Mapping Heterogeneous Population Structure of Mannheimia haemolytica Associated with Pneumonic Infection of Sheep in Southern State Karnataka, India.

Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even though the disease causes huge economic losses, epidemiology of M. haemolytica is less studied, hindering the formulation of effective control strategies. Current study aimed to highlight molecular characterisation of M. haemolytica strains isolated from ovine pneumonic infection. M. haemolytica 27 isolates with two reference strains were characterised using capsular and virulence gene typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. M. haemolytica serotype A2 recognized as predominant serotype (74%) followed by A6 (11%) and A1 (5%) serotypes. Virulence gene profiling by PCRs showed dominance of all five virulent genes [such as adh and gcp (100% each)] followed by gs60 (88.8%), lktC (85.2%), tbpB (51.9%) and least nmaA gene (14.8%). MLST profiling delineated M. haemolytic isolates into 11 sequence types (STs) with most prevalent being ST37 (27.9%) and ST16 (23%) and nine new STs (ST37, 38, 39, 40, 41, 42, 47, 48, and 49). These new STs did not belong to any of the three clonal complexes (CC4, CC8 and CC28). ST16 was exclusively noted in A1 and A6 serotypes. Amongst 25 isolates, 22 pulsotypes (GD 0.88) recorded indicated variability of the M. haemolytica isolates in PFGE analysis. In conclusion, the study suggested dominance of M. haemolytica serotype A2 harbouring different virulent genes, diverse STs and pulsotypes responsible for pneumonic pasteurellosis frequently encountered in sheep.

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.

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.

Prevalence and antibiotic susceptibility of Mannheimia haemolytica and Pasteurella multocida isolated from ovine respiratory infection: A study from Karnataka, Southern India.

BACKGROUND AND AIM: Respiratory infection due to Mannheimia haemolytica and Pasteurella multocida are responsible for huge economic losses in livestock sector globally and it is poorly understood in ovine population. The study aimed to investigate and characterize M. haemolytica and P. multocida from infected and healthy sheep to rule out the involvement of these bacteria in the disease. MATERIALS AND METHODS: A total of 374 healthy and infected sheep samples were processed for isolation, direct detection by multiplex PCR (mPCR), and antibiotic susceptibility testing by phenotypic and genotypic methods. RESULTS: Overall, 55 Pasteurella isolates (27 [7.2%] M. haemolytica and 28 [7.4%] P. multocida) were recovered and identified by bacteriological tests and species-specific PCR assays. Significant correlation between the detection of M. haemolytica (66.6%) with disease condition and P. multocida (19.1%) exclusively from infected sheep was recorded by mPCR. In vitro antibiotic susceptibility testing of 55 isolates revealed higher multidrug resistance in M. haemolytica (25.9%) than P. multocida (7.1%) isolates. Descending resistance towards penicillin (63.6%), oxytetracycline (23.6%), streptomycin (14.5%), and gentamicin (12.7%) and absolute sensitivity towards chloramphenicol were observed in both the pathogens. The antibiotic resistance genes such as strA (32.7%) and sul2 (32.7%) associated with streptomycin and sulfonamide resistance, respectively, were detected in the isolates. CONCLUSION: The study revealed the significant involvement of M. haemolytica together with P. multocida in ovine respiratory infection and is probably responsible for frequent disease outbreaks even after vaccination against hemorrhagic septicemia in sheep population of Karnataka, southern province of India.

Risk factors for Brucella spp. and Coxiella burnetii infection among small ruminants in Eastern India.

Small ruminants are the main reservoirs for brucellosis and coxiellosis, two zoonotic diseases affecting livestock production, and posing a public health threat in India. Understanding disease prevalence and risk factors associated with small ruminant infection can help mitigate disease transmission. We report a cross-sectional survey in the states of Assam and Odisha in Eastern India. We interviewed 244 farmers to assess knowledge, attitude and practices relevant to brucellosis and coxiellosis infection. Serum samples from 411 goats and 21 sheep were analysed using enzyme-linked immunosorbent assay and Rose-Bengal Brucella agglutination plate test. Higher Brucella and Coxiella burnetii seroprevalence were found in Odisha (22% and 11.5%, respectively) than Assam (9.8% and 1.6%, respectively), and certain districts in Odisha were at higher risk. No association was found between seropositive animals and clinical signs, a challenge when attempting to identify seropositive animals in the herd. None of the farmers interviewed were aware of brucellosis, its aetiology, clinical form, or zoonotic risk. This study acts as a first indication of the extent of these diseases among small ruminants in these Indian states, highlighting how farming practices are associated with increased risk of infection. More research is urgently needed to mitigate zoonoses transmission in this region.

