conducted from November 2011 through March 2014 to address the occurrence
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7 29 and molecular identity of these bacteria in ixodid ticks infesting domestic animals in Oromia,
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10 30 Ethiopia. A total of 767 ixodid ticks collected from domestic animals were screened for
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12 31 Borrelia DNA by quantitative (q) real-time PCR followed by standard PCR and sequencing
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15 32 to identify the species. Overall, 3.8% (29/767) of the tested ticks were positive for Borrelia
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17 33 DNA, including 8/119 (6.7%) Amblyomma cohaerens, 1/42 (2.4%) Am. gemma, 3/53 (5.7%)
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34 Am. variegatum, 5/22 (22.7%) Amblyomma larvae, 3/60 (5%) Amblyomma nymphs, 2/139
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22 35 (1.4%) Rhipicephalus (Boophilus) decoloratus, 2/31 (6.4%) Rh. decoloratus nymphs, and
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24 36 5/118 (4.2%) Rh. pulchellus using 16S genus-specific qPCR. The prevalence of Borrelia
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27 37 DNA was significantly higher in genus Amblyomma (20/298, 6.7%) than in genus
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29 38 Rhipicephalus (9/417, 2.1%) ticks (P=0.001). Sequencing of PCR products from the flaB and
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32 39 16S genes of Borrelia spp. from Amblyomma ticks showed the presence of a new species
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34 40 between the recurrent fever and Lyme disease groups. However, Borrelia sp. detected in
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41 Rhipicephalus ticks clustered with B. theileri/B. lonestari. The human pathogenicity of the
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39 42 Borrelia sp. detected in Amblyomma ticks from Ethiopia has not yet been investigated,
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41 43 whereas the Borrelia sp. detected in Rhipicephalus ticks in our study is the causative agent of
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44 44 bovine borreliosis in cattle and may have veterinary importance in different parts of Ethiopia.
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46 45 Furthermore, the detection of previously unrecognized Borrelia in Amblyomma and
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49 46 Rhipicephalus ticks generates additional questions concerning the bacterial fauna in hard
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51 47 ticks and will prompt researchers to perform detailed studies for better understanding.
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54 48
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56 49 Keywords: Ticks; Amblyomma; Rhipicephalus; Borrelia species; domestic animals; Oromia,
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50 Ethiopia
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 51 1. Introduction
1
2 52 Ticks are vectors of several pathogens that cause diseases of medical and veterinary
3
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5 53 importance in many countries around the world (Jongejan and Uilenberg, 2004; Parola and
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7 54 Raoult, 2001). Ticks rank second after only mosquitoes as vectors of human infectious
8
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10 55 diseases. Recent investigations have indicated escalating risks from ticks and tick-borne
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12 56 diseases due to expanding tick populations, global warming and increased contact between
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15 57 humans, animals and ticks (Beugnet and Chalvet-Monfray, 2013; Dantas-Torres et al., 2012;
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17 58 De la Fuente and Estrada-Pena, 2012).
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59 In Ethiopia, ticks are very common and widely distributed in all agro-ecological zones
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22 60 (Kumsa et al., 2014a; Kumsa et al., 2012). Species that belong to the Amblyomma,
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24 61 Rhipicephalus, Hyalomma and Haemaphysalis genera of ixodid ticks have been reported to
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27 62 exist in the country and are usually considered to have veterinary significance because they
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29 63 negatively affect the health and productivity of domestic animals as a consequence of direct
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32 64 parasitism and disease transmission (Kumsa et al., 2014c).
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34 65 Although there have been a number of studies on the prevalence, distribution and
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66 epidemiology as well as the impact on the commercial value of animal skins and hides of
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39 67 ixodid ticks in Ethiopia (Kumsa et al., 2012; Kumsa et al., 2014c), the importance of ixodid
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41 68 ticks in human health is poorly understood. The majority of the few studies of the role of
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44 69 ixodid ticks as vectors of human pathogens in Ethiopia were conducted before the discovery
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46 70 of advanced molecular tools during the 1950s-60s (Philip et al., 1966a; Philip et al., 1966b).
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49 71 The genus Borrelia belongs to the order spirochetes (Spirochaetales) and the family
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51 72 Spirochaetaceae. Borrelia species are mainly known for causing Lyme borreliosis (LB) and
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54 73 relapsing fever (RF) in different countries of the world (Cutler, 2010; Takano et al., 2011).
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56 74 Members of the genus Borrelia have been categorized into three major groups. The first, the
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75 Lyme group, comprises 18 named spirochete species and an un-named subgroup,
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 76 genomospecies 2, which includes B. burgdorferi sensu stricto, B. afzelii, B. garinii and
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2 77 species not known to be pathogenic, such as B. andersonii (Rudenko et al., 2011). These
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5 78 Borrelia are transmitted by hard ticks of the Ixodes species (Stanek et al., 2012). The second
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7 79 relapsing fever group comprises Borrelia hermsii, Borrelia parkeri and Borrelia turicatae,
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10 80 which are common in America, and Borrelia duttonii, Borrelia hispanica and Borrelia
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12 81 crocidurae, which are common in Africa and southern Europe. They are transmitted by soft
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15 82 ticks of the family Argasidae (Cutler et al., 2012; Cutler, 2010; Parola et al., 2011), and the
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17 83 human body louse is the principal vector of Borrelia recurrentis in Africa (Cutler et al.,
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84 2010).
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22 85 The third group has been classified as being more closely related to the relapsing fever group
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24 86 due to their molecular similarity revealed by phylogenetic analysis. Most species are
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27 87 associated with ixodid ticks. They include B. theileri, B. lonestari, B. miyamotoi and Borrelia
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29 88 turcica. B. theileri is transmitted by Rhipicephalus spp. Borrelia lonestari is transmitted by
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32 89 Amblyomma americanum in the United States, and B. miyamotoi is transmitted by Ixodes
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34 90 persulcatus in Asia and other Ixodes spp. in Europe. Borrelia turcica is transmitted by
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91 Hyalomma aegyptium in Turkey (Geller et al., 2012; Guner et al., 2004). Furthermore,
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39 92 previous investigators (Yparraguirre et al., 2007) hypothesized that there would be emerging
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41 93 new Borrelia species associated with Lyme-like diseases caused by relapsing fever group
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44 94 spirochetes in tropical and southern hemisphere countries where non-Ixodes species are the
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46 95 most common human biting hard ticks (Cutler et al., 2012; Cutler, 2010).
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49 96 All previous studies on Borrelia spp. in Ethiopia have concentrated on relapsing fever caused
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51 97 by Borrelia recurrentis transmitted by human lice (Boutellis et al., 2013). However recently,
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54 98 a new Borrelia sp. was detected in 7.3% of Amblyomma cohaerens collected from cattle in
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56 99 southwest Ethiopia (Mediannikov et al., 2013); additionally, Borrelia anserina was reported
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100 in 7.5% (3/40) of pools of Argas persicus and Borrelia theileri was found in 12.5% (2/16) of
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 101 pools of Amblyomma and Rhipicephalus spp. in Dire Dawa and Jimma in Ethiopia (Cutler et
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2 102 al., 2012). Thus, the current study was intended to address the occurrence and molecular
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5 103 identity of Borrelia spp. in several species of ixodid ticks that were actively feeding on
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7 104 domestic animals in nine districts of the Oromia Regional State in Ethiopia.
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 126 2. Materials and methods
1
2 127 2.1. Study areas and animals
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5 128 Ticks for the present study were collected from cattle, sheep, dogs and cats from nine districts
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7 129 in Arsi, Wolmara, Kimbibit Ada’a, Abdela, Gachi, Arero, Moyale and Yabelo located in the
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10 130 Oromia Regional State as described in the previous publication (Kumsa et al., 2014c). The
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12 131 districts are located in 6 zones in the central, southwestern and southeastern parts of the
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15 132 country with various climates and agroecology. In all study districts, the main rainy season is
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17 133 from mid-June to September, and the dry season extends from November to May (CSA,
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134 2008).
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22 135 All study animals were selected irrespective of their sex and age. The sex (female, male) and
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24 136 age (young, adult) were recorded for each study animal. The livestock in the study areas are
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27 137 traditionally managed under extensive production systems (Kumsa et al., 2014c).
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29 138 2.2. Collection and identification of ticks
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32 139 Ticks were collected from September through November 2011. All the body surfaces of each
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34 140 study animal were thoroughly examined visually to establish the presence or absence of ticks.
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141 Ticks attached to the skin of each animal were carefully removed manually using forceps or
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39 142 by hand to avoid any damage to the body and placed into separate pre-labeled small plastic
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41 143 tubes containing 70% ethanol for subsequent identification as has been done previously
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44 144 (Kumsa et al., 2014c). All ticks from the same animal were put in the same vial and
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46 145 transported to the Laboratory of the World Health Organization Collaborative Center for
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49 146 Rickettsial Diseases and Arthropod-borne Bacterial Diseases located in Marseille, France.
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51 147 Morphological identification of ticks and molecular studies were performed from January
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54 148 2012 through March 2014. All the adult ticks were identified to the species level, whereas
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56 149 larvae and nymphs were identified to the genus level under a microscope as per the
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150 morphological identification keys described previously (Hoogstraal, 1956; Walker et al.,
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 151 2003; Walker et al., 2000). The sex and life stage of each tick were determined, and then
1
2 152 photographs of the dorsal and ventral body parts of each tick specimen were captured. Tick
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5 153 genera are abbreviated as has been described by Dantas-Torres (Dantas-Torres, 2008).
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7 154 2.3. DNA extraction from ticks
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10 155 Prior to DNA extraction, each tick specimen was rinsed twice in sterile water for 15 minutes
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12 156 and then dried on sterile filter paper. Each specimen was longitudinally cut into two equal
13
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15 157 halves. One half of each specimen was kept as reserve sample to avoid the risk of losing
16
17 158 samples for any reason during or after DNA extraction. Genomic DNA was individually
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159 extracted from a total of 767 tick specimens using the QIAamp DNA tissue extraction kit
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22 160 (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA from
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24 161 each tick specimen was eluted in 100 µl of Tris EDTA (TE) buffer and stored at -20°C under
25
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27 162 sterile conditions to preclude contamination until the sample was used for PCR (Kumsa et al.,
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29 163 2014a; Kumsa et al., 2014b; Kumsa et al., 2012). To avoid cross-contamination among
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32 164 samples during DNA extraction, all parts of the DNA extracting EZI Advanced XL Robot
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34 165 were disinfected after each batch of extractions as per the recommendations of the
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166 manufacturer. The second half of each tick specimen was stored at -80°C as a backup sample.
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39 167 2.4. Molecular detection of Borrelia species by quantitative PCR targeting the 16S
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41 168 rDNA gene
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44 169 As a first step, all DNA samples were individually tested for the presence of Borrelia spp.
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46 170 using a 16S rDNA-based genus-specific quantitative (q) PCR system as has been described
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49 171 previously (Cutler et al., 2012; Mediannikov et al., 2013; Socolovschi et al., 2012b). Sterile
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51 172 water was used as negative control; Borrelia crocidurae DNA was used as a positive control.
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54 173 The samples were considered positive when cycle thresholds (Ct) were <35.
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56 174
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 176 2.5. Molecular identification of Borrelia spp. using the flaB (flagellin) and 16S genes
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2 177 Samples positive for Borrelia spp. by qPCR were further subjected to standard PCR in
3
4
5 178 automated DNA thermal cyclers to amplify a portion of the flaB (flagellin) and 16S genes of
6
7 179 Borrelia spp. as described previously (Cutler et al., 2012; Mediannikov et al., 2013) PCR was
8
9
10 180 performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, Mass).
11
12 181 The amplified products were detected by electrophoresis on 2% agarose gels in 0.5x TBE
13
14
15 182 buffer, stained with ethidium bromide and visualized using ultraviolet (UV)
16
17 183 transillumination. A DNA molecular weight marker (Boehringer-Mannheim VI, Germany)
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19
184 was used to estimate the size of the products. Water and B. crocidurae DNA were used as
20
21
22 185 negative and positive controls, respectively.
23
24 186 The PCR products were cleaned of excess primers and nucleotides using a QIAquick Spin
25
26
27 187 PCR Purification Kit (Qiagen) as per the manufacturer’s instructions. Purified DNA was
28
29 188 sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI PRISM,
30
31
32 189 PE Applied Biosystems, Foster City, CA). All obtained sequences were assembled and edited
33
34 190 using Chromas Pro1.34 (Technelyium Pty. Ltd., Tewantin). The sequences of the Borrelia
35
36
37
191 flaB and 16S genes were then subjected to BLAST analysis to determine the nucleotide
38
39 192 similarities to those available in GenBank and to construct a phylogenetic tree using Mega 5
40
41 193 software (Molecular Evolution Genetic Analysis; The Biodesign Institute, Tempe, AZ).
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43
44 194 2.6. Ethical statement
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46 195 Ethical approval for the collection of ticks from domestic animals was obtained from the
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49 196 animal research ethics board (Agreement # 14/160/550/2011) of the College of Veterinary
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51 197 Medicine and Agriculture of Addis Ababa University. All necessary oral permits were
52
53
54 198 obtained for the field studies, including permission from the administration and agricultural
55
56 199 office of each district and from each animal owner. Collection of ticks from animals was not
57
58
200 harmful and not contrary to the welfare of the animals.
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 201 2.7. Data analysis
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2 202 Microsoft Excel was used for data management. Descriptive statistics, such as percentages
3
4
5 203 and means, were employed to summarize the proportions of ticks positive for Borrelia DNA.
6
7 204 Statistical analysis was performed with EpiInfoTM7. Associations between the prevalence of
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9
10 205 Borrelia DNA among different tick species and genera and districts of collection were
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12 206 determined using the Mantel-Haenszel (MH) test with EpiInfoTM7. A p-value of <0.05 was
13
14
15 207 considered significant.
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17 208
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209
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 210 3. Results
1
2 211 In this study, a total of 767 ixodid ticks comprising 13 different species belonging to four
3
4
5 212 genera, Amblyomma spp. (298 ticks: 216 adults, 22 larvae and 60 nymphs), Rhipicephalus
6
7 213 spp. (404 ticks: 360 adults, 4 larvae and 40 nymphs), Hyalomma spp. (57 adult ticks) and
8
9
10 214 Haemaphysalis spp. (8 adult ticks) (Table 1), were screened for Borrelia DNA using
11
12 215 molecular methods (details of each tick species and their hosts are given in the previous
13
14
15 216 publication (Kumsa et al., 2014c)).
16
17 217 Overall, 3.8% (29/767) of the tested ticks were positive for Borrelia DNA. Pictures of the
18
19
218 tick species that tested positive for Borrelia DNA are depicted in Fig. 1. Borrelia DNA was
20
21
22 219 detected in 8/119 (6.7%) Am. cohaerens, 1/42 (2.4%) Am. gemma, 3/53 (5.7%) Am.
23
24 220 variegatum, 5/22 (22.7%) larva of Amblyomma spp., 3/60 (5%) nymphs of Amblyomma spp.,
25
26
27 221 2/139 (1.4%) Rh. (Bo.) decoloratus, 2/31 (6.4%) nymphs of Rh. (Bo.) decoloratus and 5/118
28
29 222 (4.2%) Rh. pulchellus using a 16S-based genus-specific qPCR system (Table 1). The overall
30
31
32 223 prevalence of Borrelia DNA was significantly higher (Mantel-Haenszel (MH), P=0.001) in
33
34 224 genus Amblyomma (20/298(6.7%) than in genus Rhipicephalus (9/404(2.2%) ticks. However,
35
36
37
225 Borrelia DNA was not detected in Hyalomma (0/57) or Haemaphysalis (0/8) genera ticks
38
39 226 feeding on domestic animals.
40
41 227 In this study, 3.8% (26/689) of the ticks collected from cattle were positive for Borrelia
42
43
44 228 DNA, and 6.1% (3/49) ticks collected from sheep were positive for Borrelia DNA; however,
45
46 229 Borrelia DNA was not detected in ticks taken from dogs (0/23) or cats (0/6). The highest
47
48
49 230 overall prevalence of Borrelia DNA was observed in ticks from the Abdela (11/103 (10.7%))
50
51 231 and Yabelo (4/38 (10.5%)) districts; however, the lowest percentage was seen in ticks from
52
53
54 232 the Ada’a (1/114 (0.9%)) district, and there was significant variation (MH, p=0.002).
55
56 233 A BLAST analysis of 344-bp fragments of flaB gene sequences of Borrelia spp. obtained
57
58
234 from 5 ticks (from 3 specimens of Am. cohaerens, Am. variegatum and nymphs of
59
60
61
62 10
63
64
65
 235 Amblyomma spp.) (Fig. 2) belonging to the genus Amblyomma demonstrated shared
1
2 236 nucleotide homology of only 91-92.6% with a new Borrelia sp. that was recently detected in
3
4
5 237 Am. cohaerens in Ethiopia (AC No. JX089967) and showed 85.6% nucleotide identity to a
6
7 238 Borrelia turcica sequence from GenBank that was detected in Hy. aegyptium in Turkey (AC
8
9
10 239 No. AB109245). Of these 5 sequences from Amblyomma ticks, 3 (from 2 specimens of Am.
11
12 240 cohaerens and 1 Amblyomma nymph specimen) had 100% similarity among themselves, and
13
14
15 241 the other two sequences (from Am. cohaerens and Am. variegatum) had a similarity of 99%
16
17 242 between themselves. The flaB gene sequences of Borrelia spp. obtained from the other 3
18
19
243 Amblyomma ticks (from Am. gemma, larva of Amblyomma spp. and nymph of Amblyomma
20
21
22 244 spp.) (Fig. 2) showed nucleotide similarities of 97.9-100% with the new Borrelia sp. detected
23
24 245 in Am. cohaerens in Ethiopia (AC No. JX089967) and 86.9%-87.7% nucleotide homology
25
26
27 246 with officially recognized species, such as Borrelia duttoni, Borrelia recurrentis and Borrelia
28
29 247 crocidurae. These 3 sequences (from Am. gemma, larva of Amblyomma spp. and nymph of
30
31
32 248 Amblyomma spp.) from Amblyomma ticks had 98-99% similarity among themselves.
33
34 249 However, only 92-93% similarity was observed among the 3 clusters of sequences obtained
35
36
37
250 from Amblyomma ticks, suggesting the presence of 3 closely related Borrelia species in
38
39 251 Ethiopia.
40
41 252 A phylogenetic analysis of a 344-bp fragment of the flaB gene demonstrated that the Borrelia
42
43
44 253 spp. detected in genus Amblyomma ticks from Ethiopia clustered together with Borrelia
45
46 254 turcica detected in Hy. aegyptium in Turkey and formed a separate branch on the
47
48
49 255 phylogenetic tree between the clusters of Lyme disease Borrelia and relapsing fever
50
51 256 associated Borrelia spp. (Figure 2). FlaB gene sequences obtained from the Amblyomma ticks
52
53
54 257 representative of the 3 clusters in Ethiopia were deposited in GenBank and given accession
55
56 258 numbers.
57
58
59
60
61
62 11
63
64
65
 259 However, a BLAST analysis of a 344-bp fragment of the flaB gene sequence of Borrelia spp.
1
2 260 obtained from 2 ticks belonging to the genus Rhipicephalus (Rh. (Bo.) decoloratus and Rh.
3
4
5 261 pulchellus) from Ethiopia showed 97.5-98.7% nucleotide homology to Borrelia sp. BR
6
7 262 (EF141022), which belongs to the genetically related B. theileri and B. lonestari group,
8
9
10 263 detected in Rh. (Bo.) microplus in Brazil. Phylogenetic analysis of a 344-bp portion of the
11
12 264 flaB gene sequence also demonstrated that the Borrelia sp. detected in genus Rhipicephalus
13
14
15 265 ticks from Ethiopia clustered with genetically related B. theileri and B. lonestari, which
16
17 266 belong to the relapsing fever-associated Borrelia group (Figure 2).
18
19
267 Our BLAST search and phylogenetic analysis results for the 344-bp fragment of the flaB
20
21
22 268 gene sequence from Borrelia spp. obtained from positive ticks was further confirmed by a
23
24 269 second BLAST and phylogenetic analysis of 16S gene sequences obtained from the
25
26
27 270 representatives species in both Amblyomma and Rhipicephalus tick genera in Ethiopia.
28
29 271
30
31
32 272
33
34 273
35
36
37
274
38
39 275
40
41 276
42
43
44 277
45
46 278
47
48
49 279
50
51 280
52
53
54 281
55
56 282
57
58
59
283
60
61
62 12
63
64
65
 284 4. Discussion
1
2 285 Our molecular study targeting flaB and 16S genes demonstrated the presence of several
3
4
5 286 Borrelia species in hard ticks from Ethiopia. These genes have been previously used as
6
7 287 reliable molecular tools to investigate and clearly differentiate closely related Borrelia
8
9
10 288 species in different species of arthropods in several countries around the world (Cutler et al.,
11
12 289 2012; Guner et al., 2004; Mediannikov et al., 2013; Parola et al., 2011; Socolovschi et al.,
13
14
15 290 2012b; Yparraguirre et al., 2007).
16
17 291 The results of the phylogenetic analysis of Borrelia spp. detected in genus Amblyomma ticks
18
19
292 from Ethiopia clustered together on a new separate branch at an intermediate position
20
21
22 293 between the Lyme disease group and recurrent fever Borrelia group on the phylogenetic tree,
23
24 294 which supports the previous observation of Borrelia sp. detected in Am. cohaerens in
25
26
27 295 southwestern Ethiopia (Mediannikov et al., 2013) and B. turcica detected in Hy. aegyptium in
28
29 296 Turkey [17,32]. In this study, we described several Borrelia species detected in Amblyomma
30
31
32 297 ticks (Figure 2) in contrast to the previous report of only one Borrelia sp. in Am. cohaerens in
33
34 298 southwestern Ethiopia (Mediannikov et al., 2013). This variation is most probably
35
36
37
299 attributable to differences in the number of species and quantity of Amblyomma ticks tested
38
39 300 and geographical locations in Ethiopia. We have tested a total of 298 Amblyomma ticks
40
41 301 consisting of 4 different species in 9 districts in Oromia; however, Medianniko et al.
42
43
44 302 (Mediannikov et al., 2013) tested only 109 Amblyomma ticks of only Am. cohaerens in 1
45
46 303 district in southwest Ethiopia.
47
48
49 304 Although published reports of hard ticks infesting humans in Ethiopia are not available,
50
51 305 previous studies from other countries indicated that Am. variegatum and Am. cohaerens were
52
53
54 306 reported to bite humans (Estrada-Pena and Jongejan, 1999). In most previous studies from
55
56 307 Ethiopia, Borrelia spp. have been thought to be transmitted by human body lice (Boutellis et
57
58
308 al., 2013; Ramos et al., 2009). Moreover, the absence of a confirmatory diagnosis using
59
60
61
62 13
63
64
65
 309 culture and PCR to accurately determine the species of causative agents of unexplained
1
2 310 fevers in Ethiopia could easily lead to misdiagnosis and inappropriate treatment for malaria
3
4
5 311 and other mimicking diseases. Details of the public health significance of this new Borrelia
6
7 312 spp. in Amblyomma ticks from Ethiopia remains to be investigated.
8
9
10 313 In our study, the observation that the BLAST and phylogenetic analysis of the Borrelia sp.
11
12 314 detected in Rh. (Bo.) decoloratus and Rh. pulchellus from Ethiopia clustered with the B.
13
14
15 315 theileri and B. lonestari group is in agreement with a previous report of Borrelia sp. detected
16
17 316 in Rhipicephalus (Boophilus) microplus in Brazil (Yparraguirre et al., 2007) and Am.
18
19
317 americanum in the USA (Barbour et al., 1996). Our finding also corroborates a previous
20
21
22 318 investigation of B. theileri in pools of Amblyomma and Rhipicephalus ticks from Ethiopia
23
24 319 (Cutler et al., 2012) and the recent finding of B. theileri in Rhipicephalus geigyi in Mali
25
26
27 320 (McCoy et al., 2014). B. theileri is the causative agents of bovine borreliosis in cattle and is
28
29 321 vectored by Rhipicephalus spp. in several countries of the world (Cutler et al., 2012;
30
31
32 322 Mediannikov et al., 2013). Although information is not available from domestic animals in
33
34 323 Ethiopia, in a study that did not determine the species of ticks involved, Culter et al. (Cutler
35
36
37
324 et al., 2012) detected B. theileri in pools of Amblyomma and Rhipicephalus (Boophilus)
38
39 325 decoloratus. As a result, our study is the second to report this bacterium from Ethiopia and is
40
41 326 the first to report the identity of ixodid ticks that carry B. theileri in Ethiopia. B. lonestari, a
42
43
44 327 species that is genetically related to B. theileri, is transmitted by Am. americanum in the USA
45
46 328 and is reported to cause a Lyme-like disease in humans (Barbour et al., 1996). The overall
47
48
49 329 prevalence of 2.2% (9/404) B. theileri recorded in Rhipicephalus ticks in our study is
50
51 330 comparable to the recent report of 0.5% B. theileri in Rh. geigyi in Mali (McCoy et al., 2014)
52
53
54 331 and the report of a less than 1% infection rate of B. theileri in Rh. microplus in Brazil
55
56 332 (Yparraguirre et al., 2007).
57
58
59
60
61
62 14
63
64
65
 333 The overall prevalence of 6.7% (20/298) of Borrelia spp. recorded in Amblyomma spp. in our
1
2 334 study is in line with the previous prevalence of 7.3% new Borrelia sp. in Am. cohaerens
3
4
5 335 (Mediannikov et al., 2013) in southwestern Ethiopia; however, the absence of Borrelia spp.
6
7 336 in Hyalomma ticks in our study is not in agreement with the previous prevalence of 44%
8
9
10 337 (67/153) B. turcica detected in adult Hy. aegyptium in Turkey (Guner et al., 2003). This
11
12 338 difference may be attributable to variations in Borrelia species involved, climatic conditions
13
14
15 339 and reservoir hosts involved in various agroecological zones in different countries (Barbour
16
17 340 et al., 1996).
18
19
341 A very interesting aspect of our present study is the report of the presence of Borrelia spp. in
20
21
22 342 tick species that are reported to be the most common and widely distributed ticks with great
23
24 343 veterinary importance in Ethiopia. For instance, Am. cohaerens is the predominant tick
25
26
27 344 species in the western part of the country (Mekonnen et al., 2001), Am. variegatum and Rh
28
29 345 (Bo). decoloratus are the dominant ticks in the central, northern, southern and eastern parts of
30
31
32 346 the country (Mekonnen et al., 2001; Tafesse, 1996), and Rh. pulchellus and Am. gemma are
33
34 347 the most common ticks in southeastern part of Ethiopia, including the Borana zone and its
35
36
37
348 surroundings (Regassa, 2001).
38
39 349 Interestingly, a recent clinical case of relapsing fever with cutaneous eschar and
40
41 350 radiculopathy in a 77-year-old female French traveler returning home from Ethiopia was
42
43
44 351 reported to be transmitted by hard ticks (Socolovschi et al., 2012a). In addition, Borrelia spp.
45
46 352 DNA was detected at a rate of 2% (2/102) in dried thin blood smears prepared to test malaria
47
48
49 353 in children with febrile illnesses at Soddo Christian Hospital in Wolaitta Soddo in southern
50
51 354 Ethiopia (Aarsland et al., 2012). These recent studies further strengthen the importance of our
52
53
54 355 findings of Borrelia DNA in hard ticks in Ethiopia.
55
56 356
57
58
59
357
60
61
62 15
63
64
65
 358 5. Conclusion
1
2 359 Although the pathogenicity for humans of the Borrelia spp. detected in this work is yet
3
4
5 360 unknown, when considering our findings from the previous study, the Borrelia spp. detected
6
7 361 in Amblyomma ticks in our study is most probably linked to public health importance, and
8
9
10 362 Borrelia sp. in Rhipicephalus spp. ticks has veterinary importance. Future studies are
11
12 363 necessary to address the isolation and culture of Borrelia spp. from ixodid ticks and the blood
13
14
15 364 of human, domestic and other reservoir host animals, as well as public health and economic
16
17 365 significance in various parts of Ethiopia. Furthermore, additional research is needed to
18
19
366 elucidate the vector competence of Amblyomma and Rhipicephalus ticks for Borrelia spp.
20
21
22 367 detected in the present study
23
24 368 References
25
26 369
27 370 Aarsland, S.J., Castellanos-Gonzalez, A., Lockamy, K.P., Mulu-Droppers, R., Mulu, M.,
28 371 White, A.C., Cabada, M.M., 2012. Treatable bacterial infections are underrecognized
29 372 causes of fever in Ethiopian children. Am. J. Trop. Med. Hyg. 87, 128-133.
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373 Barbour, A.G., Maupin, G.O., Teltow, G.J., Carter, C.J., Piesman, J., 1996. Identification of
33 374 an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible
34 375 agent of a Lyme disease-like illness. J. Infect. Dis. 173, 403-409.
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36 376 Beugnet, F., Chalvet-Monfray, K., 2013. Impact of climate change in the epidemiology of
37 377 vector-borne diseases in domestic carnivores. Comp Immunol. Microbiol. Infect. Dis.
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41 379 Boutellis, A., Mediannikov, O., Bilcha, K.D., Ali, J., Campelo, D., Barker, S.C., Raoult, D.,
42 380 2013. Borrelia recurrentis in head lice, Ethiopia. Emerg. Infect. Dis. 19, 796-798.
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44 381 CSA, 2008. Ethiopian agricultural sample survey report on livestock and livestock
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46 382 characteristics vol II, 2007/08, Addis Ababa, Ethiopia.
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48 383 Cutler, S., Abdissa, A., Adamu, H., Tolosa, T., Gashaw, A., 2012. Borrelia in Ethiopian
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51 385 Cutler, S.J., 2010. Relapsing fever--a forgotten disease revealed. J. Appl. Microbiol. 108,
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53 386 1115-1122.
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55 387 Cutler, S.J., Bonilla, E.M., Singh, R.J., 2010. Population structure of East African relapsing
56 388 fever Borrelia spp. Emerg. Infect. Dis. 16, 1076-1080.
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58 389 Dantas-Torres, F., 2008. Towards the standardization of the abbreviations of genus names of
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60 390 ticks (Acari: Parasitiformes: Ixodida). Vet. Parasitol. 154, 94-97.
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 391 Dantas-Torres, F., Chomel, B.B., Otranto, D., 2012. Ticks and tick-borne diseases: a One
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12 398 Geller, J., Nazarova, L., Katargina, O., Jarvekulg, L., Fomenko, N., Golovljova, I., 2012.
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14 400 miyamotoi in Estonian ticks. PLoS One 7, e51914.
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16 401 Guner, E.S., Hashimoto, N., Kadosaka, T., Imai, Y., Masuzawa, T., 2003. A novel, fast-
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18 402 growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey.
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21 404 Guner, E.S., Watanabe, M., Hashimoto, N., Kadosaka, T., Kawamura, Y., Ezaki, T.,
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406 isolated from the hard tick Hyalomma aegyptium in Turkey. Int. J. Syst. Evol.
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27 408 Hoogstraal, H., 1956. African Ixodidae. 1. Ticks of the Sudan (with special reference to
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33 412 Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129 Suppl,
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414 Kumsa, B., Parola, P., Raoult, D., Socolovschi, C., 2014a. Bartonella melophagi in
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41 417 Kumsa, B., Parola, P., Raoult, D., Socolovschi, C., 2014b. Molecular Detection of Rickettsia
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432 Parola, P., Diatta, G., Socolovschi, C., Mediannikov, O., Tall, A., Bassene, H., Trape, J.F.,
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8 435 Parola, P., Raoult, D., 2001. Ticks and tickborne bacterial diseases in humans: an emerging
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12 437 Philip, C.B., Hoogstraal, H., Reiss-Gutfreund, R., Clifford, C.M., 1966a. Evidence of
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16 440 Philip, C.B., Lackman, D.B., Bell, E.J., Hughes, L.E., 1966b. Laboratory identification of
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18 441 typhus isolated by Reiss-Gutfreund from Ethiopian livestock ticks. Am. J. Trop. Med.
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21 443 Ramos, J.M., Malmierca, E., Reyes, F., Tesfamariam, A., 2009. Louse-borne relapsing fever
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25 445 Regassa, A., 2001. Tick infestation of Borana cattle in the Borana Province of Ethiopia.
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28 447 Rudenko, N., Golovchenko, M., Grubhoffer, L., Oliver, J.H., Jr., 2011. Updates on Borrelia
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449 2, 123-128.
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33 450 Socolovschi, C., Honnorat, E., Consigny, P.H., Dougados, J., Passeron, A., Parola, P., Raoult,
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35 452 Ethiopia. J. Travel. Med. 19, 261-263.
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453 Socolovschi, C., Kernif, T., Raoult, D., Parola, P., 2012b. Borrelia, Rickettsia, and Ehrlichia
39 454 species in bat ticks, France, 2010. Emerg. Infect. Dis. 18, 1966-1975.
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41 455 Stanek, G., Wormser, G.P., Gray, J., Strle, F., 2012. Lyme borreliosis. Lancet 379, 461-473.
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43 456 Tafesse, B., 1996. Survey on the distribution of ticks of domestic animals in the eastern zone
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457 of Ethiopia. Trop. Anim Health Prod. 28, 145-146.
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47 458 Takano, A., Fujita, H., Kadosaka, T., Konnai, S., Tajima, T., Watanabe, H., Ohnishi, M.,
48 459 Kawabata, H., 2011. Characterization of reptile-associated Borrelia sp. in the vector
49 460 tick, Amblyomma geoemydae, and its association with Lyme disease and relapsing
50
461 fever Borrelia spp. Environ. Microbiol. Rep. 3, 632-637.
51
52
53 462 Walker, A.R., Bouattour, A., Camicas, J.L., Estrada-Pena, A., Horak, I.C., Latif, A.A.,
54 463 Pegram, R.G., and Preston, P.M., 2003. Tick of domestic Animals in Africa: a Guide
55 464 to Identification of species. Bioscience Reports, Edinburgh Scotland,U.K.
56
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465 Walker J.B., Keirans J.E., Horak I.G., 2000. The Genus Rhipicephalus (Acari, Ixodidae): A
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59 466 Guide to the Brown Ticks of the world. Cambridge Univeristy Press, Cambridge.
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62 18
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 467 Yparraguirre, L.A., Machado-Ferreira, E., Ullmann, A.J., Piesman, J., Zeidner, N.S., Soares,
1 468 C.A., 2007. A hard tick relapsing fever group spirochete in a Brazilian Rhipicephalus
2 469 (Boophilus) microplus. Vector. Borne. Zoonotic. Dis. 7, 717-721.
3
4 470
5 471 Table 1. Prevalence of Borrelia spp. using qPCR based on the 16S gene in ixodid ticks in 9
6 472 districts in Oromia, Ethiopia.
7 Zones IluAba Bora East Showa West North Arsi Borana
8 Showa
Showa
9
10
District Abdela Gachi Ada’a Wolmara Kimbibit Arsi Arero Moyale Yabelo Total
11
12
Am. cohaerens 5/49(10.2% 3/51 - - - 0/2 0/12 (0%) 0/3 (0%) 0/2 (0%) 8/119 (6.7%)
13 ) (5.9%)
14
Am. gemma - - 0/1 - - - 1/13(7.7%) 0/20 0/8 1/42(2.4%)
15
Am. lepidium
16 - - - - - - 0/1 0/1 - 0/2
17
Am. variegatum
18 - 0/5 0/13 0/7 - 3/25(12%) 0/2 - 0/1 3/53(5.7%)
19
20
Amblyomma larva 3/10(30%) 2/5(40%) 0/4 0/2 - - - - 0/1 5/22(22.7%)
21
22
Amblyomma 3/26(11.5% 0/13 0/7 0/1 - 0/10 0/1 0/1 0/1 3/60(5%)
nymph
23 )
24
Overall 11/85(12.9 5/74(6.7%) 0/25 0/10 - 3/37(8.1%) 1/29(3.4%) 0/25 0/13 20/298(6.7%)
25
Amblyomma spp. %)
26
Rh (Bo). 0/14 0/15 1/11(9.1%) 1/31(3.2 0/23 0/31 0/10 0/3 0/1 2/139(1.4%)
27
decoloratus %)
28
Rh (Bo). - - 0/2 - 0/2 - - - - 0/4
29
decoloratus larva
30
Rh (Bo). 0/3 1/6(16.7%) 0/4 0/11 1/6(16.7 - 0/1 - - 2/31(6.4%)
31
decoloratus %)
32
nymph
Rh. e. evertsi
33 0/1 0/4 0/8 0/5 0/7 0/10 0/1 0/1 - 0/37
34
35
Rh. praetextatus - - 0/9 - 0/35 0/8 - - - 0/52
36
37
Rh. pulchellus - - 0/4 - - - 0/52 1/38(2.6%) 4/24(16.7%) 5/118(4.2%)
38
39
Rh. sanguineus - - 0/14 - - - - - - 0/14
40
41
Rhipicephalus - - 0/5 - - - 0/2 0/2 - 0/9
42
spp. nymph
43
Overall 0/18 1/25(4%) 1/57(1.7%) 1/47(2.1 1/73(1.4 0/49 0/66 1/44(2.3%) 4/25(16%) 9/404(2.2%)
44
Rhipicephalus %) %)
45
spp.
Hae. leachi
46 - - 0/6 - - - - - - 0/6
47
48
Hae. spinulosa - - 0/2 - - - - - - 0/2
49
50
Overall 0/8 - - - - - - 0/8
Haemaphysalis
51
spp.
52
Hy. m. rufipes - - 0/1 0/2 0/1 - 0/2 0/5 - 0/11
53
54
Hy. truncatum - - 0/23 0/6 0/2 0/15 - - - 0/46
55
56
Overall
57 - - 0/24 0/8 0/3 0/15 0/2 0/5 - 0/57
Hyalomma spp.
58
Overall tick spp.
59 11/103(10.7 6/99(6.1%) 1/114(0.9 1/65(1.5 1/76(1.3 3/101(3%) 1/97(1%) 1/74(1.3%) 4/38(10.5%) 29/767(3.8%)
%) %) %) %)
60
61
62 19
63
64
65
 473 Fig 1. Picture of ixodid ticks positive for Borrelia spp. collected from domestic animals in 9
1
2 474 districts in Oromia, Ethiopia.
3
4
5 475
6
7 476 Fig 2. Phylogenetic tree of the flaB gene sequences from Borrelia spp. detected in ixodid
8
9
10 477 ticks collected from domestic animals in 9 districts in Oromia, Ethiopia.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 20
63
64
65
Figure

