Flock level socio-economic and other associated risk factors for Peste des petits ruminants (PPR) exposure in sheep and goats in Madhya Pradesh state, India.

To effectively control and eradicate PPR, the comprehensive understanding of risk factors associated with PPR exposure is vital. Hence, this study investigated socioeconomic and other associated risk determinants for PPR exposure at flock level in sheep and goats in a non-vaccination programme implemented Madhya Pradesh state India. A total of 410 sheep and goat flocks, comprised mostly of goats but also some mixed flocks, were surveyed during 2016 using a multistage random sampling procedure. Further, 230 blood samples were also collected from the farmers-reported PPR affected flocks and sera were tested using c-ELISA to confirm PPR exposure. The primary data on socioeconomic factors, farm management factors, health status, vaccination details and other epidemiological risk factors were collected from flock owners and descriptive statistics, chi-square analysis and logistic regression models were fitted to identify the significant risk factors for PPR incidence. The farmer's education, flock size, rearing pattern, and awareness of PPR vaccination were found to be significant pre-disposing risk factors for PPR exposure in the flocks. Hence, the control and eradication strategy need to be designed comprehensively considering the key social factors like education and vaccination awareness along with other flock level risk factors to eradicate PPR by 2030 in consonance with the global plan.

Potential diagnostic application of the baculovirus-expressed recombinant truncated nucleocapsid protein of peste des petits ruminants virus in ELISA.

The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of ∼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.

Comparative diagnostic efficacy of Avidin-Biotin recombinant nucleoprotein competitive ELISA for serosurveillance and monitoring of peste des petits ruminants in sheep and goats.

In this study extensive evaluation of Avidin-Biotin recombinant nucleoprotein competitive ELISA (ABrC-ELISA) was carried out by mass screening of a large number of sera to make use of this assay for serosurveillance and seromonitoring of peste des petits ruminants (PPR) in sheep and goats to evaluate its diagnostic efficacy value and strengthen findings associated with the assay. The recombinant PPR virus (PPRV) nucleoprotein was over-expressed in E. coli, Ni-NTA affinity-purified, and characterized and used as coating diagnostic antigen in ABrC-ELISA, and evaluated using the field sera from animals. On evaluation of the diagnostic performance or efficacy of this assay using the pre-vaccinated and post-vaccinated sera of sheep and goats (n = 1437), the ABrC-ELISA showed a relative diagnostic sensitivity of 87.2% (95% CI: 84.1-90%) and diagnostic specificity of 92.0% (95% CI: 90-93.7%), against well-established existing indigenous H protein-specific PPR competitive ELISA kit with an accuracy of 90.1% (95% CI: 88.5-91.7%) and good or substantial agreement of Cohen's Kappa value of 0.79 ± 0.017 SE (95% CI: 0.76 to 0.82). These findings suggest that the ABrC-ELISA is a potential additional diagnostic tool of a rapid, sensitive, and specific assay for the detection of the PPRV nucleoprotein antibodies in sera of sheep and goats. This PPR Ab Chek kit can be used extensively under field conditions for serosurveillance, and seromonitoring of PPR in sheep and goats at the eradication /post-eradication phase in disease-controlled countries or PPR non-enzootic countries.

Assessment of post-vaccination immune response to peste des petits ruminants virus in small ruminants in the central and western regions of India.

The cross-sectional serosurvey for post-vaccination assessment of peste des petits ruminants (PPR) virus (PPRV) antibodies in sheep and goats was carried out in different states in the central and western regions of India after the implementation of vaccination under the PPR control programme. The serum samples (n = 4687) were collected from sheep (n = 1539) and goats (n = 3148) from August 2017 to March 2018 at various epidemiological units (n = 301) of the studied regions using a stratified random sampling method and PPR competitive ELISA kit was employed to detect PPRV antibodies. The results revealed 34, 21, 52, 74, 68, and 65% of prevalence of PPRV antibodies in small ruminants in Madhya Pradesh, Goa, Chhattisgarh, Maharashtra, Gujarat, and Rajasthan states, respectively, with a difference in seropositivity in sheep and goats across the states in sheep (p < 0.01) and goats (p < 0.01). Further, this serosurvey revealed that 60% of the epi-units (n = 185) had > 50% prevalence of post vaccination PPRV antibodies across states due to variations in vaccination rates and patterns. The vaccination coverage and the reported outbreaks varied between the states in the studied regions. Due to continuous vaccination under the control program, the reported PPR outbreaks have progressively declined in most of the studied states, and the PPR risk areas are confined to a few districts and sporadically, outbreaks are reported indicating the effectiveness of vaccination. These findings provide valuable information on potential PPRV episystems, and will assist with activities regarding intensive surveillance, vaccination, biosecurity, and modification of policy decisions towards designing and implementing control and eradication measures. Further, the present situation necessitates continuous mass vaccination and active surveillance programs to make these regions free from PPR in consonance with the PPR Global Control and Eradication Strategy under the PPR Global Eradication Program. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00796-6.

Bovine babesiosis in India: Estimation of prevalence by systematic review and meta analysis.

Bovine babesiosis is a serious threat to the livestock sector especially in tropical countries like India. Understanding the epidemiology of the disease in the country is essentially important in strategizing the available methods to effectively control the disease. Keeping this as the background, the present study was undertaken to estimate the pooled prevalence of bovine babesiosis in India. The relevant literature pertaining to bovine babesiosis was identified and a total of 49 studies published between 1983 and 2018 were included in the final systematic review and meta-analysis. Meta-analysis was conducted using meta-package of R software and prevalence estimates were calculated. Bovine babesiosis was reported from 21 states of India with pooled prevalence estimate of 6% (95% CI = 4%-9%) using random effect model. Zone wise analysis revealed highest pooled prevalence in the west zone and north zone (8%) followed by east zone (7%), central zone (6%), south zone (4%) and northeast zone (4%). The results of meta-analysis indicated high variability between studies. In addition, the pooled seroprevalence was high (29%) compared to prevalence of active infection (5%) of bovine babesiosis in India. Further, the pooled prevalence estimate of B. bigemina infection in India was more (7%) compared to B. bovis infection (1%). The estimation of prevalence of active infection and seroprevalence separately will helps to understand the actual disease prevalence in the country. The study indicated the wide prevalence of bovine babesiosis in India which urges for immediate mitigation strategies.

Prevalence of Toxoplasma gondii, Leptospira spp., and Coxiella burnetii-associated antibodies in dairy cattle with reproductive disorders.

Background and Aim: In cattle dairy farms, abortions and other reproductive problems due to major infectious diseases are overlooked, and identifying their causative agents is very challenging without a confirmatory diagnosis. Further, a prevalence study in animals will provide important hints of pathogen reservoirs and provide necessary direction to disease burden with appropriate control and biosecurity measures at the farm level. This study aimed to estimate the prevalence of Toxoplasma gondii antibodies in dairy cattle associated with reproductive problems along with coexisting antibodies against abortifacient zoonotic (Coxiella burnetii and Leptospira spp.) pathogens. Materials and Methods: Cattle sera (n = 246) from dairy farms (n = 35) situated in different locations in India were screened for anti-T. gondii and C. burnetii antibodies with enzyme-linked immunoassay and Leptospira spp. antibodies with microscopic agglutination test. Results: The overall prevalence of 11.4% (95% confidence intervals [CIs]: 7.99%-15.96%) antibodies in cattle associated with reproductive problems (p < 0.021) with farm-level seropositivity of 43% was observed. Further, on analysis of screened sera, 49.8% (95% CI: 42.6%-55%) and 77.6% (95% CI: 72%-82.4%) of samples were found to be positive for C. burnetii and Leptospira spp. antibodies, respectively. Moreover, the seropositivity of 91.9% (226/246) for at least one of the screened zoonotic pathogens was observed, indicating antibodies against either of these organisms in association with reproductive disorders (p < 0.005). The percentage of cattle found to have T. gondii antibodies was only 1.8%, whereas 11.5% and 41.6% of cattle were found to have C. burnetii and Leptospira spp. antibodies, respectively. Nevertheless, the predominantly mixed infections observed were of Leptospira and C. burnetii (34.5%), followed by all three infections (4.9%); toxoplasmosis and leptospirosis (3.5%); and toxoplasmosis and Q fever (2.2%). Conclusion: The serological detection of antibodies against these pathogens in cattle may have significant implications for the livestock industry and public health, suggesting the need for continuous surveillance and monitoring of these infections to prevent their spread.