A qualitative study on antibiotic use and animal health management in smallholder dairy farms of four regions of India.

BACKGROUND: Antimicrobial resistance (AMR) is a major global public health issue. In India, access to medicines is poorly regulated and therefore antibiotics in dairy cattle are commonly used by farmers without consulting with veterinarians. This study was conducted to understand practices and knowledge related to antibiotic use and AMR among dairy farmers and veterinary professionals in selected urban and peri-urban areas of India. METHODS: A total of 28 focus group discussions with farmers and 53 interviews with veterinary professionals were carried out. RESULTS: Mastitiswas identified as the main animal health challenge. Antibiotic consultation behavior of farmers depended on the availability of veterinarians. Except in Bangalore, farmers were found to often treat animals on their own. They were found unaware of the concept of AMR, but knew the importance of vaccination. Veterinarians included in the study had a good understanding of antibiotics, AMR, and zoonotic diseases. CONCLUSION: The knowledge level and practices observed in the study related to the use/abuse of antibiotics can potentially increase the risk of development of AMR and its transfer in the community. Our findings can help support AMR - mitigation efforts in the country, including the design of better policies on antibiotic use in dairy.

Coxiella seroprevalence and risk factors in large ruminants in Bihar and Assam, India.

Coxiellosis is a zoonotic disease caused by the ubiquitous bacteria Coxiella burnetii, which can be spread either through ticks or through body fluids. In humans the infection is characterized by a febrile disease; ruminants may abort and reduce their milk yield, causing serious production losses for the farmer. In India, the disease has been known to be present since the 1970s, but little is known about the epidemiology in most states. In this cross-sectional survey in the two states of Assam and Bihar, 520 households were interviewed for risk factors, and serum samples from 744 dairy animals were analyzed using ELISA as well as PCR. Out of the farms, 17.4% had at least one seropositive animal, with significantly higher seroprevalence in Bihar (27.1%) than Assam (5.8%); and significantly more sero-positive farms in urban areas (23.1%) than rural (12.2%). On an individual animal level, 14.1% were seropositive, with higher prevalence among buffaloes than cows (28.0% versus 13.6%). Out of the seropositive animals, 10.6% had aborted during the last three years, and 37.5% had experienced problems with repeat breeding: both higher than in non-seropositive animals. In conclusion, this study indicates that coxiellosis is potentially an important cause of reproductive failures and production losses in dairy animals. The high prevalence, especially in urban areas, is a public health risk. Further research is needed to elucidate the epidemiology and identify mitigation options that could work in the different settings of different Indian states.

Seroprevalence of hemorrhagic septicemia in dairy cows in Assam, India.

Hemorrhagic septicemia (HS) is a highly fatal disease caused by Pasteurella multocida that often cause outbreaks in buffalo and cattle in India, and thus is a major cause of production losses. It is one of the livestock diseases with the highest mortality, and despite available vaccines, outbreaks still occur. To assess the seroprevalence in the state of Assam, Northeast India, 346 serum samples from cows from 224 randomly selected households, from both urban and rural areas of three districts, were tested with a commercial ELISA. In total 88 cows were seropositive (25.4%), and indigenous cattle were significantly more seropositive (33.5%) compared to the crossbred cattle (18.5%) (p = 0.002). Herd prevalence was 35.7%, and more rural farms (47.4%) were positive compared to the urban farms (23.6%) (p < 0.001). No other risk factors were identified in this study. Only one farm had vaccinated against HS, but there were no seropositive animals detected in that herd. This study shows that HS is highly prevalent in Assam. Considering the importance of dairy production in India, and the dependence of the rural Assam population on farming and livestock keeping, more extensive vaccination campaigns would be important.

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.

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.

Evaluation of lateral flow assay as a field test for investigation of brucellosis outbreak in an organized buffalo farm: A pilot study.

AIM: The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm. MATERIALS AND METHODS: A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard. RESULTS: Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT. CONCLUSIONS: The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited.