Management of bovine brucellosis in organized dairy herds through the identification of risk factors: A cross-sectional study from Karnataka, India.

Background and Aim: Brucellosis is an infectious disease caused by Brucella species. This study aimed to identify the risk factors associated with bovine brucellosis seropositivity in organized dairy farms to control the disease in unvaccinated adult bovine herds in Karnataka, India. Materials and Methods: In total, 3610 samples (3221 cattle and 389 buffaloes) were subjected to parallel testing using the Rose Bengal plate test and protein G-based enzyme-linked immunosorbent assay, followed by analyses of animal- and farm-level epidemiological datasets to identify the risk factors. Results: The apparent brucellosis prevalence at the animal level was higher in buffaloes (8.2%, 95% confidence interval [CI] = 5.9-11.4) than in cattle (6.1%, 95% CI = 5.3-7.0). In a multivariable logistic model, animals calved 3-5 times (odds ratio [OR] = 2.22, 95% CI = 1.50-3.1, reference [ref]: animals calved <2 times); animals with a history of abortion (OR = 54.73, 95% CI = 33.66-89.02), repeat breeding (OR = 19.46, 95% CI = 11.72-32.25), and placental retention (OR = 13.94, 95% CI = 4.92-39.42, ref: no clinical signs); and dogs on farms (OR = 2.55, 95% CI = 1.48-4.40, ref: absence of dogs); disposal of aborted fetus in open fields (OR = 4.97, 95% CI = 1.93-12.84) and water bodies (OR = 2.22, 95% CI = 1.50-3.1, ref: buried); purchase of animals from other farms (OR = 6.46, 95% CI = 1.01-41.67, ref: government farms); hand milking (OR = 1.98, 95% CI = 1.02-10.0, ref: machine milking); and use of monthly veterinary services (OR = 3.45, 95% CI = 1.28-9.29, ref: weekly services) were considered significant risk factors for brucellosis in organized bovine herds (p < 0.01). Conclusion: The study identified that the animals calved 3-5 times or with a history of abortion/repeat breeding/placental retention, and disposal of aborted fetus in open fields/water bodies as the potential risk factors for bovine brucellosis. These risk factors should be controlled through the implementation of best practices to reduce the brucellosis burden in bovine farms.

Characterization of humoral and cellular immune responses elicited by reduced doses of Brucella abortus S19 (calfhood) vaccine in cattle calves of India.

Brucella abortus S19 vaccine is a stable attenuated smooth strain, globally used as calfhood vaccine for the prevention of bovine brucellosis. Various agencies demonstrated different doses for vaccinating cattle and buffalo calves leading to ambiguity in selecting a suitable immune vaccine dose. The current study aimed at evaluating four graded doses of S19 vaccine to arrive at the dose which could produce comparable effectiveness as that of full dose prescribed by Indian Pharmacopeia among the Indian calves. Four vaccine doses of which the first dose consisted of full dose (40 × 109 CFU/dose) and the other three were 1/10th, 1/20th, 1/100th reduced doses along with control were tested. Each vaccine dose was administered to 13 cattle calves of 4-5 months of age maintained in separate groups. The blood samples were collected on 0 to 240 days post-vaccination (DPV) at the intervals of 0, 14, 28, 45, 60, 90, 150, 180 and 240 for assessment of vaccine-induced innate, humoral and cell-mediated immune responses. The sero-conversion of all vaccinated animals on DPV 45 and persistence of antibody till DPV 240 were noticed. No significant differences were observed in antibody response between animal groups that received full and 1/10th reduced doses. Innate and cell-mediated response by IL-6, TNF-α¸ IFN-γ, CD4+ and CD8+ cell counts showed dose-dependent responses with no significant difference between full dose and 1/10th reduced doses. The results suggest a possible one log reduction of full dose without compromising immune responses to aid larger vaccination coverage for creating herd immunity.

Countrywide cross-sectional study of swine brucellosis sero-prevalence in Indian subcontinent during 2018-2019.