An integrated surveillance network for antimicrobial resistance, India.

OBJECTIVE: To assess the preparedness of veterinary laboratories in India to participate in an integrated antimicrobial resistance surveillance network and to address gaps in provision identified. METHODS: The Indian Council of Medical Research and the Indian Council of Agricultural Research collaborated: (i) to select eight nationally representative veterinary microbiology laboratories whose capacity for participating in an integrated antimicrobial resistance surveillance network would be assessed using a standardized tool; (ii) to identify gaps in provision from the assessment findings; and (iii) to develop a plan, and take the necessary steps to address these gaps in consultation with participating organizations. FINDINGS: The main gaps in provision identified were: (i) a lack of dedicated funding for antimicrobial resistance surveillance; (ii) the absence of standard guidelines for antimicrobial susceptibility testing; (iii) a shortage of reference strains for testing and quality assurance; and (iv) the absence of mechanisms for sharing data. We addressed these gaps by creating a veterinary standard operating procedure for antimicrobial susceptibility testing, by carrying out a validation exercise to identify problems with implementing the procedure and by conducting capacity-building workshops for veterinary laboratories. CONCLUSION: Antimicrobial resistance surveillance networks depend on the availability of accurate, quality-controlled testing. The challenges identified in creating an integrated surveillance network for India can be overcome by developing a comprehensive plan for improving laboratory capacity in human, veterinary and environmental sectors that is supported by the necessary funds. The study's findings may provide guidance for other low- and middle-income countries planning to develop a similar network.

Avidin-Biotin recombinant nucleoprotein competitive ELISA for the detection of peste des petits ruminants virus antibodies in sheep and goats.

The present study describes the development of a truncated recombinant peste des petits ruminants virus (PPRV) nucleoprotein (rPPRV-NPN) and its polyclonal antibodies-based immuno-diagnostic assay, Avidin-Biotin (AB) recombinant nucleoprotein competitive ELISA (ABrC-ELISA) for the detection of PPRV antibodies in the sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of nucleoprotein (NPN) coding sequence was amplified and cloned into the pETite vector. The rPPRV-NPN with a molecular weight of ∼ 30 kDa was expressed in E. coli, purified, and characterized by SDS-PAGE and immunoblot using standard PPRV specific sera. The Ni-NTA affinity-purified rPPRV-NPN as coating antigen and its hyperimmune serum as competitive antibodies raised in guinea pigs were evaluated as diagnostic reagents in ABrC-ELISA using the known standard panel of sera. The threshold (cut-off) Percentage Inhibition (PI) value was determined as 45 (mean ± 3 SD) based on the reactivity of the known sheep and goats sera to PPRV antibodies [negative (n = 140) and positive (n = 98)] and the assay had a sensitivity of 97 % (95 % Confidence Interval (CI): 91.3-99.4 %) and specificity of 100 % (95 % CI: 97.4-100 %) with an excellent Area under curve (AUC) of 0.997 (95 % CI: 0.99-1.0). On evaluation of diagnostic performance of the assay using the sheep and goats sera (n = 391) from vaccinated, infected, and non-vaccinated animals, the ABrC-ELISA showed the relative diagnostic sensitivity of 95.88 % (95 % CI: 92.56-98.01 %) & 98.77 % (95 % CI: 96.43-99.74 %) and diagnostic specificity of 97.97 % (95 % CI: 94.19-99.58 %) & 90.54 % (95 % CI: 84.64-94.73 %) against indigenous PPR competitive ELISA kit & IDvet Screen® PPR Competition kit, respectively. The study showed that ABrC-ELISA is rapid, sensitive, and specific and can be a better alternative assay for the detection of the PPRV antibodies in the sera of small ruminants for serosurveillance / seromonitoring of PPR not only at the eradication and post-eradication phases in the disease-controlled endemic countries but also in the PPR non-endemic countries.

