OSTEOMYELITIS CAUSED BY SALMONELLA ENTERICA

SEROVAR DERBY IN BOA CONSTRICTOR

Author(s): Suyene O. de Souza, D.V.M., Renata A. Casagrande, D.V.M., M.Sc.,
Priscila R. Guerra, D.V.M., M.Sc., Cludio E. F. Cruz, D.V.M., Ph.D., Evandro
Veit, B.C.B., Marisa R. I. Cardoso, D.V.M., Ph.D., and David Driemeier,
D.V.M., Ph.D.
Source: Journal of Zoo and Wildlife Medicine, 45(3):642-644. 2014.
Published By: American Association of Zoo Veterinarians
DOI: http://dx.doi.org/10.1638/2013-0050R2.1
URL: http://www.bioone.org/doi/full/10.1638/2013-0050R2.1

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Journal of Zoo and Wildlife Medicine 45(3): 642644, 2014

Copyright 2014 by American Association of Zoo Veterinarians

OSTEOMYELITIS CAUSED BY SALMONELLA ENTERICA SEROVAR

DERBY IN BOA CONSTRICTOR
Suyene O. de Souza, D.V.M., Renata A. Casagrande, D.V.M., M.Sc., Priscila R. Guerra, D.V.M.,
M.Sc., Claudio E. F. Cruz, D.V.M., Ph.D., Evandro Veit, B.C.B., Marisa R. I. Cardoso, D.V.M., Ph.D.,
and David Driemeier, D.V.M., Ph.D.

Abstract: After demonstrating chronic weight loss, prostration, and muscle flaccidness, a captive-bred 9-moold boa constrictor (Boa constrictor constrictor) died and was submitted for necropsy. Along the spinal column there
were multiple, yellowish white, macroscopic nodules of 15 mm in diameter in the ventral side of the vertebral
body and in the intervertebral spaces. Severe multifocal necrotizing osteomyelitis associated with granulomatous
inflammation was the main histologic finding in the vertebral column. In the liver, there was discrete but similar
granulomatous changes. Positive anti-Salmonella immunostaining was observed in the spinal column and in the
liver. Salmonella enterica serovar Derby was isolated from fragments of the spinal column. These bacteria are
important cause of disease in captive reptiles.
Key words: Immunohistochemistry, osteomyelitis, pathology, salmonellosis, snake.

BRIEF COMMUNICATION
Salmonella are gram-negative facultative anaerobes. In 2007, a total of 2,579 serotypes have been
accounted in the genus Salmonella.4 Affected
animals usually do not show clinical signs, or
they may present weakness and sudden death.4,6
Reptiles are among the main natural reservoirs of
Salmonella worldwide.3,5,7
Salmonella enterica subsp. arizonae and Salmonella enterica subsp. diarizoniae are the subspecies
most often isolated from reptiles.5 Salmonellosis
is the most recognized reptilian zoonosis,7 and
reptiles kept as pets represent an important
source of Salmonella transmission to humans,
which generally results from precarious hygiene
conditions and involves fecal-oral transmission.
This communication describes a case of osteomyelitis caused by Salmonella enterica serovar Derby
in a captive-bred boa constrictor (Boa constrictor
constrictor).
Two captive bred 3-mo-old boa constrictors
were acquired from a snake-breeding facility and
then kept as pets in individual plastic boxes with
From the Departamento de Patologia Cl nica, Faculdade de Veterinaria, Universidade Federal do Rio Grande
do Sul (UFRGS), Avenida Bento Gonc alves 9090,
Agronomia 91540-000, Porto Alegre-RS, Brasil (Souza,
Casagrande, Cruz, Driemeier); Setor de Medicina Veterinaria Preventiva, Faculdade de Veterinaria, UFRGS, Av.
Bento Gonc alves 9090, Agronomica 91540-000, Porto
Alegre-RS, Brasil (Guerra, Cardoso); and Faculdade de

Ciencias
Biologicas, Universidade do Vale dos Sinos,
Figueiras CEP. 90.470Avenida Luiz Manoel, 744, Tres
280, Porto Alegre, Brasil (Veit). Correspondence should be
addressed to Dr. Cruz (claudio.cruz@ufrgs.br).

60 3 40 3 40 cm. The substrate consisted of sheets

of newspaper, and the temperature was kept at
between 22 and 288C. The pet owner had additional snakes: three Boa constrictor constrictor and
eight Boa constrictor amarali (short-tailed boa), all
kept in individual enclosures. Since its acquisition, the affected snake showed lethargy and
anorexia. Weight loss and muscular flaccidness
were evident from the fourth month of age, when
it began to be fed dead mice (Mus musculus). At 9
mo of age, having fed only five times, the reptile
was thin and was found dead. No clinical alterations were observed in the remaining snakes
from this owner, including the additional B.
constrictor constrictor acquired from the same
origin.
Upon necropsy evaluation of the spinal column,
multiple yellowish white nodules, 15 mm in
diameter, were observed in the ventral side of
the vertebral body and in the intervertebral spaces
along the whole length of the column (Fig. 1A).
The histopathologic examination of the spinal
column revealed severe multifocal necrotizing
osteomyelitis and granulomas characterized by a
central area of necrosis, sometimes associated
with bacterial myriads, in the form of rods,
surrounded by heterophilic inflammatory infiltrate, macrophages, epithelioid macrophages,
multinucleate giant cells, and a thin capsule of
connective tissue (Figs. 1B, 1C). Diffuse accentuated fibrovascular proliferation and areas of
collagen deposition were also observed. In the
liver, there were similar but discrete multifocal
granulomatous changes as well as diffuse moderate congestion and diffuse accentuated hepatocellular degeneration.

642

SOUZA ET AL.SALMONELOSIS IN BOA CONSTRICTOR

643

Figure 1. A. Multiple yellowish white nodules with 15 mm of diameter in ventral side of the vertebrae and in
the intervertebral spaces along the whole length of the column (arrows) are shown. B. Vertebrae with severe
necrotizing osteomyelitis and granulomas characterized by a central area of necrosis (asterisk), spinal cord
(arrowhead), and trabecular bone (arrows). H&E stain. 310. C. Vertebrae with severe necrotizing osteomyelitis
and granulomas characterized by a central area of necrosis (asterisk), surrounded by inflammatory infiltration,
macrophages, multinucleate giant cells and a thin capsule of connective tissue from the spinal cord (arrowhead).
H&E stain. 320. D. Positive anti-Salmonella immunostaining within the cytoplasm of macrophages scattered in the
granulomas from the spinal column (arrowhead). Streptavidin-biotin-peroxidase. 320.

Immunohistochemical (IHC) studies were performed on the spinal column and liver for
detection of Salmonella using the polyclonal
antibody anti-Salmonella (Biogenesist Salmonella
8209-4006 Rabbit Anti-Ig, Biogenesis Ltd., Poole,
Dorset, BH17 7DA, United Kingdom; dilution
1: 1,000) with 0.05% protease XIV (Sigma Chemical Company, Poole, Dorset, BH12 4QH, United
Kingdom) for 15 min for antigen retrieval, as well
as a modified avidin-biotin peroxidase complex
method using diaminobenzidine (Dako, Carpinteria, California 93013, USA) as the chromogen.
Positive anti-Salmonella IHC staining was observed in the spinal column, within the cytoplasm
of macrophages, and the necrotic areas of the
granulomas (Fig. 1D); these cells were also
observed in the liver.

Fragments of the vertebrae with whitish nodules

were aseptically collected at necropsy for aerobic
microbiologic culture at 378C. Suspected colonies
were isolated and identified through traditional
biochemical reactions, agglutination with polyvalent Salmonella O antisera (Difco, Detroit, Michigan 48201, USA). The bacterial cultures were
then submitted to the Oswaldo Cruz Institute, Rio
de Janeiro State, for strain characterization. In the
fragments of vertebrae, pure cultures of S. enterica
serovar Derby were identified.
Reptiles are important asymptomatic reservoirs
of Salmonella spp. in nature, and snakes are among
their main carriers.3 Salmonella spp. may cause
important diseases in captive reptiles.7 Diphtheritic necrotizing gastritis in a rosy boa (Lichanura
trivirgata),8 necrotizing tracheitis in a two-headed

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JOURNAL OF ZOO AND WILDLIFE MEDICINE