Amblyomma cohaerens male 
Amblyomma cohaerens female 

Amblyomma gemma male 
Amblyomma gemma female 

Amblyomma variegatum male  Amblyomma variegatum female 
 
 Amblyomma spp. nymph 
Amblyomma spp. larva 

Rhipicephalus pulchellus male  Rhipicephalus pulchellus female 
 Rhipicephalus (Bo.) decoloratus female 
Rhipicephalus (Bo.) decoloratus male 

Rhipicephalus (Bo.) decoloratus larva  Rhipicephalus (Bo.) decoloratus nymph 
Figure
100 Borrelia hermsii-AY597796-western North America
49 Borrelia hermsii-AY597795-western North America
Relapsing fever
49 Borrelia coriaceae-D82864-Japan related Borrelia spp.
Borrelia-anserina-DQ849626-Brazil
35
90 B.anserina-X75201-SW EDEN
Borrelia turicatae-AY934630-Florida-USA
42
99 Borrelia parkeri-AY934623-Florida-USA
Roe deer-Borrelia miyamotoi-FJ874925-Poland
Amblyomma americanum-Borrelia-lonestari-AY850064-USA
82
61 Rhipicephalus (Boophilus) microplus-Borrelia-sp.-EF141022-Brazil
100
54 81-Rh.pulchellus-Yabelo-Ethiopia
Borrelia spp. in
89 456Rh(Bo).decoloratus-W olmara-Ethiopia
Ethiopian
Homo sapiens-Borrelia persica-HM194741-Israel
Ornithodoros sonrai-Borrelia crocidurae-JX292921-Mali Rhipicephalus ticks
71
Borrelia duttonii-U28497-USA
100
Ornithodoros-moubata-GU357617-Borrelia-duttonii-Spain
45 Relapsing fever related
B.crocidurae-X75204-Sweden
40
21 Borrelia recurrentis-DQ346831-UK
Borrelia spp.
68 Borrelia recurrentis-D86618-Japan

100 Hyalomma aegyptium-Borrelia turcica-AB109245-Turkey

Hyalomma aegyptium-Borrelia turcica-AB109246-Turkey
98 237-Am.cohaerens-Abdella-Ethiopia
50 610-Am.variegatum-Arsi-Ethiopia
366-nymph-Amblyomma-Abdella-Ethiopia
100
98 370-Am.cohaerens-Abdella-Ethiopia Borrelia spp. in Ethiopian
364-Am.cohaerens-Gachi-Ethiopia
97 Amblyomma-cohaerens-JX089967-Etiopia
Amblyomma ticks
66
2-Am. gemma Arero-Ethiopia
98 222-nymph.Amblyomma-Abdella-Ethiopia
97 335-larva.Amblyomma-Gachi-Ethiopia

100 B.afzelii-X75202-SW EDEN

Borrelia afzelii-D63365-Japan

99 Borrelia burgdorferi-X15661-USA
85 Borrelia burgdorferi-X15660-USA Lyme disease
100
Ixodid ticks-Borrelia andersonii-D83764-Japan
related Borrelia spp. 
65 Borrelia valaisiana-AB022132-Japan
65
Borrelia tanukii-D85070-Japan

29 Borrelia japonica-D82853-Japan
Borrelia sinica-AB022138-Japan
20
Borrelia turdi-D85071-Japan
35
Borrelia garinii-AB035608-Japan
29
99 Borrelia garinii-D82846-Japan
96 Ixodes ricinus-Borrelia garinii-AB178326-Moscow-Russia

0.02
3.4. Article 5

Morphological, MALDI TOF Mass

spectrometry and molecular identification
of ixodid tick species in Oromia, Ethiopia

Bersissa Kumsa1, Lionel Almeras¹, Amina Yussuf1

Oleg Mediannikov1,2, Didier Raoult¹, Philippe
Parola¹*

1. Aix Marseille Université, URMITE, UM63, CNRS 7278,

IRD 198, Inserm 1095, 13005 Marseille, France.
2. Campus Universitaire IRD de Hann, Dakar, Senegal

To be submitted to Veterinary Parasitology

193 
 
194 
Morphological, MALDI TOF Mass spectrometry
and molecular identification of ixodid tick species
in Oromia, Ethiopia

Bersissa Kumsa1, Lionel Almeras¹, Amina Yssouf¹, Oleg

Mediannikov1,2, Didier Raoult¹, Philippe Parola¹*

1
Aix Marseille Université, Unité de Recherche en Maladies
Infectieuses et Tropicales Emergentes (URMITE), UM63,
CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO
Collaborative Center for Rickettsioses and Other Arthropod-
Borne Bacterial Diseases, Faculté de Médecine, 27 bd Jean
Moulin, 13385 Marseille cedex 5, France.
2. Campus Universitaire IRD de Hann, Dakar, Senegal

*Address for correspondence: Philippe Parola, Unité de

Recherche en Maladies Infectieuses et Tropicales
Emergentes (URMITE), Faculté de Médecine, 27 bd Jean
Moulin, 13385 Marseille cedex 5, France. Phone: 33 (0) 4 91
32 43 75. Fax: 33 (0) 4 91 38 77 72.
E-mail address: philippe.parola@univ-amu.fr

Proposed Journal for submission: Veterinary Parasitology

195 
 
196 
Abstract

The Matrix Assisted Laser Desorption/Ionization Time-of-

Flight Mass Spectrometry (MALDI-TOF MS) technology
was recently reported to be a promising method for accurate
identification of arthropods. However, storage conditions of
arthropod specimens, such as ethanol, could alter MS spectra
leading to decreasing confidence scoring identification
compared to fresh specimens. Thus, the present study aimed
to determine the discriminatory power of MALDI TOF MS
to correctly identify at the species level, ticks preserved in
70% ethanol during field collection in Ethiopia for some of
which molecular data are lacking. Following a classification
based on morphological criteria enabling to distinguish 12
tick species, legs from the 85 tick specimens were subjected
to MALDI-TOF-MS. Spectral analysis revealed an intra-
species reproducibility and inter-species specificity,
consistent with the morphological classification. To assess
MALDI-TOF-MS tick species identification accuracy, 41
tick specimens comprising 3 to 5 specimens per tick species
were used to create a reference spectra database which was
evaluated with the spectra of the 44 remaining tick
specimens. The blind tests revealed that 100% of the tick

197 
specimens were correctly identified. With the exception of
specimens from Rh. praetextatus species, relevant Log score
values (LSVs>1.8) of identification were obtained. The
morphological and MALDI-TOF MS identification were
confirmed by sequencing the 12S rRNA gene of 40 tick
specimens representing the 11 ixodid species. Taken
together, these results comfort that MALDI-TOF MS is a
reliable tool for quick tick species identification also
preserved in ethanol on condition that reference spectra
database was built with respective species specimens stored
in the same conditions.