Brucellosis in swine is a contagious disease with greater zoonotic potential caused by Brucella suis. The study describes PAN India swine brucellosis sero-prevalence in 5431 stratified random serum samples collected during 2018-2019 from 26 out of 29 states and two out of seven union territories. The serum samples were tested for anti-Brucella antibodies by indirect ELISA and overall, 4.33% apparent prevalence (AP) was recorded. The AP is ≥ 10% in five states among 26 states, P ≥ 50% in four districts out of 117 districts screened and cent percent prevalence in two epi units out of 264 sampled. Significantly high seropositivity (p < 0.05) in male (6.08%) than female pigs (3.46%) and in ≥ 24-month-old pigs indicated older and male pigs as potential carriers of the disease. The study recorded endemicity of the swine brucellosis in few regions of India requiring periodical surveillance for control of the disease. Brucella testing of boars before breeding and awareness among farmers and veterinarians will aid in reduction of disease burden in the absence of vaccination policy.

Seroprevalence and risk factors for brucellosis in small ruminant flocks in Karnataka in the Southern Province of India.

BACKGROUND AND AIM: Brucellosis is a zoonotic disease of high economic and public health importance in large and small ruminant populations worldwide. A cross-sectional study was conducted to determine the seroprevalence and risk factors of brucellosis in small ruminants in organized farms in the southern region of India. MATERIALS AND METHODS: Farms exclusively rearing sheep and goats were selected based on the number of animals (small, medium, or large) and the location of the farm (urban, periurban, or rural). A total of 1499 serum samples; 1001 from sheeps and 498 from goats were sourced from six sheep and four goat farms and tested using Rose Bengal Plate and indirect Enzyme-Linked Immunosorbent Assay tests. RESULTS: The apparent prevalence of brucellosis was higher in sheep (8.29%, 95% CI 6.7-10.1) than goats (5.82%, 95% CI 4.0-8.2). The true adjusted population level seroprevalence was also higher in sheep, at 7.7% (95% CI 6.0-9.6) than in goats, at 5.1% (95% CI 3.2-7.6). According to bivariate categorical analysis, six highly significant (p<0.001) animal- and farm-level risk factors for sheep were age, breed, number of lambings, history of abortion, rural farms, and presence of dogs on the farm. In goats, five significant risk factors were found: History of abortion, separate sheds, dogs on the farm, weekly veterinary consultation, and lack of brucellosis awareness. In a logistic regression model, abortion (OR adjusted 10.8, 95% CI 1.2-96.12), rural farms (OR adjusted 8.5, 95% CI 3.6-20.0), and absence of separate sheds on the farms (OR 1.9, 95% CI 1.1-3.5) were found to be significant risk factors for ovine brucellosis. CONCLUSION: The use of complementary measures to tackle the multiple animal- and farm-level risk factors may help to reduce the disease burden in the absence of a vaccination policy for small ruminants in India.

Spatial sero-prevalence of brucellosis in small ruminants of India: Nationwide cross-sectional study for the year 2017-2018.

Brucellosis in small ruminants caused mainly due to Brucella melitensis is an important zoonotic disease characterized by abortion, retained placenta, infertility, orchitis, epididymitis and rarely arthritis. Small ruminants are the main source of economy for the rural and marginally poor farmers and brucellosis is resulting in huge economic losses due to abortions and infertility and causing public health concern among the small ruminant keepers. Bovine brucellosis control programme has been implemented in India and small ruminants are left out of the programme mainly due to paucity of brucellosis status. The present cross-sectional study based on stratified random sampling was undertaken during 2017-18 to provide the nationwide brucellosis sero-prevalence in small ruminants. A total of 24,056 small ruminant serum samples (sheep samples = 8,103 [male-2,440 and female-5,663] and goat samples = 15,953 [male-4,331 and female-11,622]) sourced from 27 out of 29 states and two out of seven union territories (UTs), 350 districts of total 640 districts (54.68% of the Indian districts) and from 1,462 villages out of 6,40,867 villages (43.83% of the Indian villages). The serum samples were tested by indirect ELISA and overall brucellosis apparent and true prevalence of 7.45 (95% CI: 7.13-7.79) and 3.79 (95% CI: 3.44-4.17) was recorded. Significantly higher brucellosis sero-prevalence (p < .0001) was observed in sheep (11.55%) than goats (5.37%). Similarly, brucellosis seropositivity was highly significant in females compared to males in both sheep and goats. Countrywide, greater than 5% brucellosis sero-prevalence in sheep and goats was recorded in 14 and 10 states, respectively, indicating endemicity of the disease. The study provided the latest update on nationwide spatial sero-prevalence of small ruminant brucellosis which will aid government to strengthen regular surveillance and vaccination to reduce the disease burden and public health problems in the country.