king snake (Lampropeltis hondurensis),9 granulomatous systemic disease associated with fibrinonecrotic pericarditis in Madagascar Dumerilis boa
(Acrantophis dumerili),12 and septicemia in three boa
constrictors1 have been described, and all of them
were linked to S. enterica subsp. arizonae infections.
Salmonella spp. have also been isolated from 6 out
of 15 snakes with clinical evidence of segmental,
proliferative osteoarthropathy.6 In a 5-yr prospective study in a 19-member colony of the ridgenose
rattlesnake (Crotalus willardi), seven snakes were
affected by osteomyelitis in a vertebral column
associated with Salmonella enterica arizonae serotype 56:Z4, Z23 infections. In some snakes,
granulomatous hepatitis, oophoritis, orchitis, and
fibrinonecrotic enteritis were also observed.11
In the case reported herein, S. enterica serovar
Derby caused diffuse osteomyelitis in a young boa
constrictor. The bacteria can penetrate the spinal
column by direct extension, due to a trauma in the
soft adjacent tissues, or via hematogenic dispersion, due to septicemia,7 which probably was the
case here, because the bacteria was demonstrated
in the liver through IHC and there was no history
of trauma. Bacterial osteomyelitis can disseminate
through the cortical bone, especially in young
animals, in which this cortex does not form a
complete bone wall.2 Salmonella spp. can be present
in the animals digestive tract, from which it may
disseminate after an episode of lowering immunity
and may cause septicemia. Neither a bacterial nor a
histopathologic examination of the intestine was
carried out to test this hypothesis, because intestine samples were not sent for analysis.
The source of the S. enterica serovar Derby
infection in the case reported herein was not
determined. However, the box substrate, fomites,
and the mice used for feeding should be considered plausible possibilities.11 Because the snake
was young and it had shown clinical signs from
the fourth month of age, the vertical transmission
could also be the contamination route, as described previously in domesticated birds5 and
snakes (Bitis).13 Colonization of offspring during
pregnancy and birth is a significant way of
transmission, causing 65% of the newborn to be
positive for Salmonella.13
To the authors knowledge, there are no reports
of reptilian disease due to S. enterica serovar
Derby infection, which was isolated from fecal
samples of humans that were in contact with
snakes.10 Although some serotypes Salmonella
spp. are species specific, most, if not all of them,
should be considered as potential pathogenic
agents in humans and animals.

LITERATURE CITED
1. Boever WJ, Williams J. Arizona septicemia in
three boa constrictors. Vet Med Small Anim Clin.
1975;70:13571359.
2. Carlson CS, Weisbrode SE. Bones, joints, tendons, and ligaments. In: Zachary JF, McGavin MD
(eds.). Pathologic basis of veterinary disease, 5th ed. St
Louis (MO): Elsevier Mosby; 2012. p. 920971.
3. Geue L, Loschner U. Salmonella enterica in
reptiles of German and Austrian origin. Vet Microbiol.
2012;84:7991.
4. Grimont PAD, Weill FX. Antigenic formulae of
the Salmonella serovars. 9th ed. Paris, France: World
Health Organization Collaborating Centre for Reference and Research on Salmonella; 2007; 166 p.
5. Hirsh DC. Enterobacteriaceae: Salmonella. In:
Hirsh DC, MacLachlan NJ, Walker RL (eds.). Veterinary microbiology. 2th ed. Oxford, United Kingdom:
Blackwell Publishing; 2004. p. 6974.
6. Isaza R, Garner M, Jacobson E. Proliferative
osteoarthritis and osteoarthrosis in 15 snakes. J Zoo
Wildl Med. 2000;31:2027.
7. Mitchell MA. Salmonella: diagnostic methods for
reptiles. In: Mader DR (ed.). Reptile medicine and
surgery. 2nd ed. St. Louis (MO): Saunders Elsevier;
2006. 1,242 pp.
8. Oros J, Rodrgues JL, Herraez P, Santana P,
Fernandez A. Respiratory and digestive lesions caused
by Salmonella arizonae in two snakes. J Comp Pathol.
1996;115:8589.
ros J, Rodrguez JL, de los Monteros AE,
9. O
Rodrguez F, Herraez P, Fernandez A. Tracheal
malformation in a bicephalic Honduran milk snake
(Lampropeltis hondurensis) and subsequent fatal Salmonella arizonae infection. J Zoo Wildl Med. 1997;28:331
335.
10. Prapasarakul N, Pulsrikarn C, Vasaruchapong T,
Lekcharoen P, Chanchaithong P, Lugsomya K, Keschumras N, Thanomsuksinchai N, Tanchiangsai K,
Tummaruk P. Salmonella serovar distribution in cobras
(Naja kaouthia), snake-food species, and farm workers
at Queen Saovabha Snake Park, Thailand. J Vet Diagn
Invest. 2012;24:288294.
11. Ramsay EC, Daniel GB, Tryon BW, Merryman
JI, Morris PJ, Bemis DA. Osteomyelitis associated
with Salmonella enterica SS arizonae in a colony of
ridgenose rattlesnakes (Crotalus willardi). J Zoo Wildl
Med. 2002;33:301310.
12. Schillinger L, Vanderstylen D, Pietrain J, Chetboul V. Granulomatous myocarditis and coelomic
effusion due to Salmonella enterica arizonae in a
Madagascar Dumerilis boa (Acrantophis dumerili, Jan.
1860). J Vet Cardiol. 2003;5:4345.
13. Schroter M, Speicher A, Hofmann J, Roggentin
P. Analysis of the transmission of Salmonella spp.
through generations of pet snakes. Environ Microbiol.
2006;8:556557.
Received for publication 19 March 2013