Keywords: ticks; morphology; MALDI-TOF-MS; ethanol;

Oromia; Ethiopia

198 
1. Introduction
Ticks are obligate blood feeding arthropods belonging to the
phylum Arthropoda, class Arachnida and to the order Acari
(Jongejan and Uilenberg, 2004). They are grouped into three
families: Ixodidae (hard ticks) with more than 700 officially
recognized species, Argasidae (soft ticks) comprising
roughly 200 species and Nuttalliellidae with a single species
(Walker, 2003). Ticks can feed on every known class of
vertebrate (Estrada-Pena and De la Fuente, 2014). The blood
from mammals, birds, reptiles and amphibians is the only
critically important source of nutrition for growth,
development of organs for male, female, larvae and nymphs
of ticks (Parola and Raoult, 2001).
Ticks are capable of transmitting a broad range of human
and animal pathogens including viruses, bacteria, protozoa
and helminths. Some of these pathogens are of exceptional
importance because of high morbidity and mortality in both
humans and animals (Parola and Raoult, 2001). The
identification of tick species is a fundamentally important
task in epidemiological studies for examples to establish tick
species distribution maps, to characterize tick fauna and
seasonal pattern, to determine trophic preference of each tick
species and finally to assess tick species responsible of

199 
pathogens transmission to humans and domestic animals
(Dantas-Torres et al., 2012).
Morphological identification of ticks at the genus and
species level using standard taxonomic keys is still a
reference method used by entomologists around the world
(Mediannikov and Fenollar, 2014). Acarologists have
continued to improve tick identification taxonomic keys for
different geographical regions of the worldwide (Jongejan
and Uilenberg, 2004). However, tick species identification at
immature developmental stages (e.g., eggs, larvae and
nymphs) is very difficult and sometime impossible due to the
lack of species specific morphological criteria or keys
(Mediannikov and Fenollar, 2014). The absence of tick
distinctive characters was also reported for sub-species from
the same group (Pegram et al., 1987). In addition, during tick
removing from host, important body parts for anatomical
identification of the specimen could be damaged notably
head parts like basis capituli, chelicerae and hypostome
(Karger et al., 2012; Yssouf et al., 2013a).
Thus, to resolve the aforementioned problems, alternative
methods like molecular tick identification were developed
(Mediannikov and Fenollar, 2014). Although mitochondrial
markers (12S and 16S ribosomal DNAs, cytochrome oxidase

200 
subunit 1 (COX1) or cytochrome b) are frequently used for
tick species identification (Burger et al., 2014a; Burger et al.,
2014b), they are sometimes inefficient to distinguish closely
related tick species (Chitimia et al., 2009; Dvorak et al.,
2014; Murrell et al., 2000). Nuclear markers (18S, 28S,
internal transcribed spacers 1 and 2) were also described for
tick identification, but once more, they failed to discriminate
tick species that belong to the same group or complex
(Burger et al., 2013; Dobson and Barker, 1999). The
development of a universal gene sequence serving as a
“barcode” for identification of all tick species is not yet
available. Thus, this technically time-consuming and
expensive molecular approach is also confronted by the
absence of gene sequences for some tick species in the
GenBank database limiting its used (Mediannikov and
Fenollar, 2014).
During the last decade, matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) was successfully applied for the
identification and phylogenetic classification of
microorganisms (Neupert et al., 2005). This proteomic
approach was also used as taxonomic identification tool for
Drosophila species (Feltens et al., 2010). Recently, MALDI-

201 
TOF MS was successfully applied for the identification of
different types of arthropods (Dvorak et al., 2014; Feltens et
al., 2010; Hoppenheit et al., 2014; Hoppenheit et al., 2013;
Kaufmann et al., 2012a; Kaufmann et al., 2012b; Kaufmann
et al., 2011; Steinmann et al., 2013; Yssouf et al., 2014a;
Yssouf et al., 2013b; Yssouf et al., 2014b). This proteomic
approach was also applied for tick identification using whole
body of fresh specimens (Karger et al., 2012). Yssouf et al
demonstrated that protein spectra from tick legs of fresh or
frozen specimens were sufficient for accurate tick species
identification by MALDI TOF protein profiling (Yssouf et
al., 2013a).
In Ethiopia ticks are very common and widely distributed
throughout all agroecological zones (Kumsa and Mekonnen,
2011). Ticks are routinely identified by morphological
method of identification by using taxonomic keys available
in the literature. Previous studies documented that the genus
Amblyomma (8 spp.), subgenus Boophilus, (2 spp.),
Haemaphysalis (4 spp.), Hyalomma (9 spp.), Rhipicephalus
(15 spp.), Ixodes (1 sp.), Argaus (1 sp.) and Ornithodorous
(2 spp.) exist in the country (Mekonnen et al., 2001). As
these ticks live in sympatry, misidentification has been
frequently reported due to difficulty in correct identification

202 
leading to equivocal position (Mekonnen et al., 2001). Then,
MALDI-TOF MS could be a pertinent alternative to other
routine methods used for tick identification. To facilitate
sample storage and transportation, ticks collected in the field
are generally preserved in 70% ethanol in Ethiopia.
However, this mode of sample preservation was already
reported that it could alter MALDI-TOF MS profiles leading
to decrease of identification rate until the failing of paired
spectra matching with the reference MS database.
Thus, the aim of the present study was to determine the
discriminatory power of MALDI TOF MS for correct
identification of ixodid ticks preserved in 70% ethanol for
two years in Ethiopia.

203 
2. Materials and Methods
2.1. Morphological identification of ticks
Ticks were collected from September through November of
2011 from domestic animals in Ethiopia by a veterinarian.
Ticks were carefully removed using forceps or by hand to
avoid any damage to their body, and the specimen were
placed into small pre-labelled plastic tubes containing 70%
ethanol for subsequent identification (Mekonnen et al.,
2001). Ticks from the same animal were placed into the
same vial and transported to the Laboratory of the World
Health Organization Collaborative Center for Rickettsial
Diseases and Arthropod-borne Bacterial Diseases in
Marseille, France. Ticks were morphologically identified at
the species level under microscope using previously
established taxonomic identification keys (Hoogstraal, 1956;
Walker, 2003; Walker, 2000). For each tick, photographs of
the dorsal and ventral sides were captured and the gender
was determined. The tick genera and species are abbreviated
as previously described (Dantas-Torres, 2008). Then adult
tick specimens were selected for MALDI-TOF protein
profiling study.

204 
2.2. MALDI-TOF MS data and sample preparation
2.2.1. Sample preparation
All the ixodid ticks studied by MALDI TOF MS in the
present study were preserved in 70% ethanol for about 2
years. Each specimen was kept for 10 min in decreasing
ethanol concentration solutions from 70% to 10% (v/v),
followed by a wash in distilled water prior to a sample
drying on sterile filter paper as previously described (Yssouf
et al., 2013a). Then, 4 legs per tick specimen were cut with
sterile scalpels, and the remaining body parts were kept for
subsequent DNA extraction. Legs from individual tick were
manually crushed and homogenized in 40 μl of 10% formic
acid and 40 µl of 50% acetonitrile in 1.5-mL micro-
centrifuge tubes using pellet pestles (Fischer Scientific). A
quick spin was performed to discard debris and 1 µl of the
supernatant was deposited onto a spot on an MSP 96 target
polished steel micro Scout target plate (Bruker Daltonics,
Wissembourg, France), in quadruplicate. After air-drying, 1
µL of matrix solution composed of saturated α-cyano-4-
hydroxycynnamic acid (Sigma, Lyon. France), 50%
acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich,
Dorset, UK) and HPLC-grade water was directly overlaid on
each spot sample on the target plate, dried for several

205 
minutes at room temperature and introduced into the
MALDI-TOF-MS machine. To control loading on mass
spectra steel, matrix quality and MALDI-TOF apparatus
performance, matrix solution was loaded in duplicate onto
each MALDI-TOF plate with or without a bacterial test
standard (Bruker protein Calibration Standard I).

2.2.2. MALDI-TOF MS data and spectra analysis

Protein mass profiles for each tick sample was acquired a
Microflex LT spectrometer (Bruker Daltonics) with Flex
Control software (Bruker Daltonics). The spectra were
recorded in a linear positive-ion mode with an acceleration
voltage of 20 kV, within a mass range of 2-20 kD. Each
spectrum corresponded to an accumulation of 240 laser shots
performed in six regions of the same spot. The resulting
spectra were visualized with Flex Analysis 3.3 software and
exported to ClinProTools version v.2.2 and MALDI-
Biotyper v.3.0 (Bruker Daltonics, Germany) for analysis
(Fotso et al., 2014).

2.2.3. Spectra analysis and reference database creation

The spectra reproducibility from specimens of the same tick
species and the spectra specificity from specimens of

206 
different tick species were determined by analyzing and
comparing the average spectral profiles obtained from the
four spots for each individual tick specimen using the
ClinProTools 2.2 software (Bruker Daltonics). After the
validation of reproducibility and specificity of spectra, they
were loaded into the MALDI Biotyper 3.0 to create a
spectral database. The database was composed of a total of
11 species (Am. cohaerens n=4, Am. gemma n=5,
Am. variegatum n=4, Hy. truncatum n=4, Hy. m. rufipes n=3,
Rh (Bo).decoloratus n=3, Rh. e. evertsi n=4, Rh. pulchellus
n=4, Rh. sanguienus n=3, Rh. bergeoni n=4 and
Rh. praetextatus n=3) of ixodid ticks preserved in 70%
ethanol with 3 to 5 specimens for each species including
both gender. These specimens were used to create the
database 2 (Table 1).

2.2.4. Evaluation of tick identification by blind tests

For the blind tests, a total of 45 ticks with a minimum of
2 specimens per tick species were tested successively against
home-made reference spectra database (Database 1 and
Database 2) (Table 1). Database 1, created with fresh or
frozen arthropod specimens, includes the leg protein spectra
of 6 tick species (Amblyomma variegatum infected by

207 
R. africae, Rh. sanguineus, Hyalomma marginatum rufipes,
Ixodes ricinus, D. marginatus and D. reticulatus ),
30 mosquito species (Anopheles gambiae molecular form
M and An. gambiae molecular form S, An. funestus,
An. ziemanni, An. arabiensis, An. wellcomei, An. rufipes,
An. pharoensis, An. coustani, An. claviger, An. hyrcanus,
An. maculipennis, Culex quinquefasciatus, Cx. pipiens,
Cx. modestus,Cx. insignis, Cx. neavei, Ae. albopictus, Aedes
excrucians, Ae vexans, Ae. rusticus, Ae. dufouri,
Ae. cinereus, Ae. fowleri, Ae. aegypti, Ae. caspius, Mansonia
uniformis, Orthopodomyia reunionensis, Coquillettidia
richiardii and Lutzia tigripes,), and other arthropods
including louse (Pediculus humanus corporis ), triatomine
(Triatoma infestans) and bedbugs (Cimex lectularius), as
well as the spectra obtained from the bodies (without the
abdomens) of 5 flea species (Ctenocephalides felis, Ct.
Ct. canis, Archaeopsylla erinacei, Xenopsylla cheopis and
Stenoponia tripectinata) (Yssouf et al., 2013a; Yssouf et al.,
2014a; Yssouf et al., 2013b; Yssouf et al., 2014b). Database
2 includes Database 1 which was created from the leg
protein spectra of 11 tick species stored in ethanol (Table 1).
The reliability of the identification was estimated based on
the Log Score values (LSVs) exhibited by the MALDI-

208 
Biotyper software, ranged from 0 to 3. These LSVs
correspond to the degree of homology between the query
mass spectra and the reference spectra. An LSV was
obtained for each spectrum of the samples tested.

2.2.5. Cluster analysis

Cluster analysis was used to generate MSP dendogram based
on the comparison of the MSP spectra given by MALDI-
Biotyper software, clustering them according to the protein
mass profile (i.e., their mass signals and intensities). MSP of
the 12 tick species included in the database creation, were
used to determine how organisms are related to one another.

2.2.6. DNA extraction and molecular identification of

ticks by 12S gene
Prior to DNA extraction, each tick specimen was rinsed
twice in sterile water for 15 minutes, dried on sterile filter
paper (Kumsa et al., 2014a). Genomic DNA was
individually extracted from a total of 40 tick specimens
using the QIAamp DNA tissue extraction kit (Qiagen,
Hilden, Germany) according to the manufacturer‟s
instructions. The DNA from each tick specimen was eluted
in 100 µl of Tris-EDTA (TE) buffer and stored at -20°C

209 
under sterile conditions to preclude contamination until the
sample was used for PCR as has been used previously
(Kumsa et al., 2014a; Kumsa et al., 2014b). For molecular
identification of tick species, DNA samples of 1-7 individual
ticks per each tick species that were used to create the
database were subjected to standard PCR in an automated
DNA thermal cycler to amplify a fragment of the 360-base
pair from the mitochondrial 12S RNA gene as described
before (Beati and Keirans, 2001). The detection of amplified
products, the removal of excess primers and nucleotides
from the DNA, sequencing, the assembly and edition of
sequences and BLAST analysis were all performed as
previously described (Kumsa et al., 2014b; Kumsa et al.,
2012b). Phylogenetic tree was constructed using Mega 5
software (Molecular Evolution Genetic Analysis; The
Biodesign Institute, Tempe, AZ).

Ethical statement
Ethical approval for the collection of ticks from domestic
animals was obtained from the animal research ethics board
(Agreement # 14/160/550/2011) of the College of Veterinary
Medicine and Agriculture of Addis Ababa University. All
necessary permits were obtained from the administration and

210 
agricultural office of each district and from each animal
owner. The collection of ticks in the field did not involve
privately owned, wildlife, national park or other protected
areas and endangered or protected species.

211 
3. Results
3.1. Morphological identification of ticks
Ticks for the present study were collected from domestic
animals in Gachi, Abdalla, Wolmara, Ada‟a, Kimbibit, Arsi,
Arero, Moyale and Yabelo districts in Oromia regional state
in Ethiopia. A total of 2334 ixodid ticks collected from
domestic animals were morphologically identified using
taxonomic keys in to 12 species including Am. cohaerens
(n=496:364 male(m), 132 female(f)), Am. gemma
(n=122:70m, 52f), Am. variegatum (n=157: 146m, 11f), Hy.
truncatum (n=79: 62m, 17f), Hy. m. rufipes (n=29: 18m,
11f), Rh (Bo). decoloratus (n=495: 70m, 425f), Rh. e. evertsi
(n=65: 36m, 29f), Rh. pulchellus (n=751: 300m, 451f),
Rh. sanguienus (n=14: 9m, 5f), Rh. bergeoni (n=76: 44m,
32f), Rh. praetextatus (n=47: 33m, 14f) and Hae. leachi
(n=3: 0m, 3f). The photographs of each tick species
identified in the present study are depicted on figure 1. From
these morphologically identified ticks, a total of 85 ticks
from 3 to 11 specimens from each of the 12 tick species were
studied by MALDI TOF MS and molecular methods.

212 
3.2. MALDI-TOF MS data
The legs from 85 tick specimens identified morphologically
in 12 species were subjected to MALDI-TOF MS to assess
intra-species reproducibility and inter-species specificity
(Table 1). MALDI-TOF MS analysis yielded unique
reproducible protein profile spectra for each of the 12 tick
species tested with the mass ranging from 2-20 kDa (Figure
1). Likewise, male and female ticks belonging to the same
tick species revealed visually similar protein profiles. To
confirm the intra-species reproducibility and inter-species
specificity of MS spectra and to visualize the distances
between the different tick species, MS protein profiles of 3
to 5 specimens per tick species from both genders were used
to generate a dendrogram (Figure 2). Clustering analysis
revealed that all specimens of the same species were grouped
on distinct branches. In addition, all specimens from the
same genus gathered in the same part of the dendrogram,
excepted ticks from the Rh. (Bo). decoloratus which were
not grouped with the species from Rhipicephalus geneses,
but were found in the same part as specimens from the
Amblyoma geneses (Figure 2).

213 
3.3. Blind tests
MS spectra from the 11 tick species were queried
successively against the MS reference Database 1 and
Database 2 (i.e., Database 2 = Database 1 plus the leg spectra
from these 11 tick species stored in ethanol, for details see
M&M). The blind test of the 41 MSP from the legs of the
ticks preserved in ethanol against Database 1 produced the
LSVs ranging from 1.144 to 1.705. Interestingly, the tick
specimens which tick species counterparts were in the
Database 1 had higher LSVs. Nevertheless, these LSVs were
to low to be considered as reliable (LSVs < 1.8, threshold
value of identification).
Then, a total of 44 leg MS spectra, from 11 tick species with
3 to 5 specimens per species preserved in ethanol, were used
to implement MS reference Database 1, which was then
renamed Database 2 (Table 1). As solely 3 Ha. leachi
specimens were selected, they were used to control
misidentification rather than added them in the database. The
41 remaining MS spectra were then tested against Database
2, yielding to 100% of the MSP tested correctly identified at
the species level (Table 1). All queried specimens matched
with their counterpart species specimens preserved in ethanol
as first top-ranking hits. LSVs ranged from 1.698 to 2.626

214 
(Table 1). With the exception of the specimens from the Rh.
praetextatus species, all the other specimens queried against
the Database 2 were identified with a LSVs of greater than
1.8 (this score was previously defined as the threshold values
of confident identification in ticks, Yssouf 2013).

3.4. Molecular identification of ticks using 12S rRNA

gene
To validate the correct identification of tick species using
morphological keys and MALDI-TOF MS analysis, the
molecular identification of tick species based on the
sequencing of the 12S rRNA gene was performed.
A total of 40 specimens were selected including 1 to 8
specimens per tick species of the 11 tick species added in the
Database. BLAST analysis revealed that 12S gene sequences
were available only for 7 tick species (Am. variegatum,
Hy. truncatum, Hy. m. rufipes, Rh. (Bo.) decoloratus, Rh.
e. evertsi, Rh. pulchellus and Rh. sanguienus). BLAST
analysis demonstrated high intraspecies similarity among the
speciemens for each of these 7 tick species ranging from
96.5 to 100% supporting our morphological identification of
these ticks (Table 2). BLAST analysis also showed that these
7 tick species have high sequence similarity to their

215 
respective homologies available in the GenBank (Table 2).
However, the remaining other four tick species (Am.
cohaerens, Am. gemma, Rh. bergeoni and Rh. praetextatus)
their corresponding 12S RNA gene sequence homology is
not available in the GenBank. BLAST analysis of the 12S
RNA gene indicated that these 4 tick species have high
intraspecies similarity among the specimens ranging from
98.9-100% (Table 2). However, these 4 tick species showed
only 92.9 to 94.7% sequence similarity to their closet tick
species available in the GenBank (Table 2). Thus, these 4
12S RNA gene sequences of Ethiopian ixodid ticks were
submitted to the GenBank for the first time as new sequences
under accession numbers, respectively.
A phylogenetic tree was constructed from the 12S rRNA
gene sequences of ticks of the present study and the
sequences of their homology in the GenBank using the
Neighbor-joining statistics of Mega 5. Result of this
phyogenetic analysis revealed that all the individual ticks
that belong to the same species cluster together with their
respective homology sequences in the GenBank for all the
11 ixodid species of Ethiopian ticks (Figure 3).

216 
4. Discussion
Result of the morphological identification of ixodid ticks
revealed the presence of Am. cohaerens, Am. variegatum,
Am. gemma, Hy. truncatum, Hy. m. rufipes, Rh. (Bo.)
decoloratus, Rh. e. evertsi, Rh. pulchellus, Rh. sanguienus,
Rh. bergeoni and Rh. praetextatus (Fig 3) in Ethiopia. This
morphological finding was supported by MALDI TOF MS
technique that yielded reproducible protein profiles spectra
specific for each of the 11 tick species and was further
confirmed by molecular identification by amplification and
sequencing of the 12S rRNA gene of each tick species and
phylogenetic analysis. In support of our observation, several
previous morphological studies have reported the presence
of many species of ixodid ticks collected from domestic
animals in Ethiopia (Bekele, 2002; Mekonnen et al., 2001;
Regassa, 2001).
Recently, in our laboratory MALDI-TOF MS technique was
used to identify fresh tick (Yssouf et al., 2013a) and fresh
mosquitoes (Yssouf et al., 2014a; Yssouf et al., 2013b)
species from the spectra of protein extracted from their legs.
This prompted us to use MALDI-TOF MS technique to
identify Ethiopian ixodid ticks preserved in 70% ethanol.
Result of the first blind test against database 1 suggest that it

217 
is not applicable to identify tick species or other arthropods
by MALDI-TOF MS technique when the reference database
is created from fresh samples is tested against 70% ethanol
preserved ticks. This observation was further strengthened
by the result of blind test 1 of less than 1.8 LSV recorded for
all the of the 70% ethanol preserved 3 species of Ethiopian
ticks (Amblyomma variegatum, Hyalomma m. rufipes,
Rhipicephalus sanguineus) that were tested against database
1 which was previously created in our laboratory from
spectra of legs of fresh arthropods including these 3
aforementioned species of ticks. Even though it was lower
than the set cutoff value for species differentiation by
MAMDI TOF, the LSVs for these 3 tick spp. is better than
all the other LSVs recorded in the blind test 1 suggesting the
ticks belong to the same species. This finding is consistent
with previous report of absence of similarities in the spectral
profile level between the fresh specimens and those stored in
70% ethanol of the same species of fleas (Yssouf et al.,
2014b) and mosquitoes (Kaufmann et al., 2011).
Results of the blind test 2 against database 2 (which contains
all the 12 Ethiopian tick species preserved in 70% ethanol)
demonstrated that all the 11 ixodid tick species produced
distinct, consistent and reproducible species-specific protein

218 
spectra obtained from the legs of ticks preserved in 70%
ethanol. The tick reference database of our laboratory is now
upgraded by adding 11 different Ethiopian ixodid tick
species preserved in 70% ethanol. Overall, 10 tick species
specimens were identified by MALDI-TOF MS with spectra
scores >1.8 which is considered as reliable scores value for
the identification of bacterial genera by MALDI-TOF-MS as
has been used previously (Yssouf et al., 2013a). The finding
of species-specific protein spectra for both male and female
ticks that belong to the same species is also consistent with
the previous report from our laboratory (Yssouf et al.,
2013a). In support of our findings MALDI-TOF MS data
analysis has recently been used to identify various types of
arthropods including ticks (Hoppenheit et al., 2014;
Hoppenheit et al., 2013; Kaufmann et al., 2012b; Kaufmann
et al., 2011; Muller et al., 2013). The slightly lower LSV of
1.698-1.740 than the cutoff value of 1.8 recorded for the Rh.
praetextatus in our study could be attributable to low
spectral quality or strain variation among ticks of different
districts in Ethiopia. However, this LSVs still seems good
when compared to the lower LSVs of 1.3- 1.7 recorded for
up to 76% of ticks removed from patients previously (Yssouf
et al., 2013a).

