Brucellosis- A neglected zoonosis

By Ajay Pathak

What is brucellosis ?
Brucellosis- important re-emerging zoonosis with a worldwide distribution

It is primarily a disease of reproductive system of sexually matured animals with concomitant loss in productivity of affected animals Causative agent Brucella spp.

Brucella spp.- Gram negative, aerobic, non spore forming, nonencapsulated, coccobacilli Facultative intracellular pathogen

The Many Names of Brucellosis

Human Disease Malta Fever Undulant Fever Mediterranean Fever Rock Fever of Gibraltar Gastric Fever Animal Disease Bangs Disease Enzootic Abortion Epizootic Abortion Slinking of Calves Ram Epididymitis Contagious Abortion

Taxonomic History of Brucella

1887: David Bruce 1897: B. Bang 1920: K.F. Meyer et. al. 1929: I.F. Huddleston 1956: M.B. Buddle 1957: H.B. Stoenner 1968: Carmichael & Bruner 2007: Foster et. al. 2008: Scholz et. al. 2008-10: De et. al. & Scholz et. al. 2010: Tiller et. al. Micrococcus melitensis Bacillus abortus Brucella gen. nov. B. suis B. ovis B. neotomae B. canis B. ceti & B. pinnipedialis B. microti B. inopinata Two new Brucella spp. Strain BO2 from a human patient (Australia) 7 rodent Brucella strains from Australia

Genus Brucella
Class -Alphaproteobacteria Order -Rhizobiales Family -Brucellaceae Genus Brucella- 10 species affecting distinct primary hosts B. abortus preferentially infects cattle, B.melitensis sheep and goats, B. suis pigs, B.ovis sheep and B. canis dogs.

Above species infect humans with B.melitensis and B. abortus being the most common.

Species B. melitensis B. abortus B. suis

Biovars 1-3 1-6, 9 1, 3 2 4 5

Preferential host(s) sheep, goat Cattle Pig wild boar, hare reindeer, caribou Rodent desert rat Ram Dog Seals Cetaceans soil, vole, fox Human

Pathogenicity in humans high high high low high no moderate No moderate ? ? ? ?

B. neotomae B. ovis B. canis B. pinnipedialis B. ceti B. microti B. inopinata

 In humans undulant fever, weight loss, muscular pain, arthritis, excessive sweating, malaise, anorexia, headache, back pain, orchitis Complications- osteoarthritis, endocarditis and several neurological disorders

Symptoms

In animals abortion in third trimester of pregnancy, stillbirth, orchitis in males, retained placenta, endometritis, hygroma, occurance of Brucella organisms in secretions(milk, vaginal fluid and other body fluids)

General characteristics
The genomes of Brucella abortus, B. melitensis and some biotypes of B. suis contain two distinct chromosomes Brucella spp. lacks most of the traditional virulence factors like capsules, secreted proteases, exotoxins, endotoxins, pili and/or fimbriae or virulence plasmids, seen in other pathogens The lipopolysaccharide (LPS) layer of pathogenic Brucella is less immunogenic than that of most Gram-negative organisms because it does not bear a marked PAMP Many strains require supplementary CO2 for growth Usually oxidase and urease positive

Global scenario
The zoonotic pathogens B. abortus, B. melitensis, and B. suis were designated as Select Agents of Category B by CDC, USA.

Brucellosis is widely distributed all over the world and it is one of the world's major zoonotic problems accounting for the annual occurrence of more than 500,000 cases (Pappas et al. 2006).
Worldwide, reported incidence of human brucellosis in endemic disease are as varies widely, from <0.01 to >200 per 100,000 population

Worldwide Incidence of Human Brucellosis

Pappas et al. The new global map of human brucellosis: Lancet Infect Dis, 2006;6(2):91-9.