Evaluation of the immune responses against reduced doses of Brucella abortus S19 (calfhood) vaccine in water buffaloes (Bubalus bubalis), India.

BACKGROUND: Brucella abortus S19 is the most widely used vaccine for the prevention of bovine brucellosis which remains the reference vaccine to which many other vaccine/s are compared. Considering the larger vaccination coverage by reduced dose of vaccine, the study aimed to compare reduced graded doses (1/10th, 1/20th and 1/100th) with standard dose of S19 vaccine (40 × 109CFU /dose) to determine the effective immunizing dose in water buffaloes. METHODS: A total of 25 female buffalo calves (Bubalus bubalis) in the age group of 4-5 months were equally grouped into five animals each in four test and one control groups and given with specified vaccine dose. The blood samples were collected on post vaccination days 14, 28, 45, 60, 90 and 120 for assessing innate (TNF-α and IL-12), humoral (IgG antibodies against Brucella LPS) and cell mediated immune responses (IFN-γ, CD4 + and CD8 + counts). RESULTS: The full dose, 1/10th and 1/20th reduced doses of S19 vaccine was capable of eliciting pathogen-specific antibody response, vaccine induced secretion of IL-12, TNF-α and IFN-γ with CD4 + and CD8 + effector T cell responses. Persistence of antibody and magnitude of immune responses were found dose dependent. CONCLUSION: Comparable immune responses were noticed with 1/10th reduced dose similar to standard dose. With this observation, decline of antibody titre will reduce the number of false positives and reduced dose of vaccine will facilitate larger vaccination coverage in the country.

Comparative evaluation of fluorescence polarization assay and competitive ELISA for the diagnosis of bovine brucellosis vis-a-vis sero-monitoring.

Brucellosis is an important zoonosis that constitutes a serious public health hazard which is caused by a bacterium belonging to the genus Brucella. In the present study, two highly specific serological tests for brucellosis diagnosis, fluorescence polarization assay (FPA) and competitive ELISA (cELISA) were standardized in the laboratory, evaluated and compared with rose bengal plate test (RBPT), indirect ELISA (iELISA) and commercial cELISA kit. For test evaluation, 1386 serum samples [apparently healthy animals (n = 260), samples from Brucella infected farms (n = 701) and B. abortus S19 vaccinated animals (n = 425)] were analyzed to assess suitable diagnostic test in B. abortus S19 post vaccinated bovine population. In apparently healthy brucellosis free farms, RBPT, iELISA, in-house FPA and cELISA were found to be highly specific than commercial cELISA. Commercial cELISA kit was comparatively more sensitive than other serological tests in samples collected from infected farms. The FPA showed sensitivity nearly equal to RBPT and in-house cELISA showed greater sensitivity than RBPT in infected farms. In animals with persistent vaccinal antibodies, only in-house FPA and cELISA recorded higher specificity of 87.64 and 90.27%, respectively. The other tests, RBPT and iELISA displayed similar reactivity with vaccine antibodies to that of infection antibodies whereas commercial cELISA kit showed an intermediate specificity of 47.69%. With these findings, RBPT, iELISA and cELISA are suggested for screening infected herds, and in-house developed FPA and cELISA tests with a proven specificity can be used for confirmatory diagnosis of brucellosis in B. abortus S19 post vaccinated animal populations.

Serological and molecular analysis for brucellosis in selected swine herds from Southern India.