219 
This study is the first to report the use of MALDI-TOF-MS
and molecular tools for identification of ixodid ticks
preserved in 70% ethanol in Africa.
The observation of the hierarchical grouping of the mass
spectra of each individual tick specimen clustered according
to their respective genera and species on the dendrogram
(Fig. 2) using the MSP dendrogram function of the MALDI-
Biotyper 3.0 is in line with previous findings in several
arthropods (Karger et al., 2012; Kaufmann et al., 2011;
Yssouf et al., 2013a; Yssouf et al., 2014a; Yssouf et al.,
2013b; Yssouf et al., 2014b). In this study the clustering of
the specimens of the Rh. (Bo). decoloratus together with the
specimens from the Amblyomma genus in steady of the
grouping together with Rhipicephalus genus most probably
suggests that MSP dendrogram function of the
MALDIBiotyper 3.0 may not be good method for tick
phylogenetic study as has been argued previously (Karger et
al., 2012; Yssouf et al., 2013a).
The demonstration of satisfactory and reproducible data for
species identification of ixodid ticks from samples preserved
in 70% ethanol by MALDI-TOF MS is important for the fact
that 70% ethanol is the most widely utilized convenient
medium for storing samples for entomological surveys and

220 
for processing by molecular techniques under field condition
around the world and in African countries including
Ethiopia. This finding is in line with the recent report of
identification of mosquitoes species preserved in alcohol
(Kaufmann et al., 2012a; Kaufmann et al., 2011).
The use of legs of adult ticks to identify tick species by
MALDI-TOF MS further confirms the previous report in our
laboratory where legs of fresh ticks and mosquitoes were
used for species identification (Yssouf et al., 2013a; Yssouf
et al., 2014a; Yssouf et al., 2013b). The use of protein profile
spectra extracted from legs of ticks for species identification
by MALDI-TOF MS has the advantage over the use of the
whole body or the abdomen of ticks due to the already
established fact that in hematophagous arthropods the
presence of blood has a considerable negative impact on
MALDI-TOF patterns and reduce the intensity of the
biomarker masses (Kaufmann et al., 2012a; Kaufmann et al.,
2011). However, our observations during the study period
(data not shown) show that the use of legs is more feasible
for large and medium body sized genera of ticks. In the case
of larvae and nymphs of ticks and small size genera of ticks
like Haemaphysalis species ticks the other body parts
excluding the abdomen seem to be suitable.

221 
Among the ixodid ticks we have identified by MALDI-TOF
MS and 12S rRNA gene the species Am. cohaerens, Am.
variegatum, Rh (Bo). decoloratus, Rh. pulchellus, Rh. e.
eversti, Rh. praetextatus, and Hy. m. rufipes are widely
distributed ticks with great economic and public health
importance in different parts of Ethiopia (Mekonnen et al.,
2001; Stephany et al., 2009). For instance, recently the
detection of emerging and remerging human pathogenic
bacteria including spotted fever group rickettsiae, Coxiella
burnetii and new Borrelia spp. were reported from these
ticks (Kumsa et al., 2014c).
The 12S rRNA gene has been previously used as a reliable
tool for identification of different species of ixodid ticks
including Rhipicephalus ticks (Beati and Keirans, 2001). The
phylogenetic tree constructed from the 12S rRNA gene
sequences of tick and homology sequences of ticks in the
GenBank presented herein is consistent with previous studies
(Beati and Keirans, 2001). The phylogenetic tree constructed
from the 12S rRNA gene sequences of ticks of this study
presented herein is in agreement and confirmed our
morphological identification of ticks and MALDI-TOF MS
tick identification. Interestingly, all the tick spp. for which
nucleotide homology is available in the GenBank showed

222 
high nucleotide identities of 99-100% with their respective
reference species in the GenBank and had high bootstrap
values in the 12S gene phylogenetic tree (Fig 3) (Beati and
Keirans, 2001). However, on the contrary in all the 4 tick
species (Am. cohaerens, Am. gemma, Rh. bergeoni and
Rh. praetextatus) for which identical 12S gene is missing in
the GenBank demonstrated low nucleotide homology with
reference 12S gene sequence in the GenBank to the closet
tick spp. and low bootstrap value in the 12S gene
phylogenetic tree and cluster near to the related tick spp.
(Fig. 3).
In conclusion the present study shows that protein profiling
by MALDI-TOF MS is suitable for identification of ixodid
ticks preserved in 70%. Further studies are needed to extend
the MALDI-TOF MS method of species identification to
other arthropods of public and economic importance.

Acknowledgements
The authors greatly acknowledge domestic animal owners in
the different districts of Oromia for their kindness in
allowing us to collect ticks from their animals.
 

223 
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230 
Figure Legends

Figure 1. Alignment of MALDI-TOF MS profiles of 12 tick

species from 4/5 geneses using Flex analysis 3.3 software
showing species-unique peaks for conclusive species
identification. An arbitrary color code was used for spectra
from specimens of the same genese. a.u., arbitrary units; m/z,
mass-to-charge ratio.

Figure 2. Dendrogram constructed from 2 to 5

representative spectra of 12 tick species from 4/5 geneses.
The dendrogram was calculated by Biotyper 3.0 software
and the distance units correspond to the relative similarity of
MS spectra. All specimen presented here were used to
implement our laboratory reference Database 1 called now
Database 2. The same color code as used for figure 1 was
used for specimens from the same genus. F, female; M,
male;

Figure 3. Phylogenetic tree construct from 12S gene to show

the correct molecular identification of ticks used for
reference database creation.

231 
Table 1. Ixodid tick species used to create the reference database 2 of MALDI-TOF spectra and
arthropods used in the blind test.

No. of
No. of
specimens
specimens ID log score-
used for No. No.
Species Origin used to values*
the blind female male
create the [low-High]
test
database
procedure
Am. cohaerens Oromia 4 4 3 5 [1.823 - 1.963]
Am. gemma Oromia  5 2 2 5 [2.222 - 2.418]
Am. variegatum Oromia  4 4 2 6 [1.833 - 2.023]
Hy. truncatum Oromia  4 3 2 5 [1.965 - 2.003]
Hy. m. rufipes Oromia  3 4 1 6 [1.821 - 2.244]
Rh (Bo).
Oromia  3 5 7 1 [1.808 - 2.368]
decoloratus
Rh. e. evertsi Oromia  4 3 2 5 [2.013 - 2.077]
Rh. pulchellus Oromia  4 4 4 4 [1.843 - 2.173]
Rh. sanguienus Oromia  3 3 1 5 [2.051 - 2.134]
Rh. bergeoni Oromia 4 7 4 7 [1.801 - 2.626]
Rh. praetextatus Oromia 3 5 2 6 [1.698 - 1.740]
Ha. leachi# / 3 3 0 [0.538 -1.064]
Total 41 44 30 55
*Range of log score values. #Species not included in the database.
207 
 Table 2. Result of BLAT analysis of the 12S RNA gene of ticks
showing the sequence similarities among each specimen of the same
species and sequence similarity to their homology in the GenBank.
Tick species Number Intraspecies % Nearest homology sequence in % similarity to

sequenced for 12S tested similarity the GenBank homology

RNA gene among (GenBank Accession Number) sequence in the

speciemens GenBank

Am. cohaerens 4 99.4-100% Am. variegatum (HQ856538) 92.9%

Am. gemma 5 99.4-100% Am. hebraeum (AF150049) 93.5%

Am. variegatum 4 99.2-100% Am. variegatum (HQ856466) 100%

Hy. truncatum 4 98.9-100% Hy. truncatum (AF150031) 96.5%

Hy. m. rufipes 1 - Hy. m. rufipes (KC817412) 100%

Rh. decoloratus 1 - Rh. decoloratus (KF569940) 99.4%

Rh. e. evertsi 4 100% Rh..e.evertsi (AF150052) 99.4%

Rh. pulchellus 3 99.4%-00% Rh. pulchellus (AF150024) 100%

Rh. sanguienus 2 100 Rh. sanguineus (DQ002997) 99.7%

Rh. bergeoni 3 99.7%-00% Rh. turanicus (JQ480849) 94.7%

Rh. praetextatus 9 99.4-100% Rh. muhsamae (FJ536558) 97.6%

208 
209 
210 
211 
4. FLEAS

237 
 
238 
Fleas (order Siphonaptera) are holometabolous obligate

blood-feeding ectoparasites of birds and mammals; the

immature stages (egg, larva and pupa) are found in burrows

or nests, whereas adult fleas are usually found on animal

hosts (10,40). Fleas are normally considered as ectoparasites

of dogs and cats, however, they readily infest and feed on

other species of domestic animals and humans (10). Fleas are

important vectors and reservoirs of several pathogens that

cause emerging or re-emerging infectious diseases such as

Yersinia pestis (plague), Rickettsia typhi (murine typhus),

Rickettsia felis (flea-borne rickettsiosis), Bartonella henselae

(cat-scratch disease) and Bartonella clarridgeiae (10,25,31).

The most common flea species on domestic animals and

humans in Ethiopia are Ctenocephalides felis felis (cat flea),

Ctenocephalides canis (dog flea), Pulex irritans (human

flea), Echidnophaga gallinacea (sticktight poultry flea) and

Xenopsylla cheopis (rat flea) (20). Despite the nationwide

239 
distribution and great economic significance of fleas in

Ethiopia information on the role of fleas as vectors of

pathogens of veterinary and medical importance is scarce

(17). To address this lack of information fleas collected from

dogs and cats in Ethiopia were tested for the presence of

bacteria pathogenic for humans. The DNA extracted from

these fleas were screened for human pathogenic bacteria first

by genus or species-specific genes and then when it was

found important were subjected to standard PCR to amplify

the targeted genes and then the identity of the bacteria was

determined by sequencing and phylogenetic analysis. Results

of our study showed the presence of R. felis in fleas collected

from dogs and cats. In addition Bartonella henselae was

reported for the first time in fleas of cats from Ethiopia.

These findings are presented herewith as a published paper.

240 
4.1. Article 6

Molecular detection of Rickettsia felis

and Bartonella henselae in dog and

cat fleas in central Oromia, Ethiopia.

Kumsa, B., Parola, P., Raoult, D., Socolovschi, C.,

Am. J. Trop. Med. Hyg., 90(3), 2014, pp. 457–462

241 
  248 
Am. J. Trop. Med. Hyg., 90(3), 2014, pp. 457–462
doi:10.4269/ajtmh.13-0010
Copyright © 2014 by The American Society of Tropical Medicine and Hygiene

Molecular Detection of Rickettsia felis and Bartonella henselae in Dog and Cat Fleas
in Central Oromia, Ethiopia
Bersissa Kumsa, Philippe Parola, Didier Raoult, and Cristina Socolovschi*
Aix Marseille Université, Marseille, France; Department of Parasitology, College of Veterinary Medicine and Agriculture,
Addis Ababa University, Bishoftu, Ethiopia

Abstract. Fleas are important vectors of several Rickettsia and Bartonella spp. that cause emerging zoonotic diseases
worldwide. In this study, 303 fleas collected from domestic dogs and cats in Ethiopia and identified morphologically as
Ctenocephalides felis felis, C. canis, Pulex irritans, and Echidnophaga gallinacea were tested for Rickettsia and Bartonella
DNA by using molecular methods. Rickettsia felis was detected in 21% of fleas, primarily C. felis, with a similar prevalence
in fleas from dogs and cats. A larger proportion of flea-infested dogs (69%) than cats (37%) harbored at least one C. felis
infected with R. felis. Rickettsia typhi was not detected. Bartonella henselae DNA was detected in 6% (2 of 34) of C. felis
collected from cats. Our study highlights the likelihood of human exposure to R. felis, an emerging agent of spotted fever,
and B. henselae, the agent of cat-scratch disease, in urban areas in Ethiopia.

INTRODUCTION Bartonella spp. are hemotropic, facultative, gram-negative,

Fleas (order Siphonaptera) are holometabolous blood-
feeding insects; the immature stages (egg, larva, and pupa)
are found in burrows or nests, whereas adult fleas are usually
found on animal hosts.1 These insects are considered ectopara-
sites of dogs and cats2 and also infest and readily feed on other
species of domestic animals and humans.3 Ctenocephalides felis
felis (cat flea), C. canis (dog flea), Pulex irritans (human flea),
Echidnophaga gallinacea (sticktight poultry flea), and
Xenopsylla cheopis (rat flea) are the most commonly reported
species on dogs and cats.2,4 In infested dogs and cats, flea bites
cause dermatologic problems such as severe pruritus, self-
inflicted trauma, and flea allergy dermatitis.5
Fleas are important vectors and reservoirs of several Rickettsia
and Bartonella spp. that cause emerging or re-emerging zoo-
notic infectious diseases in humans.6,7 Rickettsia spp. (order
Rickettsiales, family Rickettsiaceae) are small, gram-negative,
obligate, intracellular fastidious bacteria.8,9 Flea-transmitted
Rickettsia spp. that are pathogenic to humans include Rickettsia
typhi, which belongs to the typhus group and is the causative
agent of murine typhus; and R. felis, which belongs to the spotted
fever group. Laboratory studies have indicated that both
Rickettsia species are maintained in fleas by vertical and horizon-
tal transmission and can persist for life without causing damage.11
Rickettsia felis is transmitted to humans by flea bites and contact
with flea feces. Recently, R. felis was detected in patients with
fever of unknown cause, with a prevalence of 4.4% in Senegal11
and 3.7–7.2% in Kenya.12 In addition, 3.4% of the afebrile
patients in western Kenya were positive for R. felis by molecular
methods.13 Although C. felis is considered to be the only known
biological vector of R. felis,10 R. felis has recently been detected
in different flea species in many countries in Europe,14–16 Asia,17
north and sub-Saharan Africa18–20 and North and Latin
America.21,22 However, in Ethiopia, few reports are available,23
although R. felis has recently been reported in a small number
of P. irritans and C. felis specimens collected from human
dwellings in southwestern regions of this country.19
intracellular bacteria that are usually transmitted by arthro-
pod vectors, such as fleas, lice, flies, and ticks. These bacteria
are highly adapted to their mammalian hosts and infect eryth-
rocytes and endothelial cells. To date, the genus Bartonella
comprises more than 20 species and subspecies, includ-
ing B. henselae, B. quintana, B. alsatica, B. clarridgeiae,
B. elizabethae, B. grahamii, B. vinsonii subsp. arupensis, B.
vinsonii subsp. berkhoffii, B. washoensis, B. rochalima, and
B. koehlerae, which are pathogenic to humans and detected
in fleas.24 Cats are considered to be the main reservoir of
B. henselae and can transmit the pathogen to humans via
scratches or bites, although naturally infected cats are asymp-
tomatic. Although several countries worldwide have reported
B. henselae in cats and in fleas,25,26 the presence of Bartonella
spp. in fleas was not reported in Ethiopia until recently.
However, an 11% (5 of 46) prevalence of antibodies against
Bartonella spp. was reported in cats from Addis Ababa,27
and Bartonella spp. related to B. elizabethae were reported
in small mammals in northern Ethiopia.28
All reports of fleas on animals in Ethiopia resulted from
studies of other ectoparasites, specifically ticks, mange mites,
and lice,1 and these studies focused on the epidemiology,
species composition, and impact of ectoparasites on the skin
and hides of food animals. As a result, studies of the role of
fleas as vectors of pathogens of veterinary and medical impor-
tance in Ethiopia are lacking. Despite the nationwide distri-
bution and great economic significance of fleas, information
on the occurrence of Bartonella spp. in fleas collected from
domestic animals in this country is not available. In addition,
little is known about Rickettsia species in fleas collected from
domestic animals in Ethiopia. Therefore, the current study
was conducted to determine the presence of Bartonella and
Rickettsia species in fleas collected from domestic dogs and
cats in central Oromia, Ethiopia.
MATERIALS AND METHODS

Study areas and animals. In this study, domestic dogs and

*Address correspondence to Cristina Socolovschi, Faculté de
Médecine, Aix Marseille Université, 27 Boulevard Jean Moulin, 13385
Marseille Cedex 5 France. E-mail: cristina.socolovschi@univ-amu.fr
cats in the town of Bishoftu, Ethiopia (8 °44¢37.69²N,
38 °59¢19.28²E) were studied for flea infestation. Bishoftu,
the capitol of the Ada’a District, is located 47 km east of
Addis Ababa and has an elevation of 1,880 meters above sea
level. The town has a population of 95,000 persons. The area

457
458 KUMSA AND OTHERS
receives an average annual rainfall of 8.00 mm3, has average
maximum and minimum temperatures of 30.7 °C and 8.5 °C,
respectively, and a mean relative humidity of 61.3%.29
Fleas were collected during September–November 2011.
In the study area, the main rainy season is during mid-June–
September, and the dry season is during November–May. All
study animals were selected irrespective of sex, age, and breed
by using a simple random sampling method. The sex (female,
male) and age (young, adult) were recorded for each study
animal. The animals were categorized into two age groups,
young (£ 1 year) and adult (> 1 year) according to Kumsa and
Mekonnen.1 In the town of Bishoftu, dogs generally stay in
gardens and their owners provide food. Cats also usually stay
in gardens and are provided with food, water, and sleeping
areas by the owners. In the present study, stray dogs and cats
were not sampled.
Study methods. A cross-sectional study type was used to
determine the species of fleas infesting dogs and cats in Bishoftu
by using a house-to-house survey. All collected fleas were tested
by using molecular methods for Bartonella spp. and spotted
fever and typhus group Rickettsia spp.
Flea collection and identification. The skin of each study
dog and cat was first rubbed with a piece of cotton soaked in
ether and then combed for 10–15 minutes by using a one-
toothed standard flea comb (0.08 cm distance between teeth),
which other previous investigators recommended for flea col-
lection from dogs and cats.1,30 After combing, the flea comb
was held over a white plastic tray, and the fleas were collected
from the tray by using forceps. Echidnophaga gallinacea fleas,
usually found firmly attached to the skin around the eyelids,
snout, ears, lips and heads of the infested animals, were manu-
ally collected y using forceps and gloved hands. The collected
fleas (minimum = 1 and maximum = 23) from each infested
dog and cat were placed into pre-labeled small plastic tubes.
All collected fleas from each animal were placed in separate
vials containing 70% ethanol and transported to the Labora-
tory of the World Health Organization Collaborative Center
for Rickettsial Diseases and Arthropod-borne Bacterial Dis-
eases in Marseille, France.
Morphologic identification of fleas and molecular studies
were performed from the end of 2011 through 2012. The flea
species and sex were determined by using a microscope
according to standard morphologic identification keys as
described.1 The head shape, length of the first and second
spines of the genal comb, number of setae-bearing notches of
the dorsal aspect of the hind tibiae, and the number of setae on
the first segment of the neck were used to differentiate C. felis
from C. canis as described.1 Photographs of body parts of each
flea were made.
DNA extraction from fleas. Before DNA extraction, each
flea specimen was rinsed twice in sterile water for 15 minutes
and dried on sterile filter paper. Each specimen was dissected
laterally into two halves. Genomic DNA was extracted from
each specimen by using the QIAamp DNA Tissue Extraction
Kit (QIAGEN, Hilden, Germany) according to the manufac-
turer’s instructions. The DNA from each flea was eluted in
100 mL of Tris-EDTA buffer and stored at −20 °C under ster-
ile conditions to preclude contamination until the sample was
used for polymerase chain reaction. The second half of each
flea was stored at −80 °C as a backup sample.
Molecular detection of Rickettsia and Bartonella species.
The DNA extracted from the fleas was individually tested by
using genus-specific quantitative PCR (qPCR) for spotted
fever group Rickettsia by targeting the gltA gene (RKND03
system)19,20 in accordance with the manufacturer’s recommen-
dations and protocols (Applied Biosystems, Foster City, CA).
In addition, each DNA sample was tested for the bioB gene by
using a second qPCR with primers and a probe specific for R.
felis.20,25,31 All DNA samples positive for either the gltA or
bioB genes were further confirmed by a third qPCR specific
for the orfB gene, which is highly sensitive and specific for R.
felis, by using described primers and probes.20,31 A sample was
considered positive for R. felis when it was showed a positive
result for at least two of the genes described above.
In addition, 21 randomly selected R. felis DNA-positive
samples were subjected to a standard PCR analysis specific
for the ompA gene with primers 190.70, 190.180, and 190.701
(Eurogentec, Seraing, Belgium), which amplify a 629–632-
basepair fragment.20,31 Sterile water was used as a negative
control and DNA from R. montanensis was used as a positive
control. Sequencing was performed as described.32 Sequences
were aligned by using ChromasPro version 2.31 (http://en.bio-
soft.net/dna/chromas.html) and then analyzed and compared
with the Rickettsia sequences available in GenBank by using
the BLAST search tool.
All flea samples were also individually tested by genus-
specific qPCR for typhus group Rickettsia spp. by using a
PCR specific for the Rpr274P20,32 gene according to the man-
ufacturer’s protocol using a 7900HT Fast Real-Time PCR
System (Applied Biosystems, Foster City, CA). Sterile water
was used as a negative control and DNA from an R. typhi cell
culture was used as a positive control.
All samples were individually tested for the presence of the
16S–23S ribosomal RNA intergenic spacer gene by qPCR
with a 21-basepair probe specific for the genus Bartonella as
described.8,25 Sterile water was used as a negative control and
DNA from B. elizabethae was used as a positive control. All
DNA samples positive for the intergenic spacer gene were
further tested for B. henselae by qPCR specific for the pap31
gene, which is specific for this species, as described.8,25
Ethics. Ethical approval for the collection of fleas from
domestic animals was obtained from the animal research ethics
board (Agreement # 14/160/550/2011) of the College of Veter-
inary Medicine and Agriculture of Addis Ababa University.
All necessary oral permits were obtained for the described field
studies, including permission from the administration and agri-
cultural office of the district and from each animal owner. Flea
collection from the animals was not harmful to the animals.
Data analysis. Microsoft (Redmond, WA) Excel was used
for data management. Descriptive statistics, such as percentages
and means, were used to summarize the proportions of flea
infestations among the animals and infection of fleas with
R. felis. Statistical analysis was performed by using EpiInfo 7
(Centers for Disease Control and Prevention, Atlanta, GA),
Associations between the sex and age groups of the dogs and
cats, species and sex of fleas, and prevalence of flea infestation
and R. felis in fleas were determined by using the Mantel-
Haenszel test and EpiInfo 7. A P value £0.05 was con-
sidered significant.
RESULTS
Identification of fleas. In this study, 64% (30 of 47) of dogs
and 50% (8 of 16) of cats were infested with at least with one
 BARTONELLA HENSELAE AND RICKETTSIA FELIS IN FLEAS FROM ETHIOPIA 459

Table 1
Species composition and overall percentage of flea infestations by sex and age of dogs and cats in Oromia, Ethiopia*
Host factors Host sex Host age