 Bovine brucellosis (B. abortus) has been eradicated from Australia, Canada, Cyprus, Denmark, Finland, The Netherlands, New Zealand, Norway, Sweden and the United Kingdom

It remains an uncontrolled problem in regions of high endemicity such as the Africa, Mediterranean, Middle East, parts of Asia and Latin America

The recent isolation of distinctive strains from marine mammals and humans has extended the ecologic range of the genus and, potentially its scope as a zoonosis

Indian scenario
Serological evidences are suggestive of high endemicity of brucellosis in India. It was first reported in 1942. B. abortus biotype-1 is the predominant infective biotypes in cattle and buffaloes while B. melitensis biotype-1 is in sheep, goats and man. In India, 5% cattle, 3% buffaloes, 7.9% sheep and 2.2% of goats are infected with brucellosis (Renukaradhya et al. 2002). Growing dairy industry in India- Increased risk due to increased trade and rapid movement of livestock

 In a detailed study of 47 organized farms in the southern State of Karnataka by Isloor et al. (1998), 207 of 4,995 (4.1%) serum samples from cattle showed titres for brucellosis. In organized farms with a history of abortion, placenta retention and repeat breeding, the prevalence rate was 17%. Chahota et al. (2003) reported an outbreak of brucellosis in an organized dairy farm, leading to abortions, retained placenta and stillbirths in cows at Himachal pradesh. In a dairy herd of about 290 animals 24 pregnant cows in the farm at the time of outbreak were affected. The Brucella abortus biotype-I was isolated from infected animals. India has an enormous cattle wealth. The disease has been repeatedly reported in the animals in India. However, very few human studies have been undertaken. It is estimated that the true incidence is 25 times higher than the reported cases due to underdiagnosis

Author Mathur (1964)

Reports Reported seroprevalence of 8.5% among dairy workers and 4.2% in aborted women. He also isolated Brucella spp. from the blood of 7 human cases from north India Reported seroprevalence of 6.46% in aborted women

Panjarathinam (1984)

Rana et al. (1985)

Reported brucellosis (20.7%)among workers of veterinary hospitals and slaughter house of Union Territory of Delhi
Reported seroprevalance of 20.6, 12.75, 25.45% by RBPT, STAT,ELISA among abattoir personnel of Delhi Reported 11.11% seroprevalence in north India

Kumar et al.(1997)

Handa et al.(1998)

Kadri et al.(2000) Kalla et al.(2001)

Reported seroprevalnce in 0.8% patients of pyrexia of unknown origion (PUO) in Kashmir Reported outbreak of polyarthritis with pyrexia related to brucellosis in Western Rajasthan (48 cases)

Author Thakur and Thapliyal (2002) Sen et al.(2002) Mudaliar et al. (2003) Mantur et al.(2007)

Reports Reported a seroprevalence of 17.39% in field veterinarians and abattoir workers Seropositive cases were seen in 28 of 414 (7.0%) patients of PUO in Varanasi Reported 5.33 % seropositivity in animal handlers of Maharashtra Reported seroprevalence of 1.6% by STAT and isolated B. melitensis in 43 pediatric patients during a period of 13 years. During the same period they diagno sed 492 adult patients from Belgaum, Karnataka Reported 31.28% seropositivity and obtained 8 isolates of Brucella melitensis biotype 1 from Varanasi

Jain et al.(2008)

Sathyanarayanan et al. (2011) Reported 61 out of 68 seropositive cases by STAT from a tertiary care center in karnataka Appannanavar et al. (2012) Reported 9.98% seropositivity by STAT from tertiary care centre in north India

Samples
Blood (with and without anticoagulant) Milk Vaginal Swabs Vaginal Discharge

Placental tissue

Aborted fetus- stomach content, spleen, lung, liver, heart blood, peritoneal fluid Human serum in plain tube

Processing of samples
Serological tests Rose Bengal Plate test (RBPT)38 animal samples were found positive by RBPT

Negative Positive RBPT

Standard tube agglutination Test (STAT)

Indirect ELISA
IgG IGM ELISA

Isolation of Brucella
Isolation was performed according to standard procedures described by Alton et al. (1975) Milk Centrifuged at 8000 rpm Pellet & cream Inoculated on Brucella agar Incubated for 3-4 days at 370C Vaginal swabs enrichment in supplemented Brucella broth for 48 hrs Streaked on Brucella agar with selective supplement Incubated for 3-4 days at 370C

Identification
Gram staining- Small, gram negative cocobacilli in clumps Oxidase Test- positive Catalase test- positive Rapid Urease test positive Nitrate reduction- positive

Dye inhibition test using thionin and basic fuchsin

PCR detection of the bcsp31 and IS711 genes

Thank you