BACKGROUND: Swine brucellosis is a zoonotic disease caused by Brucella suis. The study describes the occurrence of brucellosis in two organized piggeries in Southern India. METHODS: A total of 585 serum samples comprising 575 from pigs and 10 from animal handlers were collected and tested by serological tests and PCR. Tissue samples were collected for isolation of the pathogen. RESULTS: Out of 575 serum samples screened, 236 (41.04%) were positive for brucellosis by both Rose Bengal plate test (RBPT) and indirect ELISA (iELISA) and 47 (8.17) samples showed Brucella DNA amplification by genus specific PCR. The sows those aborted and 19 boars with orchitis were seropositive for brucellosis indicating association of clinical symptoms with brucellosis seropositivity. Two of 10 pig handlers were positive by RBPT and showed significant serum agglutination test (SAT) titres of >1:160 and 1:320. B. suis bvI was isolated and identified by biochemical tests and confirmed by amplification Brucella genus and Bruce ladder PCRs from vaginal and testicular samples. CONCLUSIONS: The introduction of untested breeding boars in the farms might have resulted in the disease transmission and spread. The present study highlighted the diagnosis of B. suis bvI as a cause of abortions in the pigs and occupational exposure to farm personnel.

Fluorescence polarization assay: Diagnostic evaluation for porcine brucellosis.

Brucellosis in pigs, caused by the bacterium Brucella suis, is an important zoonotic infection. In the present study, fluorescence polarization assay (FPA) was standardized and compared with indirect enzyme linked immunosorbent assay (iELISA) and competitive ELISA (cELISA) for diagnosis of porcine brucellosis. Test performances were evaluated using representative panel (n = 100), samples from swine brucellosis outbreak (n = 300), samples from brucellosis suspected animals (n = 291) and sera samples from apparently healthy animals (n = 1121). With panel samples, the FPA cut-off ≥11ΔmP was arrived with sensitivity (Se) and specificity (Sp) of 95.00 and 98.75%, respectively. Testing of samples from swine brucellosis outbreak, the diagnostic Se and Sp of 100 and 95.14% by iELISA, 73.91 and 100% by cELISA and 86.96 and 100% by FPA, respectively were recorded. Similarly, in case of swine brucellosis suspected samples, relative performance of FPA with cELISA had revealed higher kappa value of 0.864 with an accuracy of 93.47. Indirect ELISA was found to be highly sensitive but showed cross reactivity mainly for Yersinia enterocolitica O9 antibodies than cELISA and FPA. The high specificity of FPA test recorded in various types of samples in the study indicated that, FPA could serve as confirmatory test for individual animal diagnosis, outbreak confirmation, surveillance and quarantine of swine brucellosis cases.

Assessment of fluorescence polarization assay: a candid diagnostic tool in Brucella abortus strain 19 vaccinated areas.

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals.

Lateral flow assay for brucellosis testing in multiple livestock species.

Lateral flow assay (LFA) for brucellosis was standardized and evaluated. The test showed high diagnostic sensitivity, specificity and accuracy for diagnosis of brucellosis in bovines, small ruminants and swine. The study emphasized the importance of LFA as a useful, rapid, and easy-to-perform tool for the testing of brucellosis.

Prevalence and risk factors of brucellosis among veterinary health care professionals.

The study describes prevalence, clinical symptoms and risk factors for brucellosis in personnel engaged in veterinary health care in Karnataka, India. A total of 1050 sera samples were collected from animal handlers, veterinarians, veterinary students, para-veterinarians and persons engaged in artificial insemination of animals. The sera samples were tested for brucellosis by Rose Bengal plate test (RBPT), serum agglutination test (SAT), IgG and IgM indirect ELISA and PCR. Age, sex, clinical symptoms and risk factors were recorded in structured questionnaire. Of the 1050 samples tested, 6.76, 6.38, 3.90, 2.67 and 2.0% were positive by IgG ELISA, RBPT, SAT, IgM ELISA and PCR, respectively and overall prevalence recorded was 7.04%. The prominent clinical symptoms observed were intermittent fever (71.62%) followed by joint pain and body aches. A high degree of suspicion, awareness and multimodal diagnostic approach is suggested for early diagnosis, treatment and disease follow up.

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.