Host Flea spp. Female Male Young Adult Total

Dog Ctenophalides felis 11/19 (58) (90:77) 18/28 (64) (114:38) 12/17 (71) (79:24) 17/30 (57) (112:27) 29/47 (62) (191:51)
C. canis 2/19 (10) (2:4) 3/28 (11) (4:6) 3/17 (18) (4:9) 2/30 (7) (2:1) 5/47 (11) (6:10)
Pulex irritans 2/19 (10) (1:1) 6/28 (21) (2:5) 4/17 (23) (2:3) 4/30 (13) (1:3) 8/47 (17) (3:6)
Echidnophaga gallinacea 0/19 (0) 2/28 (7) (2:0) 1/17 (6) (1:0) 1/30 (3) (1:0) 2/47 (4) (2:0)
Overall in dogs 11/19 (58) (80:18) 19/28 (68) (122:49) 13/17 (76) (86:36) 17/30 (57) (116:31) 30/47 (64) (202:67)
Cat C. felis 6/10 (60) (19:12) 2/6 (33) (2:1) 6/7 (86) (17:9) 2/9 (22) (4:4) 8/16 (50) (21:13)
*Values are no. infested/no. tested (%) (no. females:no. males).

species of flea (Table 1). Of the 269 fleas (202 females and
67 males collected from dogs, 242 C. felis (191 males and
51 females), 16 C. canis (6 females and 10 males), 9 P. irritans
(three females and six males) and 2 E. gallinacea (two
females) specimens were distinguished by morphologic iden-
tification. In female dogs, an overall prevalence of 58% (11 of
19) and 98 fleas (80 females and 18 males) was recorded. The
corresponding values for male dogs were 68% (19 of 28) and
171 fleas (122 females and 49 males), respectively (Table 1).
An overall prevalence of 76% (13 of 17) and 57% (17 of 30)
was observed in young and adult dogs, respectively (Table 1).
A total of 122 fleas (86 females and 36 males) and 147 fleas
(116 females and 31 males) were collected from young and
adult dogs, respectively (Table 1). There was no statistically
significant difference in the overall prevalence of flea infes-
tations between dogs of different ages (13 of 17 versus 17 of
30; P = 0.2, by Mantel-Haenszel test) and sex (11 of 19 versus
19 of 28; P = 0.5, by Mantel-Haenszel test) (Table 1).
A total of 34 (21 females and 13 males) C. felis were identi-
fied on cats (Table 1). In this study, C. felis was the most
prevalent flea species on both hosts; overall prevalence of
62% (29 of 47) on dogs (29 of 47 versus 8 of 47; P = 0.00002,
by Mantel-Haenszel test) and 50% (8 of 16) on cats. Pulex
irritans, with an overall prevalence of 17% (8 of 47), was the
second most prevalent species, followed by C. canis; the least
prevalent species on dogs was E. gallinacea (Table 1).
For dogs (191 of 242 versus 51 of 242; P < 0.0001, by Mantel-
Haenszel test) and cats (21 of 34 versus 13 of 34; P = 0.05, by
Mantel-Haenszel test), the number of female C. felis was sig-
nificantly higher than the number of males (Table 1). Further-
more, the prevalence of C. felis monospecies in dogs (60%) (18
of 30 versus 5 of 30; P = 0.0006, by Mantel-Haenszel test) and
cats (100%, 8 of 8) was significantly higher than all other types
of mixed species infestations (Table 2). The prevalence of
Table 2
Percentage of mono- and multiple flea species infestations in positive
dogs and cats in Oromia, Ethiopia*
Flea spp. Dog
Ctenocephalides felis 18/30 (60)† 8/8 (100)
C. felis and C. canis 2/30 (7)
C. felis and Pulex irritans 5/30 (17)†
C. felis, C. canis, and P. irritans 3/30 (10)
C. felis and Echidnophaga 1/30 (3)
gallinacea
E. gallinacean 1/30 (3)
Overall frequency 30/30 (100) 8/8 (100)
*Values are no. positive/no. tested %).
†P = 0.0006, by Mantel-Haenszel test.
Cat Overall
26/38 (68)
– 2/30 (7)
– 5/30 (17)
– 3/30 (10)
– 1/30 (3)
– 1/30 (3)
38/38 (100)
C. felis and E. gallinacea (3%, 1 of 30) mixed species infesta-
tions recorded in dogs was lower than all other types of mixed
species infestations (Table 2).
Molecular detection of Rickettsia spp. in fleas. Molecular
analysis of 303 fleas collected from dogs and cats identified
R. felis DNA in 63 fleas (21%) (Table 3). In our study, R. felis
DNA was detected in 21% (56 of 269) of fleas from dogs and
in 21% (7 of 34) of fleas from cats. The overall prevalence of
R. felis was not significantly different between fleas obtained
from dogs and cats. In dogs, 11% (1 of 9) of P. irritans and 6%
(1/16) of C. canis specimens were positive for R. felis DNA.
However, R. felis DNA was not detected in E. gallinacea from
dogs (Table 3). The overall percentage of R. felis was higher
in C. felis than in C. canis (29 of 47 versus 5 of 47; P < 0.0001).
However, no statistically significant differences were observed
in the overall prevalence of R. felis between female and male
C. felis from dogs (46 of 191 versus 8 of 51; P = 0.2, by Mantel-
Haenszel test) and cats (4 of 21 versus 3 of 13; P = 0.8, Mantel-
Haenszel test) (Table 3).
The overall prevalence of R. felis was 21% (21 of 98) and
20% (35 of 171) among fleas collected from female and male
dogs, respectively. The corresponding values for fleas col-
lected from young and adult dogs were 19% (23 of 122) and
22% (33 of 147), respectively (Table 3). No statistically signif-
icant differences were observed in the overall prevalence of
R. felis between the fleas collected from female versus male
dogs and young versus adult dogs.
Analysis of flea species positive for R. felis DNA on the
infested animals showed that 69% (20 of 29) of dogs infested
with C. felis harbored at least one flea that contained R. felis
DNA, whereas 7% (2 of 29) of dogs infested with C. felis
harbored fleas that contained R. felis DNA (Table 4). How-
ever, in the case of C. canis, P. irritans, and E. gallinacea, only
a small percentage of infested dogs harbored fleas that
contained R. felis DNA (Table 4). In contrast to dogs, only
37% (3 of 8) of cats infested with C. felis harbored at least one
flea positive for R. felis DNA (Table 4).
The mean ± SD qPCR cycle threshold (Ct) values for posi-
tive samples were 21.1 ± 6.6 for the orfB gene, 28.4 ± 3.1 for
the bioB, and 31.3 ± 2.3 for the gltA gene.
We amplified a 629–632-baspair fragment of the ompA
gene from 18 samples that were representative of all of the
species of fleas collected. A BLAST analysis of the ompA
gene sequence from all flea species showed 99.6–100% nucle-
otide homology with the GenBank reference sequence of
R. felis obtained from C. felis in Yucatan, Mexico (GenBank
accession no. AJ563398), and one sample was 100% simi-
lar to R. felis originating from Guatemala and Costa Rica
(GenBank accession no. JN990593).
460 KUMSA AND OTHERS

Table 3
Prevalence of Rickettsia felis in different flea species collected from dogs and cats in Oromia, Ethiopia*
Host factor Host sex Host age

Host Flea spp. Flea sex Female host Male host Young host Adult host Total hosts

Dog Ctenocephalides felis F 20/77 (26) 26/114 (23) 20/79 (25) 26/112 (23) 46/191 (24)
M 1/13 (8) 7/38 (18) 2/24 (8) 6/27 (22) 8/51 (16)
Total 21/90 (23) 33/152 (22) 22/103 (21) 32/139 (23) 54/242 (22)
C. canis F 0/2 (0) 0/4 (0) 0/4 (0) 0/2 (0) 0/6 (0)
M 0/4 (0) 1/6 (17) 0/9 (0) 1/1 (100) 1/10 (10)
Total 0/6 (0) 1/10 (10) 0/13 (0) 1/3 (33) 1/16 (6)
Pulex irritans F 0/1 (0) 1/2 (50) 1/2 (50) 0/1 (0) 1/3 (33)
M 0/1 (0) 0/5 (0) 0/3 (0) 0/3 (0) 0/6 (0)
Total 0/2 (0) 1/7 (14) 1/5 (20) 0/4 (0) 1/9 (11)
Echidnophaga gallinacea F – 0/2 (0) 0/1 (0) 0/1 (0) 0/2 (0)
M – – – – –
Total – 0/2 (0) 0/1 (0) 0/1 (0) 0/2 (0)
Overall: dogs 21/98 (21) 35/171 (20) 23/122 (19) 33/147 (22) 56/269 (21)
Cat C. felis F 4/19 (21) 0/2 (0) 2/17 (12) 2/4 (50) 4/21 (19)
M 3/12 (25) 0/1(0) 2/9 (22) 1/4 (25) 3/13 (23)
Total 7/31 (23) 0/3 (0) 4/26 (15) 3/8 (37) 7/34 (21)
Overall: dogs and cats 28/129 (22) 35/174 (20) 27/148 (18) 36/155 (23) 63/303 (21)
*Values are no. positive/no. tested (%).

No typhus group Rickettsia species were detected in any of been reported, a variation that is most likely attributable to
the 303 fleas collected from dogs and cats in Bishoftu in cen- differences in geographic and animal factors. The observation
tral Oromia. of a higher overall percentage of R. felis in C. felis than in
Molecular detection of Bartonella spp. in fleas. Two of 303 other flea species and detection of R. felis in C. canis and
fleas tested were positive for Bartonella spp. by Bartonella P. irritans collected from dogs in our study is consistent with
qPCR and B. henselae-specific qPCR, and had mean ± SD those of other reports.18,19 The absence of a significant differ-
Ct values of 31.0 ± 3.2 and 21.2 ± 1.1, respectively. Bartonella ence in the overall prevalence of R. felis between fleas from
henselae DNA was detected in 6% (2 of 34) C. felis collected female and male dogs and fleas from young and adult dogs in
from cats. One C. felis was coinfected with R. felis. this study suggests that the sex and age of the host does not
affect the prevalence of R. felis in fleas.
DISCUSSION Average Ct values for the orfB, bioB, and gltA genes in
detection of R. felis DNA in fleas in the present study are
We detected R. felis in C. canis collected from dogs. The consistent with reported values.31 This variation in Ct values
results of our study also provide additional molecular and most likely reflects differences in sensitivity and specificity
geographic evidence for R. felis in C. felis and P. irritans among different assays and specific gene targets for the DNA
specimens collected from dogs and cats in central Oromia. of this bacterium. In addition, fifty randomly selected second
Our molecular findings were validated by the facts that all half-cut female flea backup samples were negative for R. felis
negative control samples had negative results and all positive DNA, confirming our initial negative observations for the
control samples had positive findings in all quantitative and corresponding first half-cut female flea samples.
conventional PCRs. The finding of a higher proportion of flea-infested dogs
The detection of an overall percentage of 21% of R. felis (69%) than infested cats (37%) carrying C. felis that contain
DNA in fleas collected from dogs and cats is comparable to R. felis DNA is consistent with previous findings.22 The sig-
the prevalence of R. felis reported by investigators in several nificantly higher count, prevalence, and number of species of
countries.14,18,21,33 However, overall prevalence values that fleas on dogs than on cats in this and previous studies1 under-
are lower17,25 and higher12,22 than our results for R. felis have scores the need for additional studies to clearly determine the
relative importance of dogs and cats in the transmission of
Table 4 R. felis. In addition, we detected R. felis in one of two C. felis
Proportion of infested dogs and cats harboring fleas infected with collected from a goat in Ethiopia, but R. felis DNA was not
Rickettsia felis and Bartonella henselae in Oromia, Ethiopia* detected in one C. felis flea taken from a cow.
R. felis B. henselae Furthermore, we report detection of B. henselae in C. felis
Flea spp. Infection status Dog Cat Cat collected from cats in Ethiopia. Bartonella henselae DNA was
Ctenocephalides At least 1 infected 20/29 (69) 3/8 (37) 1/8 (12) detected in only in 6% (2 of 34) of C. felis fleas collected from
felis All infected 2/29 (7) 0/8 (0) 1/8 (12) cats in our study. This finding suggests that cats are important
None infected 7/29 (24) 5/8 (62) 6/8 (75) sources of this bacterium and is consistent with reports of
C. canis At least 1 infected 0/5 (0) – – B. henselae in fleas from several countries worldwide.18,25,26,33
All infected 1/5 (20)
Bartonella henselae is the major cause of cat-scratch disease,
– –
None infected 4/5 (80) – –
Pulex irritans At least 1 infected 0/8 (0) – – for which cats are implicated as the main animal reser-
All infected 1/8 (12) – – voir.6,7,24,25,34 The finding of a single C. felis coinfected with
None infected 7/8 (87) – – B. henselae and R. felis in this study implies the possibility of
Echidnophaga None infected 2/2 (100) – – dual infections in exposed humans. Benign lymphadenopathy,
gallinacea
low-grade fever, malaise and aching, headache, anorexia, and
 BARTONELLA HENSELAE AND RICKETTSIA FELIS IN FLEAS FROM ETHIOPIA
splenomegaly are the major clinical signs in patients affected
with cat-scratch disease.6,7,24
The high overall prevalence of flea infestation in dogs
(64%) and cats (50%) in the present study supports the obser-
vation of a prevalence of 99.5% in dogs and 91.5% in cats in
Hawassa in southern Ethiopia.1 These observations suggest
the presence of favorable climatic conditions important for
flea survival, reproduction, and development on dogs and cats
in the study area. These findings also indicate that in Ethiopia,
dogs and cats are not generally treated for any ectoparasites,
including fleas.
The absence of statistically significant variations in the
overall prevalence of flea infestation among dogs of different
age and sex groups in our study is consistent with those of
earlier observations.5,35,36 Previous investigators argued that
differences in the prevalence of flea infestation is associated
with host habitat and environmental factors rather than host-
dependent factors.36 Female C. felis were more abundant than
males in dogs and cats, a result consistent with previous find-
ings.1,3,5,35,37–39 Factors such as the longer time required by
female fleas to imbibe large quantities of blood for reproduc-
tion, higher female survival rates, and the greater ability of
females to evade capture during host grooming have been
suggested as possible explanations.1,3,5,35–39
The higher overall percentage, overall flea count. and four
flea species on dogs compared with cats (C. felis only) in the
present study is consistent with findings of a previous report1
and the following reports: three species on dogs and one spe-
cies on cats in Libya,3 three species on dogs and one species
on cats in Albania,35 and three species on dogs and two spe-
cies on cats in Hungary.5 These finding suggest that dogs are
the preferred hosts of fleas, which is most likely caused by
stronger grooming behavior of cats in removing fleas from
their bodies.36,37 In general, dogs have thicker, longer, and
denser fur than cats, which favors infestations.
The predominance of C. felis over all other flea species in
both host species is in consistent with results of an earlier
study in Ethiopia1 and with other studies worldwide.5,37–39
Ctenocephalides felis is generally considered the predominant
species found on dogs and cats, and has replaces C. canis as
the most common flea species on domestic dogs in many
countries.37 This flea species is better adapted than C. canis
to a wider range of environmental factors.38,39 Furthermore,
previous studies have shown that C. felis is the most common
flea in urban areas, whereas C. canis is the most common in
rural areas.30 The finding that C. canis was the second most
abundant species, followed by P. irritans and E. gallinacea, on
dogs in our study is consistent with results of reports from
Hungary,5 Albania,35 Spain,36 and Iran.39 Consistent with pre-
vious reports,1,3,36 E. gallinacea is a species frequently found
on birds and only occasionally reported on dogs and cats in
various regions because of transient infestations from contact
with birds. In our study, the absence of X. cheopis, the tropi-
cal rat flea, is explained by the fact that this flea species is
rarely found on carnivores except during seasons of higher rat
density and occurrence of disease epizootics. In addition,
highland agroecology is more favorable for X. cheopis than
areas such as Bishoftu, which is located in midland altitudes.
Possible explanations for the absence of C. felis strongylus in
our study are that this flea species is adapted to domestic
ruminants, as observed in Libya3 or is restricted to rural areas
and stray dogs and cats in Ethiopia.
461
In conclusion, our study shows that C. felis is the most
common ectoparasite of dogs and cats in urban central
Oromia, Ethiopia. This study provides molecular evidence of
R. felis and B. henselae in the fleas collected from dogs and
cats in this region. To our knowledge, this is the first study to
report the presence of B. henselae in fleas collected from cats
in Ethiopia. The high prevalence of flea infestations in dogs
and cats and the finding that a high percentage of fleas are
infected with these important zoonotic bacteria in this study
suggest that R. felis and B. henselae may be more important in
Ethiopia than previously believed. Our study demonstrates
that dogs, cats and their fleas may play important roles in
increasing the risk of human exposure to these pathogenic
bacteria in this country. Therefore, R. felis and B. henselae
should be considered in the differential diagnosis of any fever
of unknown cause among patients in this country. Future
studies are required to address the isolation and culture of
the bacteria, serologic and molecular prevalence, clinical
signs, treatment, and potential risk factors in humans in
Ethiopia. Additional epidemiologic studies in different animal
and arthropod species and the public health significance of
these zoonotic bacteria in different agroecologic zones are
urgently needed in this country.
Received January 8, 2013. Accepted for publication June 22, 2013.
Published online January 20, 2014.
Acknowledgments: We thank dog and cat owners in Bishoftu for
allowing us to collect fleas from their animals, and laboratory techni-
cians at URMITE (Marseille, France), particularly Veronique Brice,
Marielle Bedotto, and Annick Bernard, for technical support.
Authors’ addresses: Bersissa Kumsa, Didier Raoult, and Cristina
Socolovschi, URMITE, UMR, CNRS 6236, IRD 198, Faculté de
Médecine, Aix Marseille Université, Marseille, France, E-mails:
bersissak@yahoo.com, didier.raoult@gmail.com, and cristina
.socolovschi@univ-amu.fr. Philippe Parola, Service des Maladies
Infectieuses et Tropicales, Hôpital Nord, Marseille, France, E-mail:
philippe.parola@gmail.com.
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5. LICE AND SHEEP
KED

249 
 
Lice and sheep keds (Melophagus ovinus) are two of the most

important ectoparasites of domestic animals worldwide

(16,37). Even though several species of lice of domestic

animals are very common and widely distributed in Ethiopia

including Bovicola (Damalinia) bovis, Linognathus vituli (the

long-nosed cattle louse), Solenopotes capillatus (the little blue

cattle louse), Haematopinus quadripertusus (the cattle tail

louse) of cattle, Bovicola (Damalinia) ovis and Linognathus

ovillus of sheep and Bovicola (Damalinia) caprae, and

Linognathus stenopsis of goats, Heterodoxus spiniger,

Trichodectes canis and Linognathus steosus of dogs previous

study was not conducted to investigate the public health

importance of animal lice and Melophagus ovinus in Ethiopia

(18,19).

In Ethiopia close contact between humans and animals that are

usually infested with ectoparasites is considered as normal. For

instance, it is common to find households sharing living house

251 
with small ruminants during the night to protect them from

predators. Similar to other developing parts of the world,

generally poor hygiene and sanitation prevails in the country

(18). Therefore, it is very interesting to determine the presence

of zoonotic bacteria in lice and sheep ked infesting domestic

animals in Ethiopia using molecular tools. For this purpose the

DNA extracted from these ectoparasites were tested by genus

or species-specific genes and then the bacteria were identified

by standard PCR after sequencing and phylogenetic analysis.

Results of our study demonstrated the presence of A. soli,

A. lowffii, A. pitti and 3 new Acinetobacter spp. in the lice and

keds collected from domestic animals in Ethiopia. These

findings are presented herewith as a published paper.

252 
5.1. Article 7
 

Molecular detection of Acinetobacter species

in the lice and keds of domestic animals in

Oromia Regional State, Ethiopia.

Kumsa, B., Socolovschi, C., Raoult, D., Parola, P.,

PLoS ONE 7(12): e52377.

doi:10.1371/journal.pone.0052377.

253 
 
264 
Molecular Detection of Acinetobacter Species in Lice and
Keds of Domestic Animals in Oromia Regional State,
Ethiopia
Bersissa Kumsa1,2, Cristina Socolovschi2, Philippe Parola2, Jean-Marc Rolain2, Didier Raoult2*
1 Department of Parasitology, College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu, Ethiopia, 2 Aix Marseille Université, URMITE, UM63, CNRS
7278, IRD 198, Inserm 1095, Marseille, France

Abstract
This study was conducted to determine the presence of Acinetobacter and Rickettsia species DNA in lice and Melophagus
ovinus (sheep ked) of animals from Oromia Regional State in Ethiopia. From September through November 2011, a total of
207 cattle, 85 sheep, 47 dogs and 16 cats were examined for ectoparasites. Results of morphological identification revealed
several species of ectoparasites: Linognathus vituli (L. vituli), Bovicola bovis (B. bovis) and Solenopotes capillatus (S. capillatus)
on cattle; B. ovis and Melophagus ovinus (M. ovinus) on sheep; and Heterodoxus spiniger (H. spiniger) on dogs. There was a
significantly (p#0.0001) higher prevalence of L. vituli observed in cattle than both S. capillatus and B. bovis. Molecular
identification of lice using an 18S rRNA gene analysis confirms the identified lice species by morphological methods. We
detected different Acinetobacter species among lice (11.1%) and keds (86.4%) including A. soli in L. vituli of cattle, A. lowffii in
M. ovinus of sheep, A. pittii in H. spiniger of dogs, 1 new Acinetobacter spp. in M. ovinus and 2 new Acinetobacter spp. in H.
spiniger of dogs using partial rpoB gene sequence analysis. There was a significantly higher prevalence of Acinetobacter spp.
in keds than in lice (p#0.00001). Higher percentage of Acinetobacter spp. DNA was detected in H. spiniger than in both B.
ovis and L. vituli (p#0.00001). Carbapenemase resistance encoding genes for blaOXA-23, blaOXA-24, blaOXA-58, blaNDM-1
and blaOXA-51 were not found in any lice and keds. These findings suggest that synanthropic animals and their
ectoparasites might increase the risk of human exposure to zoonotic pathogens and could be a source for Acinetobacter
spp. infections in humans. However, additional epidemiological data are required to determine whether ectoparasites of
animals can act as environmental reservoirs and play a role in spreading these bacteria to both animal and human hosts.

Citation: Kumsa B, Socolovschi C, Parola P, Rolain J-M, Raoult D (2012) Molecular Detection of Acinetobacter Species in Lice and Keds of Domestic Animals in
Oromia Regional State, Ethiopia. PLoS ONE 7(12): e52377. doi:10.1371/journal.pone.0052377
Editor: Patrick C. Y. Woo, The University of Hong Kong, China
Received August 1, 2012; Accepted November 12, 2012; Published December 17, 2012
Copyright: � 2012 Kumsa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: didier.raoult@gmail.com

Introduction Linognathus steosus, were reported. In contrast, there are no reports

Lice and sheep keds (Melophagus ovinus) are two of the most
common and economically important ectoparasites of domestic
animals worldwide. They are responsible for a wide range of
health problems in domestic animals [1]. In infested animals they
cause losses in productivity; due to anemia, loss of wool or hair due
to scratching, biting and rubbing, disruption in feeding, hide and
skin damage, secondary skin infections and damage, reduced
newborn birth weights, abortion in pregnant animals, and damage
to fences, equipment and buildings due to excessive rubbing and
scratching [2,3].
Several species of lice have been found on Ethiopian cattle; one
species of chewing lice, Bovicola (Damalinia) bovis, and three species
of sucking lice, including Linognathus vituli (the long-nosed cattle
louse), Solenopotes capillatus (the little blue cattle louse) and
Haematopinus quadripertusus (the cattle tail louse) [4]. On sheep, the
biting lice Bovicola (Damalinia) ovis; one species of sucking lice,
Linognathus ovillus and one species of the fly, Melophagus ovinus, are
common ectoparasites [5,6]. On goats, one species of biting lice,
Bovicola (Damalinia) caprae, and the sucking lice Linognathus stenopsis
have been reported [6,7]. On dogs, the biting lice Heterodoxus
spiniger and Trichodectes canis as well as one species of sucking lice,
available on the lice from cats in Ethiopia [8].
All the previous reports on the lice and sheep keds of animals in
Ethiopia resulted from studies of other ectoparasites, specifically
ticks and mange mites [6,7,9,10,11]. The only lice-specific study in
Ethiopia was carried out by Kumsa and Bekele (2008) on the lice
of cattle in the Endegagn district. Earlier studies focus on the
epidemiology, species composition, distribution and impact of
ectoparasites on the skin and hides of food animals. Studies on the
role of lice and keds of domestic animals as vectors of pathogens of
veterinary and medical importance have not yet been conducted
in Ethiopia.
Acinetobacter species are Gram-negative coccobacilli bacteria
commonly found in water, soil, mud, living organisms, vegetables,
as well as in the feces, urine and skin of humans and animals
[12,13,14,15]. Currently, the genus comprises 23 validly named
species and 12 genomic species. Owing to difficulty of precise
identification of all members to the species level with advancement
and development of new techniques in molecular methods, the
taxonomy of the genus Acinetobacter has been continually revised
[16,17,18]. Recently, formerly Acinetobacter genomic sp. 3 was

PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52377


renamed as A. pittii, Acinetobacter genomic sp. 13TU as A. nosocomialis
and Acinetobacter genomic sp. G13 as A.lwoffii [16].
In humans, members of the genus Acinetobacter have emerged as
opportunistic pathogens, and are frequently implicated in various
types of infections, especially in immunocompromised individuals
and intensive health care units [14] throughout the world. In
tropical countries, they are associated with severe community-
acquired infections [13]. They have the capability to survive for
prolonged periods under a wide range of environmental conditions
[13]. A. baumannii is described as the most common species in this
genus frequently associated with outbreaks and has been
repeatedly reported to develop a high level of resistance against
all available classes of antimicrobial drugs in many parts of the
world [12]. A. pittii is reported as the second most commonly
isolated Acinetobacter species after A. baumannii in human patients
[16,17]. More recently other less known species such as A. lwoffii
and A. soli have been associated with serious infections and
considered as emerging pathogens [14,19,20]. For instance A.
lwoffii has been associated with acute gastroenteritis in USA [21],
multidrug resistant A. lwoffii in southern Thailand [22], A. soli
outbreak as a cause of infection in neonatal intensive health care
unit in Korea [23] and nosocomial bloodstream infections due to
A. pittii in United States were reported.
In animals infection due to Acinetobacter species is considered as
an emerging problem due to escalating in the number of reports
from many countries of the world. Nosocomial infection by A.
baumannii in dogs and cats in intensive care unit in Switzerland
[24], infection due to A. baumannii in pets and horses in Switzerland
[25], multidrug resistant Acinetobacter spp. in veterinary clinics in
Germany [12], detection of A. baumannii in samples from cattle and
pigs slaughtered for human consumption in major Scottish
abattoirs [26], carbapenemase producing Acinetobacter spp. in cattle
from France [27] and OXA-23 producing Acinetobacter spp. from
faeces of horses in Belgium [28] are some of the current reports
that notify the growing importance of Acinetobacter spp. in
veterinary medicine mirroring the situation happening in human
medicine.
In arthropods Acinetobacter spp. have been detected in different
species including tsetse flies, sand flies, mosquitoes, fleas and ticks
in many countries of the world. Details on Acinetobacter spp.
detected in arthropods, species of vectors and methods of detection
is presented (Table 1). Also, recently several investigators detected
A. baumannii in both body and head lice of humans from some
countries [15,29,30,31]. Despite the worldwide distribution and
great economic significance of these ectoparasites, information is
not available on the occurrence of Acinetobacter species in the lice
and flies found on domestic animals. In addition, there is little
known about Rickettsia species in the lice and flies of domestic
animals in Ethiopia. Therefore, the current study investigated the
presence of these bacteria in arthropods of domestic animals in six
districts in Oromia Regional State, Ethiopia.
Materials and Methods
Study areas and animals
Lice and Melophagus ovinus were collected from indigenous cattle,
sheep and dogs in six different districts in Oromia Regional State,
Ethiopia: Asalla, Walmara, Shano, Ada’a, Bedele and Gachi. The
districts are located in 5 zones in the central, southeast and
southwestern parts of the country, with various climates and
agroecology (Table 2). Ectoparasite collections were performed
from September through November of 2011. A long rainy season
from July to September, a short rainy season from March to May
and a dry season from November to April prevail in all the study
PLOS ONE | www.plosone.org
Acinetobacter Species in Ectoparasites, Ethiopia
districts. In addition, October is a post rainy month with only few
days of raining while November is a month during which the dry
season commences hence is without raining or rarely for some
days. The farming system in all the districts is characterized by a
mixed crop-livestock production system. The livestock in the study
areas are traditionally managed under extensive production
systems [32].
Collection and morphological identification of
ectoparasites
Lice and flies were carefully removed manually, using forceps or
by hand, to avoid any damage to the body and were then placed in
vials containing 70% ethanol for subsequent identification. In the
case of lice collected from dogs, the dog’s body was brushed for ten
minutes with a flea comb as previously described [8]. All lice and
flies from the same animal were put in the same vial and
transported to the Laboratory of the World Health Organization
Collaborative Center for Rickettsial Diseases and Arthropod-
borne Bacterial Diseases located in Marseille, France. Morpho-
logical identification of ectoparasites and molecular studies were
performed from the end of 2011 through 2012. All of the
ectoparasites were identified to the species level using a microscope
and the morphological identification keys described [1,3].
Photographs of the dorsal and ventral body parts of each
ectoparasite were captured (Fig. 1), and the number and sex of
each louse and fly was determined.
Molecular identification of ectoparasites
Prior to DNA extraction, each specimen was rinsed twice in
sterile water for 15 minutes and then dried on sterile filter paper.
Each specimen was longitudinally cut into two equal halves.
Genomic DNA was extracted from each specimen using the
QIAamp DNA tissue extraction kit (Qiagen, Hilden, Germany) as
per the instructions of the manufacturer. DNA from each
ectoparasite was eluted in 200 ml of TE buffer and stored at
220uC under sterile conditions to preclude any contamination
until the sample was used for PCR. The second half of each louse
and fly was kept at 280uC as a backup sample.
For molecular species identification, DNA samples of 3 to 8
randomly selected individual lice per species were subjected to
standard PCR in an automated DNA thermal cycler to amplify a
fragment of the 18S rRNA gene as described [33]. The PCR was
carried out in a Peltier PTC-200 model thermal cycler (MJ
Research Inc, Watertown, Mass.). The amplified products were
detected by electrophoresis on 2% agarose gels in TBE 0.56
buffer, stained with ethidium bromide and visualized using
ultraviolet (UV) transillumination. A DNA molecular weight
marker (Boehringer-Mannheim VI, Germany) was used to
estimate the size of the products. The positive controls consisted
of one sample from Pediculus humanus capitis collected in Mali and
one sample of P. humanus humanus collected in Algeria. These
samples were included in the PCR assay, and sterile water was
used as negative control.
The PCR products were cleaned of excess primers and
nucleotides using a QIAquick Spin PCR Purification Kit (Qiagen)
as per instructions of the manufacturer. Purified DNA was
sequenced using the BigDye Terminator Cycle Sequencing Ready
Reaction Kit (ABI PRISM, PE Applied Biosystems, Foster City,
CA). All obtained sequences were assembled and edited using
Chromas Pro1.34 (Technelyium Pty. Ltd., Tewantin). The
sequences of the 18S rRNA genes were then subjected to BLAST
analysis to determine similarities to those available in GenBank
and to construct a phylogenetic tree using Mega 5 software
2 December 2012 | Volume 7 | Issue 12 | e52377
 Acinetobacter Species in Ectoparasites, Ethiopia

Table 1. Summary of Acinetobacter spp. detected in various species of arthropods from different countries of the world.

Acinetobacter sp. Arthropod sp. Detection method Country Reference

A. radioresistans Ctneocephalides felis Culture and PCR Australia [53]

A. johnsonii Ct. felis Culture and PCR Australia [53]
A. junii Ct. felis Culture and PCR Australia [53]
A. lwoffii Ixodes holocyclus Culture and PCR Australia [53]
A. lwoffii Boophilus microplus Culture and PCR Australia [53]
A. johnsonii Boophilus microplus Culture and PCR Australia [53]
A. junii Boophilus microplus Culture and PCR Australia [53]
A. radioresistans Boophilus microplus Culture and PCR Australia [53]
A. baumannii Lutzomyia longipalpis Culture and PCR Brazil [39]
A. juni Culex quinquefasciatus PCR India [54]
A. calcoaceticus Culex quinquefasciatus PCR India [54]
A. calcoaceticus Anopheles stephensis PCR and biochemical Iran [55]
A. calcoaceticus Anopheles maculipennis PCR and biochemical Iran [55]
Acinetobacter sp. Lutzomyia longipalpis PCR Brazil [56]
Acinetobacter sp. Bemisia tabaci PCR Iran [44]
Acinetobacter sp. Glossina palpalis palpalis Culture and PCR Cameroon [57]
Acinetobacter sp. Bactericera cockerelli PCR USA [42]
A. genomosp. 3 Aedes albopictus PCR Madagascar [58]
A. genomosp. 13U Aedes aegypti PCR Madagascar [58]
A. baumannii Glossina palpalis palpalis Culture and PCR Angola [40]

doi:10.1371/journal.pone.0052377.t001

(Molecular Evolution Genetic Analysis; The Biodesign Institute,
Tempe, AZ).
Molecular detection of Acinetobacter and Rickettsia
species
All the specimens were individually tested for the presence of
Acinetobacter species DNA targeting the rpoB gene [30] by real-time
quantitative (q) PCR as per the instructions of the manufacturer
(Applied Biosystems, Foster City, CA). In addition, the ectopar-
asite DNA was tested for spotted fever group Rickettsia with primers
targeting the gltA gene specific for this group [34] and for typhus
group Rickettsia with primers targeting the Rpr274P gene, which
encodes a hypothetical protein [35]. Sterile water was used as
negative control; A. baumannii, R. montanensis and R. typhi DNA were
used as positive controls. The samples were considered positive
when cycle thresholds (Ct) were ,35.

Table 2. Description of study districts and number of study animals in Oromia Regional State.

Annual
Km from rainfall
Addis Altitude in Av. Annual No. Animals
District Zone Ababa Agroecology in m.a.s.l Coordinates mm Temp in 6C examined

Asalla Arsi 175 southeast Highland 2500 7u569589.5799N 2000–4000 20–30 Sheep = 39
39u8923.4299 E
Shano North Showa 70 Northeast Highland 2861 9u20.00.0099N 945.4 6.1–18.5 Cattle = 38
39u18900.0099E Sheep = 19
Walmara West Showa 45 west Highland 2500 9u7950.6499N 1060 4.6–23.3 Cattle = 42
38u28938.4699E
Ada’a East Showa 47 east Midland 1911 8u44937.6999N 1911 13–26.5 Cattle = 53
38u59919.2899E Dogs = 47
Cats = 16
Bedele Illubabora 483 southwest Midland 1974 8u,2791.7699N 1400 12.5–27.5 Cattle = 32
36u2195.0899E Sheep = 21
Gachi Illubabora 460 southwest Midland 1751 9u1492.5799N 1300 13–28 Cattle = 42
35u 4948.4699E Sheep = 6

m.a.s.l = meters above sea level; Av. = average; uC = degrees Celsius.

doi:10.1371/journal.pone.0052377.t002

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Figure 1. Photographs of morphologically identified 5 species of lice and Melophagaus ovinus collected from domestic animals. A,
Bovicola ovis of sheep; B, Bovicola bovis of cattle; C, Heterodoxus spiniger of dog; D, Linognathus vituli of cattle; E, Solenopotes capillatus of cattle; F,
Melophagus ovinus (sheep ked) of sheep.
doi:10.1371/journal.pone.0052377.g001
Molecular detection of carbapenemase encoding genes
All the DNA of lice (n = 82) and flies (n = 19) positive for
Acinetobacter species were tested for the presence of carbapenemase
encoding genes by qPCR targeting blaOXA-23, blaOXA-24,
blaOXA-58 and blaNDM-1 using primers, probes and all
conditions as has been described before [15,36]. In addition, a
total of 32 lice and 10 flies with sufficient amount of DNA were
evaluated for carbapenemase encoding genes by standard PCR
targeting blaOXA-51 and blaOXA-23 with primers and all
conditions as described before [15].
Molecular identification of Acinetobacter spp. by partial
rpoB gene
A total of 32 lice and 10 keds DNA of sufficient amount and
positive for Acinetobacter spp. by qPCR were further subjected to
standard PCR targeting partial rpoB gene (zone 1) to identify
Acinetobacter spp. using the primers and all conditions as described
before [17]. Sterile water and A. genomosp DNA were used as
negative and positive controls, respectively. Detection of amplified
products, cleaning of excess primers and nucleotides from DNA,
sequencing, assembling and edition of sequences, BLAST analysis
and rpoB gene phylogenetic tree construction with Maximum
likelihood statistics of Mega 5 were all performed using similar
methods as described for 18S rRNA gene for lice above.
Ethical statement
Ethical approval for the collection of lice and flies from domestic
animals was obtained from the animal research ethics board
(Agreement # 14/160/550/2011) of the College of Veterinary
Medicine and Agriculture of Addis Ababa University. All
necessary oral permits were obtained for the described field
studies, including permission of administration and agricultural
office of each Ethiopian district and from each animal owner.
PLOS ONE | www.plosone.org
Acinetobacter Species in Ectoparasites, Ethiopia
Ectoparasite collections are not harmful and are not against the
welfare of animals. No collection had been done from privately-
owned, wildlife, national park or other protected areas and
endangered or protected species.
Data analysis
Microsoft Excel was used for data management. Descriptive
statistics such as percentages and means were employed to
summarize the proportions of infestations with lice and keds.
Statistical analysis was performed with EpiInfoTM7 and a P-value
of ,0.05 was considered significant.
Results
Morphological identification of ectoparasites
A total of 207 cattle, 85 sheep, 47 dogs and 16 cats in six
districts were examined for the presence of lice and sheep keds
(Table 2). The results of the morphological identification of the
collected lice and flies from infested animals revealed a total of 408
Linognathus vituli, 4 Bovicola bovis and 3 Solenopotes capillatus from
cattle; 22 Heterodoxus spiniger from dogs; 298 Bovicola ovis and 22
Melophagus ovinus from sheep (Fig. 1). Furthermore, the study
showed that out of all of the examined cattle, 19.3% (40/207) were
infested with L. vituli, 0.5% (1/207) with S. capillatus and 0.5% (1/
207) with B. bovis. In cattle, there was a significantly higher
prevalence of L. vituli than both S. capillatus and B. bovis
(p#0.0001). Of the total examined sheep, 48.2% (41/85) were
infested with B. ovis and 21.05% (4/19) were positive for M. ovinus.
In addition, 19.1% (9/47) of the examined dogs were infested with
H. spiniger. Alternatively, lice were not detected on any of the cats
studied.
4 December 2012 | Volume 7 | Issue 12 | e52377

Molecular identification of ectoparasites
Molecular identification of the lice based on the 18S rRNA gene
analysis was used to confirm the species of lice identified by
morphological methods. A BLAST analysis of 18S rRNA gene
sequences of lice from dogs morphologically identified as H. spiniger
(n = 5) showed 100% (509/509) similarity to the GenBank
reference of H. spiniger collected from Japan (GU569166) (Fig. 2).
Likewise, a BLAST analysis of 18S rRNA gene sequences of lice
that were collected from cattle and were morphologically
identified as L. vituli (n = 7) showed 99.3% (553/557) similarity
to Linognathus vituli collected in Australia (GenBank Access. No.
AY077774). Four mutations were detected between our sequence
and the reference (AY077774) at 283 bp (T-G), 327 bp (T-A),
352 bp (T-C), and 420 bp (T-C). This sequence was submitted to
GenBank under accession number JX401573.
Interestingly, a sequence analysis of the 18S rRNA gene of 3 lice
from cattle morphologically identified as Solenopotes capillatus
showed 92.2% (536/581) homology with the GenBank sequence
of Neohaematopinus sciuri (AF423798) and 92% (520/565) homology
with Hoplopleura hirsute collected in Japan from the cotton rat
Sigmodon hispidus (GU569181).
A BLAST analysis of the 18S rRNA gene of 4 Bovicola bovis lice
from cattle showed 99.8% (494/495) similarity to B. ovis (GenBank
Access No. GU569184) and 99.4% (510/513) similarity to
Geomydoecus craigi (GenBank Access No. AF385040) (Fig. 2). There
are no 18S rRNA gene sequences available in GenBank for S.
capillatus and Bovicola bovis. Thus, these two sequences were
submitted to GenBank under accession numbers JX184910 and
JX184911, respectively. An analysis of the 18S rRNA gene of
Bovicola ovis (n = 8) from sheep revealed 100% (495/495) similarity
with Bovicola ovis detected in Japan and Australia with the
sequences GU569184 and AY077769, respectively.
An analysis of the 18S rRNA gene from Pediculus humanus
humanus (n = 1) from Mali revealed 99.4% (496/499) similarity
with Pediculus humanus corporis detected in France with Accessions
Nos. AF139479 and AF139478, respectively. An analysis of the
18S rRNA gene from Pediculus humanus capitis (n = 1) from Algeria
revealed 99.8% (518/520) similarity with the Pediculus humanus
capitis detected in Thailand, Portugal and China with Accessions
Nos. AY236418, AY236414 and AY236417, respectively.
A phylogenetic tree was constructed from the 18S rRNA gene
sequences of lice collected in this study and the most similar
sequences in the GenBank using the Neighbor-joining statistics of
Mega 5. H. spiniger reference lice and our lice sample from dogs
that belong to the family Boophidae in the suborder Amblycera
formed one group on the phylogenetic tree (Fig. 2). Reference
Linognathus vituli and our lice sample from cattle that belong to the
family Linognathidae under the suborder Anoplura clustered
together. The 3 individual S. capillatus lice from cattle, which
shared the same family and suborder with L. vituli, clustered near
the cattle louse. Both the control and the reference P. humanus
capitis and P. humanus humanus isolated from human head and body
lice, belonging to the family Pediculidae under the suborder
Anoplura, formed a separate group on the phylogenetic tree that
was close to L. vituli from cattle. The Bovicola ovis reference lice and
our lice sample from sheep that belong to the family Trichodec-
tidae in the suborder Ischnocera clustered in a separate group
close to the four B. bovis cattle lice, which belong to the same
genus, family and suborder (Fig. 2).
Detection of Acinetobacter and Rickettsia species in lice
and M. ovinus
We detected Acinetobacter spp. in a total of 82 lice (11.1%) and 19
flies (86.4%) (Table 3). In our study, there was a significantly
PLOS ONE | www.plosone.org
Acinetobacter Species in Ectoparasites, Ethiopia
higher prevalence of Acinetobacter spp. in flies than in lice (82/735
vs. 19/22; p#0.00001). The study showed that a higher
percentage of Acinetobacter spp. DNA was detected in H. spiniger
of dogs than in B. ovis and L. vituli (15/22 vs. 19/298; 47/408;
p#0.00001). The prevalence of Acinetobacter spp. in L. vituli
collected from cattle was significantly higher than in B. ovis from
sheep (47/408 vs 19/298; p = 0.02). Acinetobacter spp. was not
detected in any of the 4 Bovicola bovis collected from cattle (Table 3).
Results of our study revealed that out of the total lice infested
animals, 88.9% (8/9) of dogs were infested with H. spiniger, 45%
(18/40) of cattle were infested with L. vituli and 34.4% (14/41) of
sheep were infested with B. ovis and harbored at least 1 louse
positive for Acinetobacter spp. (Table 4). Similarly, Acinetobacter spp.
was detected at least in one M. ovinus in all the 4/4 (100%) infested
sheep. Highest proportion of Acinetobacter spp. was observed in
cattle infested with L. vituli in Walmara and Gachi districts whereas
highest percentage of Acinetobacter spp. was noted in sheep infested
with B. ovis in Shano and Asalla districts (Table 4).
A molecular investigation of the 735 lice collected from
domestic animals and 22 Melophagus ovinus collected from sheep
using qPCR did not produced any positive results for either
spotted fever or typhus group Rickettsia species.
Molecular identification of Acinetobacter spp
We succeeded in the amplification of 350 bp fragment of partial
rpoB gene from a total of 10 samples including one from L. vituli of
cattle, 3 from M. ovinus of sheep and 6 from H. spiniger of dogs
(Table 5). BLAST analysis of partial rpoB gene sequence and rpoB
phylogenetic tree showed the presence of A. soli in L. vituli of cattle,
A. lwoffii in M. ovinus of sheep, 1 new Acinetobacter sp. in M. ovinus of
sheep, A. pittii in H. spiniger of dogs and 2 new Acinetobacter sp. in H.
spiniger of dogs (Table 5 and Fig. 3). Five of these 10 sequences
were submitted to GenBank under accession numbers KC130085-
89, respectively.
Detection of carbapenemase encoding genes in
Acinetobacter species
A molecular investigation of 82 lice and 19 keds positive for
Acinetobacter spp. by qPCR did not produced any positive results for
blaOXA-23, blaOXA-24, blaOXA-58 and blaNDM-1 genes
encoding for carbapenemase resistance. Likewise, investigation
of 32 lice and 10 keds DNA by standard PCR never produced any
positive results for both blaOXA-23 and blaOXA-51 genes
encoding for carbapenemase resistance.
Discussion
In this study, we detected, for the first time, Acinetobacter spp. in
the lice and flies of animals from Ethiopia. In addition, we
obtained two new sequences of 18S rRNA genes from Bovicola bovis
and Solenopotes capillatus collected from cattle. We believe that the
results of this study are valid, as all of the negative control samples
produced negative results and all the positive control samples
tested positive for both Acinetobacter spp. and Rickettsia spp.
detection in the lice and flies of these animals. These findings
confirmed that our molecular study conditions precluded any
accidental cross-contamination of samples during the study period.
Similarly, in our 18S rRNA gene study of lice, we obtained
sequences from both positive controls, Pediculus humanus capitis and
Pediculus humanus humanus, confirming the appropriateness of our
working conditions.
In the study, the species of lice morphologically identified as L.
vituli, B. bovis and S. capillatus from cattle; H. spiniger from dogs; and
B.ovis from sheep (Fig. 1) were confirmed molecularly using 18S
5 December 2012 | Volume 7 | Issue 12 | e52377
 Acinetobacter Species in Ectoparasites, Ethiopia

Figure 2. Phylogenetic tree based on 18S gene sequences of lice species collected from domestic animals. Accession numbers in red
color are 18S gene sequence of lice of animals from Ethiopia recently deposited in the GenBank. Minimum evolution method was used to build the
phylogentic tree. Bootstrap values are indicated at the nodes.
doi:10.1371/journal.pone.0052377.g002

rRNA gene sequences. The 18S rRNA gene has been used
previously as an important tool to investigate human lice
phylogeny [33], to study the evolution of sucking lice [37] and
to study the phylogeny of lice [38]. The phylogenetic tree
constructed from the 18S rRNA gene sequences of the collected
lice and reference lice in the GenBank presented here supports the
information from previous studies [37,38].
In this study, we sequenced the 18S rRNA genes of B. bovis and
S. capillatus from cattle for the first time. B. bovis is the only biting
lice species in cattle, and it has important morphological features
such as a rounded head, reddish-brown color and dark transverse
bands on the abdomen [1] (Fig. 1). S. capillatus from cattle are the
smallest sucking lice, with morphological characteristics that
include a hexagonal sterna plate on the thorax, a weak front pair
of legs and prominent abdominal tubercles [3]. In Ethiopia, both
species are typically reported with low prevalence [4]. The finding
in this study that there was a significantly (p#0.0001) higher
prevalence of L. vituli than both S. capillatus and B. bovis in cattle is
in agreement with previous work [4]. Our findings in sheep, dogs
and cats of this study are also in line with previous reports [5,6,8].

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 Acinetobacter Species in Ectoparasites, Ethiopia

Table 3. Percentage of Acinetobacter spp. detected by qPCR in lice and flies collected from domestic animals in six districts in
Oromia.

District (Number of positive samples for Acinetobacter spp./Number of tested samples) (%)

Lice spp. Asalla (%) Shano (%) Walmara (%) Ada’a (%) Bedele (%) Gachi (%) Total (%)

B. ovis 13/234(5.5) 5/57(8.8) - - 1/7(14.3) - 19/298(6.4)

L. vituli - 7/51(13.7) 10/93(10.7) 4/84(4.8) 1/12(8.3) 25/168(14.9) 47/408(11.5)
S. capillatus - - - 1/3(33.3) - - 1/3(33.3)
B. bovis - 0/4(0) - 0/0 (0) - - 0/4(0)
H. spiniger - - - 15/22(68.2) - - 15/22(68.2)
Total lice 82/735(11.1)
Fly species
M. ovinus - 19/22(86.4) - - - - 19/22(86.4)
Total flies 19/22(86.4)

doi:10.1371/journal.pone.0052377.t003

The observation that 11.1% of the lice in the current study were
found to contain Acinetobacter spp. is lower than the earlier report of
47% in human head lice and 71% human body lice from Ethiopia
[31], 21% in human body lice [29] and 33% in human head lice
from Paris [30]. Recently, a low prevalence (4%) of A. baumannii in
head lice was reported from Senegal [15]. However, the overall
percentage of 86.4% (19/22) of Acinetobacter spp. detected in M.
ovinus of sheep in our study is greater than that found in the
aforementioned reports. The variation among study districts in the
percentage of Acinetobacter spp. in lice and keds in infested animals
is most probably attributed to differences in agroecology, animal
management and factors like age, sex, physiological status or
presence of other concurrent diseases that may favor infection of
lice or the animal by the bacteria. This observation coincides with
the findings of differences in the prevalence of infection in lice of
humans by A. baumannii among different study areas with different
altitudes in south western Ethiopia [31].
In line with our findings, Acinetobacter spp. were detected in many
species of arthropods in different parts of the world (Table 1). For
instance, Acinetobacter spp. have been detected in 13% of Lutzomyia
longipalpis (sand flies) in Brazil [39], 7.7% of Glossina palpalis palpalis
(tsetse fly) in Angola [40], 8.7% in the gut of the Prionoplus reticularis
larvae (wood feeding beetle) in New Zealand [41], up to 75% in
Bacterria cockerelli (potato psyllid) in the USA [42], 18.9% in
chewing lice of pocket gophers in the USA [43] and 1% in Bemisia
tabaci (tobacco whitefly) in India [44]. Moreover, A. baumannii have
been detected with a prevalence of 5.1% in the feces of domestic
animals in Senegal [15].
Most earlier investigators suggested that it is still yet not exactly
determined how both body and head human lice acquired A.
baumannii infection [30,31]. However, some authors argued
undiagnosed transient A. baumannii bacteremia in infested patients
as a source of infection for body lice but they stated it is not
possible to rule out the possibility of acquiring A. baumannii
infection in human body lice from external environmental
contamination [29]. Other investigators pointed out that bacterial
spp. such as Acinetobacter abundant in the environment reside in the
gut of several species of arthropods as transient or natural flora
[39] acquired commonly by vertical transmission [40] and also are
maintained and spread by several mechanisms of horizontal
transmission including mating, cofeeding, or contact with
contaminated faeces [41,42,44]. Having all these facts in mind
and due to the ubiquitous nature of Acinetobacter spp. in the
environment including on the skin and in the faces of animals [15]
lice and keds possibly had acquired Acinetobacter spp. infection from
the skin, faces or transient Acinetobacter spp. bacteremia of their host
animals. Moreover, it is not possible to rule out infection of lice
and keds by vertical route and also probably animals may play a
role as reservoirs for these bacteria.
We did not detect Rickettsia DNA in the lice and flies in our
study. This finding contrasts with previous work that reported
detecting R. helvetica in M. ovinus from sheep, in Linognathus stenopsis

Table 4. Proportion of infested animals positive for Acinetobacter spp. in lice and keds by qPCR in six districts in Oromia.

District No. animals positive for Acinetobacter spp./No. animals infested with lice or fly

B. ovis (Sheep) L. vituli (Cattle) S. capillatus (Cattle) B. ovis (Cattle) H. spiniger (Dog) M. vinus (Sheep)

Asalla 9/29 (31.0%) - - - - -

Walmara - 6/9 (66.7%) - - - -
Shano 4/10 (40%) 2/9 (22.2%) - 0/1 - 4/4 (100%)
Ada’a - 3/9 (33.3%) 1/1 (100%) - 8/9 (88.9%) -
Bedele 1/2(50%) 1/4 (25%) - - - -
Gachi - 6/9 (66.7%) - - - -
Total 14/41(34.4%) 18/40 (45%) 1/1 (100%) 0/1 8/9 (88.9%) 4/4 (100%)

doi:10.1371/journal.pone.0052377.t004

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 Acinetobacter Species in Ectoparasites, Ethiopia

Figure 3. Phylogenetic tree based on partial rpoB gene sequences of Acinetobacter species. Maximum Likelihood method was used to
build the phylogentic tree. Bootstrap values are indicated at the nodes. Bold indicates the taxonomic position of Acinetobacter species identified in
this study.
doi:10.1371/journal.pone.0052377.g003

from goats, and Rickettsia spp. in Haematopinus eurysternus from cattle
in Hungary [45,46].
The absence of any positive results for blaOXA-23, blaOXA-
24, blaOXA-58, blaNDM-1 and blaOXA-51 genes encoding for
carbapenemase resistance by both qPCR and standard PCR in
Acinetobacter spp. in the lice and keds of domestic animals in our
study is in line with the previous finding of absence of resistance to
several antimicrobials in A. soli in intensive health care units of
neonates in Brazil [23], full susceptibility to several antibiotics of A.
lwoffii from acute gastroenteritis in USA [21] and absence of
blaOXA-like genes encoding for carbapenemase resistance in A.
baumannii from faeces of domestic animals in Senegal [15]. On the

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 Acinetobacter Species in Ectoparasites, Ethiopia

Table 5. Summary of BLAST analysis of partial rpoB gene sequences obtained from lice and keds of domestic animals in six
districts in Oromia, Ethiopia.

Lice/fly spp Host spp Length (bp) Nearest match in GenBank % similarity

H. spiniger Dog 383 Acinetobacter genomosp. 3 (DQ207479) 99.2%

H. spiniger Dog 351 Acinetobacter sp.Hy-7 (FJ469982) 90.8%
H. spiniger Dog 391 Acinetobacter sp.Hy-7 (FJ469982) 90.7%
H. spiniger Dog 310 Acinetobacter sp. Hy-7 (FJ469982) 86.5%
H. spiniger Dog 387 Acinetobacter sp. Hy-7 (FJ469982) 90.5%
H. spiniger Dog 388 Acinetobacter sp. Hy-7 (FJ469982) 90.5%
L. vituli Cattle 381 Acinetobacter soli (HQ148175) 98.9%
M. ovinus Sheep 365 Acinetobacter sp.G13 (FN393754) 98.9%
M. ovinus Sheep 293 Acinetobacter sp.G13 (FN393754) 94.39%
M. ovinus Sheep 328 Acinetobacter sp-Hy-7 (FJ469982) 90.2%
P. h. humanus (Control) Man 383 Acinetobacter genomosp.3 (DQ207479) 99.2%

doi:10.1371/journal.pone.0052377.t005

other hand our findings contrast the previous multidrug resistance
in A. baumannii reported from several countries of the world
[12,36,47]. This finding also contradicts the observations of
multidrug-resistant isolates of Acinetobacter spp. from a range of
environmental sources in South Korea [20] and the presence of
resistance to antibiotics in A. baumannii and other Acinetobacter spp.
from blood cultures in Norway [48]. This variation is most
probably attributed to differences in strains of the bacteria among
various studies and other many factors contributing for emergence
of resistance.
Acinetobacter spp. identification study from DNA of lice and keds
of animals in Oromia uncovered the occurrence of 3 previously
described species and 3 new Acinetobacter spp. (Table 5 and Fig. 3).
All the 3 previously described species, we detected: A. soli from L.
vituli of cattle, A. lowffii from keds of sheep and A. pittii from H.
spiniger of dogs with high nucleotide sequence identities of 98–
100% with their respective reference species in the GenBank
(Table 5). This finding supports the criteria established for
Acinetobacter spp. identification using partial rpoB gene sequence
analysis [17,18]. In line with our observation higher predominance
of other Acinetoabacter spp. (24.8%) than A. baumannii (only 8.8%)
from human blood culture isolates was recently reported from
Norway [48]. Furthermore, these species are nowadays reported to
cause various types of human infections worldwide
[21,23,49,50,51] and are implicated as emerging Acinetobacter spp.
We also identified two new Acinetobacter species from H. spiniger of
dogs and one from ked of sheep (Table 5 and Fig. 3). All these 3
new Acinetobacter spp. demonstrated low nucleotide homology with
reference rpoB sequence in the GenBank and low bootstrap value
in the rpoB phylogenetic tree. Results of our study suggest the
presence of specific Acinetobacter species in lice and keds of domestic
animals unlike A. baumannii in human lice. We believe that
additional in depth epidemiological studies involving other species
of lice and ectoparasites of different animal species from vast areas
in the world are needed as has been done during the last decade
for Bartonella species, ectoparasites and their animal hosts [52].
Further studies are also required to isolate and determine the
human health significance of the new Acinetobacter spp. detected in
the lice and keds from animals.
To our knowledge, this study is the first to report the presence of
DNAs from different Acinetobacter spp. in various species of lice
collected from domestic animals and in flies collected from sheep.
Our study demonstrates that Acinetobacter spp. are not only
common as hospital pathogens and in human lice but they can
also be detected in the ectoparasites of animals. Our findings
suggest that synanthropic animals and their ectoparasites might
play a role to increase the risk of human exposure to zoonotic
pathogens and could be a source for Acinetobacter spp. infections in
humans. However, additional epidemiological data are required to
justify the significance of this finding and to determine whether
ectoparasites from animals can act as environmental reservoirs and
play a role in spreading these bacteria to both animal and human
hosts.
Acknowledgments
The authors greatly acknowledge all animal owners in Oromia and Yonas
Abiyi, Teferi Banti, Fikadu and Gurara Megerssa Veterinary professionals
of Oromia Regional laboratories for their help during sample collection.
Also Amina Boutellis and Seydina M. Diene PhD students at the Faculty of
Medicine of Aix Marseille University, are gratefully acknowledged for their
professional help during laboratory work of the study.
Author Contributions
Conceived and designed the experiments: DR PP. Performed the
experiments: BK CS. Analyzed the data: CS BK DR JMR. Contributed
reagents/materials/analysis tools: DR. Wrote the paper: BK CS DR PP
JMR.

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5.2. Article 8

Bartonella melophagi in Melophagus ovinus

(sheep ked) collected from sheep in northern

Oromia, Ethiopia.

Kumsa, B., Raoult, D., Parola, P., Socolovschi, C.,

Comparative Immunology, Microbiology and Infectious

Diseases 37 (2014) 69– 76.

265 
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 Author's personal copy

Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69–76

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology

and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

Bartonella melophagi in Melophagus ovinus (sheep ked)

collected from sheep in northern Oromia, Ethiopia
Bersissa Kumsa a,b , Philippe Parola a , Didier Raoult a , Cristina Socolovschi a,∗
a
Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, 13005 Marseille, France
b
Department of Parasitology, College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Bishoftu, Ethiopia

a r t i c l e i n f o a b s t r a c t

Article history: Melophagus ovinus (sheep ked) is one of the most common ectoparasites that contributes
Received 11 September 2013 to enormous economic losses in the productivity of sheep in many countries. The present
Received in revised form 28 October 2013
study was conducted from January 2012 to July 2013 on M. ovinus collected from sheep at
Accepted 1 November 2013
three sites in Ethiopia. Of the sheep studied, 65.7% (88/134) were infested with M. ovinus.
The prevalence of M. ovinus was 76% (76/100), 47% (8/17) and 23.5% (4/17) at the Kimbibit,
Keywords:
Chacha and Shano sites, respectively. An overall number of 229 M. ovinus specimens (138
Sheep
Bartonella melophagi females, 86 males and five pupae) and 554 M. ovinus specimens (272 females, 282 males)
Melophagus ovinus were collected from young and adult sheep, respectively. Bartonella DNA was detected in
Sheep ked 89% (694/783) of M. ovinus using a quantitative Bartonella genus-specific PCR assay tar-
Molecular detection geting the 16S/23S rRNA intergenic spacer region. The sequencing of the PCR products of
Oromia, Ethiopia fragments of the gltA and rpoB genes showed 99.6–100% and 100% homology, respectively,
with B. melophagi. Statistically significant variation was not noted in the overall prevalence
of Bartonella DNA between female and male M. ovinus. All of the sheep infested with M. ovi-
nus 100% (88/88) harbored at least one M. ovinus specimen that contained Bartonella DNA.
This study highlights that B. melophagi in M. ovinus from sheep in highlands in Ethiopia
possibly has certain zoonotic importance.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction Transfer from infested to non-infested sheep is by direct

Melophagus ovinus (M. ovinus) (sheep ked) is one of
the most common and economically important blood-
feeding permanent ectoparasites of sheep worldwide [1].
M. ovinus is wingless fly that belongs to the family Hip-
poboscidae, and its entire life cycle occurs in the fleece of
the sheep host. Female M. ovinus produces a single fully
developed larva every 6–8 days that firmly attaches to
the wool and becomes a puparium in 6–12 h. The pupar-
ium later develops into an adult within 19–30 days [2].
∗ Corresponding author at: URMITE, UMR CNRS 7278, IRD 198, INSERM
U1095, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5,
France. Tel.: +33 04 91 32 43 75; fax: +33 04 91 38 77 72.
E-mail address: cristina.socolovschi@univ-amu.fr (C. Socolovschi).
contact. Although M. ovinus is considered to be a sheep-
restricted ectoparasite, the organism has been found on
domestic animals (goat, rabbit (Oryctolagus cuniculus) and
dog), wild animals (European bison (Bison bonasus) and red
fox (Vulpes vulpes)) and humans [1,3,2].
In infested sheep, M. ovinus causes inflammation due to
pruritus; a loss of wool and skin damage due to rubbing,
scratching and biting; secondary microbial infections and
cutaneous myiasis; a reduction in weight gain and wool
growth; a disruption in feeding; a loss of blood; and a
reduction in the value of the hide due to cockle [1,4,2,5].
M. ovinus is a vector of Trypanosoma melophagium, which
are nonpathogenic trypanosomes, in sheep [1,4] and is
also implicated in mechanically transmitting the virus that
causes blue-tongue in sheep [6]. Furthermore, recently,
Bartonella schoenbuchensis and Bartonella chomeli in the

0147-9571/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cimid.2013.11.001
 Author's personal copy
70 B. Kumsa et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69–76
USA [7], Anaplasma ovis in Hungary [8] and Borrelia burgdor-
feri sensu lato in China [9] have been detected in M. ovinus.
Bartonella spp. are small hemotropic, facultative, Gram-
negative and arthropod-borne intracellular alphapro-
teobacteria that belong to the family Bartonellaceae.
Bartonella are aerobic bacilli, oxidative negative, fastidious,
slow growing and highly adapted to their vertebrate hosts.
The bacteria infect the erythrocytes and endothelial cells of
their mammalian hosts. To date, the genus Bartonella com-
prises more than 30 different species and subspecies that
infect a wide variety of mammalian hosts [10,11].
In humans Bartonella spp. are implicated in causing
several emerging and reemerging diseases manifested by
a wide variety of clinical signs [12,13]. At least 13 Bar-
tonella spp. were reported to cause diseases in humans,
of which B. bacilliformis (Carrion’s disease), B. quintana
(trench fever) and B. henselae (cat scratch disease) were
described as the most common species of the genus [13,14].
Other species, such as B. elizabethae, B. vinsonii subsp.
berkhoffii, B. vinsonii subsp. arupensis, B. koehlerae, B. alsat-
ica, B. washoensis, B. rochalimae, and B. tamiae, have been
associated with chronic bacteremia and/or endocarditis,
bacillary angiomatosis, peliosis hepatitis, retinitis, uveitis
and myocarditis [10,15,13,14,16].
Many species of domestic (cat, dog, cattle and sheep)
and wild (rodents, deer and elk) animals play key role
as reservoir hosts of Bartonella spp. In the mammalian
reservoir hosts, Bartonella spp. usually cause persis-
tent asymptomatic bacteremia [17]. However, recently,
ruminant-associated Bartonella spp., including B. chomeli, B.
bovis, B. schoenbuchensis and B. capreoli, have been reported
to infect cattle in France [18,19], the USA [20], Caledonia
[21], China [22] and Poland [23]. These cited investigations
in domestic ruminants are part of an escalating number of
reports of Bartonella spp. in veterinary medicine, mirroring
the situation in human medicine.
Many different species of hematophagous arthropods,
including lice (Pediculus humunus humunus), fleas (Cteno-
cephalides felis), sand flies (Lutzomyia verrucarum), biting
flies (Haematobia spp.) [24], deer keds (Lipoptena cervi)
and ticks (Ixodes ricinus), are implicated in mediating the
transmission of Bartonella spp. between reservoir hosts
and humans (and other susceptible hosts) [25,26,10,17].
Although there are several reports on the presence of Bar-
tonella spp. in lice, fleas, biting flies and ticks from domestic
animals from many countries around the world, there is
paucity of information on the occurrence of Bartonella spp.
of medical and veterinary importance in M. ovinus world-
wide. The only available reports on Bartonella spp. from
Ethiopia are 6% (2/34) B. henselae in C. felis collected from
cats [27], an 11% (5/46) prevalence of antibodies against
Bartonella spp. in cats from Addis Ababa [28] and Bartonella
spp. related to B. elizabethae in small mammals in northern
Ethiopia [29].
In Ethiopia, all previous studies on M. ovinus focused
on the organism’s prevalence and impact on the commer-
cial value of the skin of sheep [30,31]. However, studies
on the role of M. ovinus as vectors of pathogens of veteri-
nary and medical importance are remarkably scarce in the
country. In a country such as Ethiopia, where ectoparasites
of domestic animals are very common; contact between
animals and humans is normally very high; and humans
usually consume raw or insufficiently cooked meat, blood
and milk of animals, information on vector-borne bacteria
is extremely important. Thus, the current study aimed to
determine the presence of and to identify Bartonella spp.
in M. ovinus feeding on sheep at three different sites in
Ethiopia.
2. Materials and methods
2.1. Study areas and animals
In this study, sheep from three sites in Kimbibit, Shano
and Chacha were studied for M. ovinus infestation. Shano
(9 19� 60.00�� N 39◦ 18� 0.00�� E), Kimbibit (9◦ 24� 39.95�� N
◦
39◦ 21� 14.75�� E), and Chacha (9◦ 31� 20.23�� N 39◦ 27� 5.40�� E)
are located 70, 110 and 130 km, respectively, to the north of
Addis Ababa. These areas have a highland type of agroecol-
ogy, with altitudes of 1880, 3000 and 2700 meter above
sea level (m.a.s.l.), respectively. Kimbibit and Shano are
located in Oromia regional state, whereas Chacha is located
in Amhara regional state. Sheep is the major animal species
kept in Ethiopian highlands with a cool climate [32]. At
all study sites, the main rainy season is from mid-June to
September, and the dry season extends from November to
May. The farming system at all sites is characterized by a
mixed crop–livestock production system. The livestock in
the study areas are traditionally managed under extensive
production systems [32].
M. ovinus was collected from sheep in Shano, Kimbibit
and Chacha during January and February 2013. All study
sheep were selected irrespective of their sex and age. Sex
(female or male) and age (young or adult) were recorded
for each study animal. The animals were categorized into
two age groups, young (up to one year) and adult (older
than one year), according to a previous publication [30,31].
2.2. Collection and identification of M. ovinus
M. ovinus was carefully removed manually, using for-
ceps or by hand to avoid any damage to the body, and were
then placed in small plastic vials containing 70% ethanol
for subsequent identification. All collected M. ovinus spec-
imens from the same infested animal were placed into
pre-labeled, separate small plastic vials and transported to
the Laboratory of the World Health Organization Collabo-
rative Center for Rickettsial Diseases and Arthropod-borne
Bacterial Diseases in Marseille, France. The sex and stage of
M. ovinus was determined using a microscope according
to standard morphological identification keys, as previ-
ously described [30]. Photographs of the dorsal and ventral
body parts of each M. ovinus specimen were captured
(Figs. 1 and 2).
2.3. DNA extraction from M. ovinus
Before DNA extraction, each M. ovinus specimen was
rinsed twice in sterile water for 15 min and then dried
on sterile filter paper. Each specimen was longitudinally
cut into two equal halves. One-half of each specimen was
kept at −80 ◦ C as reserve sample to avoid risks of losing
 Author's personal copy
B. Kumsa et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69–76
Fig. 1. Melophagus ovinus female and male collected from sheep, Oromia, Ethiopia.
samples for any reason during or after DNA extraction.
Genomic DNA was individually extracted from a total of
783 M. ovinus one-half specimens using the QIAamp DNA
tissue extraction kit (Qiagen, Hilden, Germany) according
to the manufacturer’s instructions. The DNA from each M.
ovinus specimen was eluted in 100 �l of TE buffer and
stored at −20 ◦ C under sterile conditions to preclude con-
tamination until the sample was used for PCR [30]. To
avoid cross-contamination among samples during DNA
extraction, all parts of the DNA extracting EZI Advanced
XL Robot were disinfected after each batch of extraction
as per recommendations of the manufacturers. Further-
more, one non-infected tick from laboratory colonies of
Fig. 2. Pupae of Melophagus ovinus collected on sheep, Oromia, Ethiopia.
71
Rh. sanguineus kept at the WHO Collaborative Center for
Rickettsial Diseases (Marseille, France) was included in
each batch of extraction to rule out cross contamination
among samples. The second half of each M. ovinus specimen
was stored at −80 ◦ C as a backup sample.
2.4. Molecular detection of Bartonella spp. using ITS gene
in M. ovinus
As a first step, all DNA samples were individually
tested for the presence of the 16S/23S rRNA intergenic
spacer-region ITS gene specific for the genus Bartonella by
real-time quantitative PCR (qPCR) with a 21-bp probe, as
previously described [33,34], following the instructions of
the manufacturer (Applied Biosystems, Foster City, CA).
Sterile water and DNA extracted from non-infected Rh.
sanguineus were used as negative controls, and B. eliza-
bethae DNA was used as a positive control. The analytical
sensitivity of qPCR assay targeting ITS gene that could
detect the number of copies of B. melophagi per ked was
determined in triplicate reactions with ten-fold serial dilu-
tions of a known titration of Bartonella quintana suspension.
The samples were considered positive when the cycle
threshold (Ct) was <35, due to the data obtained after the
analysis of the analytical sensitivity of qPCR targeting ITS
gene. To verify the quality of our DNA extraction randomly
selected samples negative 50.6% (40/79) for Bartonella spp.
were further subject to standard PCR to amplify 18S rRNA
gene of M. ovnus as described before [30].
2.5. Molecular identification of Bartonella spp. using gltA
and rpoB genes
To identify Bartonella spp., a total of 50 randomly
selected M. ovinus DNA samples positive for Bartonella spp.
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based on qPCR analysis of the ITS gene were further sub-
jected to a standard PCR targeting the citrate synthase (gltA)
gene and the rpoB gene using primers and all conditions
described before [35,21,36]. Sterile water and B. elizabethae
DNA were used as negative and positive controls, respec-
tively. The detection of amplified products, the removal of
excess primers and nucleotides from the DNA, sequencing,
the assembly and edition of sequences and BLAST analysis
were all performed using similar methods described before
[30].
2.6. Data analysis
Microsoft Excel was used for data management.
Descriptive statistics, such as percentages and means, were
employed to summarize the proportions of infestations
with M. ovinus and infections of M. ovinus with Bartonella
spp. Statistical analysis was performed with EpiInfoTM 7.
Associations between the sex and age groups of the sheep,
the sites of collection and the sex of M. ovinus and the preva-
lence of M. ovinus infestation in sheep and Bartonella spp. in
M. ovinus were determined by the Mantel–Haenszel (MH)
test using EpiInfoTM 7. A p-value of <0.05 was considered
significant.
2.7. Ethical statement
Ethical approval for the collection of M. ovinus from
sheep was obtained from the animal research ethics board
(Agreement # 14/160/550/2011) of the College of Veteri-
nary Medicine and Agriculture of Addis Ababa University.
All necessary oral permits were obtained for the field
studies, including permission from the administration and
agricultural office of each site and from each animal owner.
M. ovinus collection from sheep was not harmful and not
contrary to the welfare of the animals.
3. Results
3.1. Prevalence of M. ovinus in sheep
In the present study, 88/134 (65.7%) sheep were infested
with M. ovinus (Table 1). An overall number of 783 M. ovinus
specimens (410 females, 368 males and five pupae) were
collected from infested sheep. The prevalence of M. ovi-
nus was 76% (76/100), 47% (8/17) and 23.5% (4/17) at the
Kimbibit, Chacha and Shano sites, respectively (Table 1).
The prevalence of M. ovinus was significantly higher in
Kimbibit than at both the Chacha (MH test, p = 0.01) and
the Shano (MH test, p = 0.00001) sites. In female sheep, an
overall prevalence of 63.1% (36/57) and 252 M. ovinus spec-
imens (135 f, 116 m and one pupa) were recorded. The
corresponding figures for male sheep were 65.8% (52/79)
prevalence and 531 M. ovinus specimens (275 f, 252 m and
four pupae) (Table 1). An overall prevalence of 65% (26/40)
and 64.6% (62/96) was observed in young and adult sheep,
respectively (Table 1). An overall number of 229 M. ovi-
nus specimens (138 f, 86 m and five pupae) and 554 M.
ovinus specimens (272 f, 282 m and zero pupae) were col-
lected from young and adult sheep, respectively (Table 1).
A statistically significant difference was not noted in the
overall prevalence of M. ovinus infestations between sheep
of different sex (MH, p = 0.75) and age (MH, p = 0.96) groups.
In the current study, at all three sites, there was
no statistically significant difference between the overall
numbers of female and male M. ovinus specimens collected
from female compared with male sheep (MH, p = 0.67),
young compared with adult sheep (MH, p = 0.1) and in the
overall collection (MH, p = 0.19). The female-to-male ratio
of M. ovinus was 1:02, 1:9, 1:4 and 1:1 for the Kimbibit,
Chacha and Shano sites and the overall collection, respec-
tively.
3.2. Molecular detection of Bartonella spp. using ITS gene
in M. ovinus
An overall prevalence of 88.6% (694/783) for Bartonella
spp. DNA was recorded in M. ovinus collected from sheep
in northern Oromia using molecular tools (Table 2). The
prevalence of Bartonella spp. DNA was 88.4% (586/663),
94.9% (93/98) and 68.2% (15/22) at the Kimbibit, Chacha
and Shano sites, respectively (Fig. 3) (Table 2). A statisti-
cally significant difference in the prevalence of Bartonella
spp. at the three sites (MH, p ≥ 0.05) was not observed. Sim-
ilarly, statistically significant variation was not noted in the
overall prevalence of Bartonella spp. between female and
male M. ovinus collected from sheep at the three sites or
between different sex and age groups (Table 2).
The overall prevalence of Bartonella spp. was 74.2%
(187/252) and 95.5% (507/531) among M. ovinus speci-
mens collected from female and male sheep, respectively.
The corresponding figures among M. ovinus specimens col-
lected from young and adult sheep were 96.5% (222/230)
and 85.3% (472/553), respectively (Table 2). Statistically
significant differences were not found in the overall preva-
lence of Bartonella spp. between M. ovinus specimens
collected from female compared with male sheep and
young compared with adult sheep (MH, p ≥ 0.05).
An analysis of M. ovinus specimens that were positive
for Bartonella spp. DNA on the infested sheep revealed that
100% (88/88) of the sheep infested with M. ovinus harbored
at least one M. ovinus specimen containing Bartonella spp.
DNA. The mean qPCR Ct values and standard deviations
(SDs) for Bartonella spp. DNA-positive samples using the
ITS gene were 22.2 ± 4.04 (ITS gene: mean copies/half of ked
1.7 × 105 [minimum 9 × 103 , maximum 3 × 106 ]), 23 ± 4.1
(ITS gene: mean copies/half of ked 9.6 × 104 [minimum
5 × 103 , maximum 1.8 × 106 ]), 30 ± 4.2 (ITS gene: mean
copies/half of ked 0.6 × 103 [minimum 0.3 × 102 , maximum
1.3 × 104 ]), and 22.6 ± 4.1 (ITS gene: mean copies/half of
ked 1.3 × 105 [minimum 6.7 × 103 , maximum 2.4 × 106 ])
for females, males, pupae and overall samples, respectively.
Visualization of strong DNA bands of amplified PCR
products using electrophoresis in all the 40 DNA samples
tested for 18S rRNA gene suggested that our DNA extraction
conditions were appropriate and free of PCR inhibitors.
3.3. Molecular identification of Bartonella spp. in M.
ovinus using gltA and rpoB genes
To identify Bartonella spp., we amplified a 200- to
250-bp fragment of the gltA gene and an 800- to 900-
bp fragment of the rpoB gene from each of the 50 DNA
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B. Kumsa et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69–76 73

Table 1
Overall percentage of M. ovinus infestations by sex and age of sheep at three sites in northern Ethiopia.

Site Host sex Host age Total

No. infested/No.
examined (%)
(Total M. ovinus: f, m, p)

Female Male Young Adult

No. infested/No. No. infested/No. No. infested/No. No. infested/No.
examined (%) examined (%) examined (%) examined (%)
(Total M. ovinus: f, m, p) (Total M. ovinus: f, m, p) (Total M. ovinus: f, m, p) (Total M. ovinus: f, m, p)

Kimbibit 31/41 (75.6%) 45/59 (76.3%) 21/27 (77.8%) 54/72 (75%) 76/100 (76%)
(225: 120 f, 104 m, 1 p) (438: 214 f, 222 m, 2 p) (152: 85 f, 64 m, 3 p) (511: 249 f, 262 m, 0 p) (663: 334 f, 326 m, 3 p)
Chacha 3/6 (50%) 5/11 (45.4%) 3/5 (60%) 5/12 (41.7%) 8/17 (47%)
(10: 5 f, 5 m, 0 p) (88: 58 f, 28 m, 2 p) (76: 52 f, 22 m, 2 p) (22: 11 f, 11 m, 0 p) (98: 63 f, 33 m, 2 p)
Shano 2/10 (20%) 2/9 (22.2%) 1/7 (14.3%) 3/12 (25%) 4/17 (23.5%)
(17: 10 f, 7 m) (5: 3f, 2 m) (1: 1 f, 0 m) (21: 12 f, 9 m) (22: 13 f, 9 m)

Total 36/57 (63.1%) 52/79 (65.8%) 26/40 (65%) 62/96 (64.6%) 88/134 (65.7%)
(252: 135 f, 116 m, 1 p) (531: 275 f, 252 m, 4 p) (229: 138 f, 86 m, 5 p) (554: 272 f, 282 m, 0 p) (783: 410 f, 368 m, 5 p)

Table 2
Prevalence of Bartonella spp. using ITS gene by qPCR analysis of M. ovinus collected from sheep in northern Oromia.

Host factors Host sex Host age Total hosts

No. positive M.
ovinus/No. tested M.
ovinus (%)

Site M. ovinus sex Female host Male host Young host Adult host
No. positive M. No. positive M. No. positive M. No. positive M.
ovinus/No. tested M. ovinus/No. tested M. ovinus/No. tested M. ovinus/No. tested M.
ovinus (%) ovinus (%) ovinus (%) ovinus (%)

Kimbibit F 92/120 (76.7%) 203/214 (94.8%) 84/85 (98.8%) 211/249 (84.7%) 295/334 (88.3%)
M 76/104 (73.1%) 213/222 (95.9%) 32/35 (91.4%) 228/262 (87%) 289/326 (88.6%)
Pupa 0/1 (0) 2/2 (100%) 2/3 (66.7%) 0/0 (0) 2/3 (66.7%)
Total 168/225 (74.7%) 418/438 (95.4%) 147/152 (96.7%) 439/511 (85.9%) 586/663 (88.4%)

Chacha F 4/5 (80%) 57/58 (98.3%) 51/52 (98.1%) 10/11 (90.9%) 61/63 (96.8%)
M 4/5 (80%) 27/28 (96.4%) 21/22 (95.4%) 10/11 (90%) 31/33 (93.9%)
Pupa 0/0 (0) ½ (50%) ½ (50%) 0/0 (0) ½ (50%)
Total 8/10 (80%) 85/88 (96.6%) 73/76 (96%) 20/22 (90.9%) 93/98 (94.9%)

Shano F 7/12 (58.3%) 2/3 (66.7%) 1/1 (100%) 8/14 (57.1%) 9/15 (60%)
M 4/5 (80%) 2/2 (100%) 1/1 (100%) 5/6 (83.3%) 6/7 (85.7%)
Total 11/17 (64.7%) 4/5 (80%) 2/2 (100%) 13/20 (65%) 15/22 (68.2%)

Overall 187/252 (74.2%) 507/531 (95.5%) 222/230 (96.5%) 472/553 (85.3%) 694/783 (88.6%)

samples randomly selected to represent M. ovinus col-
lected from the three sites and sheep of different sex and
age groups. A BLAST analysis of the gltA gene sequence
from all 50 M. ovinus specimens showed 99.6–100%
(240/241–241/241) nucleotide homology with the Gen-
Bank reference sequence of the candidate B. melophagi
gltA gene obtained from M. ovinus found on sheep in
the USA (GenBank accession number: AY692475). Sim-
ilarly, a BLAST analysis of the rpoB gene sequence of
Bartonella spp. obtained from all 50 M. ovinus speci-
mens showed 100% (800/800) nucleotide identity with the
GenBank reference sequence B. melophagi rpoB obtained
from M. ovinus found on sheep in the USA (GenBank
accession number: EF605288); however, published arti-
cle is not available on B. melophagi in M. ovinus of
sheep.
4. Discussion
In this study, the observation of negative results
for all negative-control samples and positive findings
for all positive-control samples in both qPCR and con-
ventional PCR assays validated our molecular findings
and confirmed that our working conditions precluded any
accidental cross-contamination of the samples during the
study period.
A molecular identification study of Bartonella spp. in M.
ovinus using the rpoB and gltA genes confirmed the pres-
ence of B. melophagi in our samples. This finding supports
the earlier argument that the rpoB and gltA genes have been
described as more potent than all other genes for the differ-
entiation of closely related Bartonella spp. [35]. B. melophagi
was isolated from sheep blood in the USA [37], and recently,
the same bacteria were isolated from the blood of two
female patients with pericarditis and skin lesions in the
USA [38]. Furthermore, have been deposited the rpoB gene
sequence of B. melophagi amplified from M. ovinus in Gen-
Bank (accession number: EF605288), but these data are
still unpublished and not accessible using PubMed search.
With all of these previously established facts in mind, we
hypothesize that M. ovinus may play an important role in
 Author's personal copy
74 B. Kumsa et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69–76
Fig. 3. Map of Ethiopia showing the study sites and the rate of Bartonella melophagi infection in Melophagus ovinus, sheep ked by site.
the transmission of B. melophagi from sheep to sheep in
Ethiopia. To our knowledge, this study is the first to report
B. melophagi in M. ovinus collected from sheep in Ethiopia.
We believe that additional in-depth studies are neces-
sary to determine the precise role of M. ovinus, sheep and
humans in B. melophagi transmission in the highlands of
Ethiopia.
An overall prevalence of 88.6% for B. melophagi DNA
in M. ovinus collected from sheep was recorded in our
study and is in agreement with the previously reported
prevalence of Bartonella spp. in other species of arthro-
pods in other countries [7]. However, overall prevalence
values that are lower [39,24] and higher than our results
for Bartonella spp. have been reported [7], variation that
is most likely attributable to differences in geographical,
animal and vector factors. The absence of a significant dif-
ference in the overall prevalence of B. melophagi in M.
ovinus from the three sites and between M. ovinus col-
lected from female compared with male sheep and young
compared with adult sheep in this study suggests that
the study site and the sex and age of the sheep do not
affect the prevalence of B. melophagi in M. ovinus. This
observation supports the role of M. ovinus as a poten-
tial vector of B. melophagi, as has been suggested before
[38].
The absence of a significant difference in the overall
prevalence of B. melophagi between female and male M.
ovinus collected from sheep in this study suggests that the
sex of M. ovinus does not affect the organism’s infection
with B. melophagi, which is in line with a previous report of
100% prevalence of Bartonella spp. in M. ovinus in the USA
[7].
The detection of B. melophagi in the non-feeding pupae
of M. ovinus suggest the possibility of vertical transmission
of B. melophagi in M. ovinus, as has been argued before [7].
The overall prevalence of 65.7% for M. ovinus recorded
in our study indicates the great importance of M. ovinus
in sheep at the study sites in northern Oromia. This high
overall prevalence is most likely attributable to several
important factors, including favorable climatic conditions
important for the survival, reproduction and development
of all stages of M. ovinus; malnutrition and poor husbandry
systems; the poor awareness of farmers; and inadequate
veterinary services at the study sites. This finding is in
line with previous reports from many sites in Ethiopia
that showed that M. ovinus is restricted to highland areas
with cool climates, where sheep are commonly raised, but
is not found in lowland areas in Ethiopia [31]. However,
this finding contrasts with the situation in most developed
European countries and other countries of the world where
M. ovinus is eradicated with effective pesticides [2].
A significantly higher prevalence of M. ovinus in Kim-
bibit than at both the Chacha and the Shano sites in our
study possibly reflects that the Kimbibit site, with an alti-
tude of 3000 m.a.s.l., is a more suitable environment for
reproduction of M. ovinus than the other two sites, which
are at lower altitudes [31]. The absence of a statistically
significant difference in the overall prevalence of M. ovinus
among sheep of different sex and age groups in our study
is in line with earlier observations that suggest that differ-
ences in the prevalence of M. ovinus infestation in sheep
are associated with variations in altitude and environmen-
tal factors more than with host-dependent factors due to
the high likelihood of the transfer of M. ovinus by direct
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B. Kumsa et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69–76
contact among sheep, including transfer from ewes to
lambs during suckling [2].
5. Conclusion
This study provides molecular evidence of B. melophagi
infection in M. ovinus collected from sheep in Ethiopia.
Given the high prevalence of M. ovinus infestations in sheep
and the very high prevalence of B. melophagi infection in M.
ovinus in our study, together with the previous isolation of
the same bacteria from human and sheep blood in the USA,
M. ovinus may be more important in Ethiopia than previ-
ously believed. This study highlights that B. melophagi in
M. ovinus on sheep in highlands in Ethiopia may have cer-
tain zoonotic importance. Overall, our present data warrant
detailed future studies on ruminant-associated Bartonella
spp. in Ethiopia, a country for which there is no informa-
tion about Bartonella spp. and associated diseases in either
humans or animals. In addition, future studies are neces-
sary to address the isolation and culture of this bacterium
from human and sheep blood and its public health signifi-
cance in Ethiopia.
Acknowledgments
The authors greatly acknowledge the sheep owners at
the study sites for allowing us to collect M. ovinus from
their sheep. We would also like to thank the laboratory
technicians from URMITE, Marseille, and particularly Jean-
Michel Bérenger, and Veronique Brice for their technical
support.
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6. CONCLUSIONS

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276 
6.1. Conclusion and perspectives

Our study to investigate zoonotic bacteria in ticks collected from

animals in Ethiopia demonstrated that spotted fever group (SFG)
rickettsiae are higher in central parts of Ethiopia coinciding with
the distribution of Amblyomma variegatum ticks while in south-
eastern Oromia in Borana zone Amblyomma gemma seems the
vector for R. africae. The new Borrelia spp. recorded in our
study seems to be associated with Amblyomma spp. ticks
especially with Am. cohaerens in the western Oromia, with Am.
variegatum in the central parts and with Am. gemma in the
south-eastern districts in Oromia. Furthermore, our study
provided clear molecular evidence for the presence of Coxiellaa
burnetii in different ixodid tick species especially in south-
eastern Oromia in Borana zone in pastoral community.
The study on the identification of ixodid ticks of Ethiopia
evidenced that protein profiling by MALDI-TOF MS could be
also used for identification of ixodid ticks species preserved in
70% ethanol based on the protein extracted from the legs of
large and medium size ticks provided that reference spectra
database is created from corresponding tick species specimens
stored in the same conditions with the blind test spectra. For the

277 
small size ticks, eggs, larvae and nymphs of ticks other body
parts without abdomen seems more applicable.
Our study on fleas provided molecular evidence that R. felis and
B. henselae in the fleas collected from dogs and cats in Ethiopia.
Likewise, our study on lice and sheep ked documented the
presence of Acinetobacter spp. in the lice and sheep ked
(Melophagus ovinus) collected from domestic animals in
Ethiopia and high prevalence of Bartonella melophagi in
M. ovinus of sheep in highlands of Ethiopia.
Overall, our findings alert physicians managing patients with
fever of unknown aetiology in Ethiopia and those who care for
travellers from Ethiopia to consider the presence of several
species of zoonotic vector-borne bacteria such as SFG
rickettsiae, C. burnetii, R. felis, B. henselae and B. melophagi as
potential causative agents in their differential diagnosis. In
addition, future studies are needed to address the isolation,
culture and genotypes of these zoonotic bacteria from different
arthropod species collected from animals and vegetation
(example questing ticks), and in blood samples from humans,
domestic and other reservoir animals and determine the public
health significance of these bacteria in Ethiopia.
Future studies are needed to address the vector competence of
Rhipicephalus (Boophilus) decoloratus and Am. gemma for

278 
R. africae by demonstrating its ability to acquire the pathogen
from an infected host and transmit it to nave susceptible animals
during the subsequent feeding. Similarly, the vector competence
of M. ovinus for Bartonella melophagi, Amblyomma spp. ticks
for the new Borrelia spp. remains to be confirmed by future
studies.
Other important questions awaiting future studies include
determination to get clear information on the comparative role
of different species of domestic and wild animals, the role of
different species of arthropods and identification of reservoir
animals and description of diseases caused by reported bacteria
in human patients. Much works is needed to increase the society
and professional awareness about vector-borne bacterial
diseases so as to mitigate the negative effects on public health.
Future studies are expected to develop revised MALDI-TOF MS
protein profiling method for tick identification by providing
standardized methods of sample preparation, protein
differentiation, parameters of spectra quality control and spectra
analysis and interpretation methods so that this technique will be
widely used uniformly around the world. In addition,
standardized MALDI-TOF MS method for identification of the
small size ticks, eggs, larvae and nymphs of ticks need to be
developed.

279 
 
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