Peste Des Petits Ruminants in Atypical Hosts and Wildlife: Systematic Review and Meta-Analysis of the Prevalence between 2001 and 2021.

Peste des petits ruminants (PPR) or goat plague is considered a leading, highly contagious, and most lethal infectious viral disease of small ruminants affecting the worldwide livestock economy and international animal trade. Although sheep and goats are the primarily affected, the PPR Virus (PPRV) host range has expanded to other livestock (large ruminants) and wildlife animals over the last few decades, resulting in serious concern to the ongoing PPR global eradication program, which is primarily optimized, designed, and targeted towards accessible sheep and goat population. A systematic review and meta-analysis study was conducted to estimate the prevalence and spill-over infection of PPRV in large ruminants (bovine and camel) and wildlife. Published articles from 2001 to October 2021 on the "PPR" were searched in four electronic databases of PubMed, Scopus, Science direct, and Google Scholars. The articles were then selected using inclusion criteria (detection/prevalence of PPRV in bovine, camel, and wildlife population), exclusion criteria (only sheep or goats, lack of prevalence data, experimental trial, test evaluation, and reviews written in other languages or published before 2001), and the prevalence was estimated by random effect meta-analysis model. In the current study, all published articles belonged to Africa and Asia. The overall pooled prevalence of PPR estimates was 24% (95% CI: 15-33), with 30% in Asia (95% CI: 14-49) and 20% in Africa (95% CI: 11-30). The overall estimated pooled prevalence at an Africa-Asia level in bovine and camel was 13% (95% CI: 8-19), and in wildlife, it was 52% (95% CI: 30-74) with significant heterogeneity (I2 = 97%) in most pooled estimates with a high prevalence in atypical hosts and wildlife across Asia and Africa. Over the last two decades, the host range has increased drastically in the wildlife population, even for prevalent PPR in the unnatural hosts only for a short time, contributing to virus persistence in multi-host systems with an impact on PPR control and eradication program. This observation on the epidemiology of the PPRV in unnatural hosts demands appropriate intervention strategies, particularly at the livestock-wildlife interface.

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.

Perceptions and preparedness of veterinarians to combat brucellosis through Brucellosis Control Programme in India.

BACKGROUND AND AIM: Brucellosis caused by bacteria belongs to the genus Brucella is an important zoonosis and constitutes a serious public health hazard worldwide including India. The present study aimed to estimate the knowledge of veterinarians on brucellosis, its public health threat, diagnosis, and vaccination. MATERIALS AND METHODS: This cross-sectional study was conducted during 2013-2015 and 453 veterinarians representing 11 states/Union Territories (UT) of India (Assam, Tripura, Meghalaya, Goa, Karnataka, Madhya Pradesh, Uttar Pradesh, Delhi, Jammu and Kashmir, Tamil Nadu, and Punjab) were interviewed using self-administered questionnaire. RESULTS: Out of 453 veterinarians, 71.74% stated handling of the animals on day-to-day basis and 28.25% were engaged in administration activities. The veterinarians ranked foot-and-mouth disease and brucellosis at the first and fourth ranks among the list of ten economic impacted diseases in the country. A significant association was observed between laboratory confirmation with those who handled brucellosis-suspected cases (p=0.000). Similarly, significant association was noted for the availability of vials/slides (p=0.114), vacutainers (p=0.008), icebox (p=0.103), and refrigerator (p=0.106) for those who preferred laboratory diagnosis. Only 20% of the veterinarians recommended vaccination against bovine brucellosis, and 17% obtained laboratory confirmation for the brucellosis-suspected cases. CONCLUSION: The study highlighted the need for awareness programs, laboratory facilities, veterinary doctors, and protective measures for the veterinarians for combating brucellosis through the control program in the country.

Pathological Changes in Experimental Intramammary Infection with Different Staphylococcus Species in Mice.

INTRODUCTION: Mastitis is caused by different Staphylococcus species, produce great economic loss to farmers. Present study was conducted to know pathological changes in mice inoculated with Staphylococcus epidermidis, S. chromogenes, S. haemolyticus and S. aureus isolated from bovine milk. MATERIALS AND METHODS: Mice were inoculated with 50 µl (2x104 cfu organisms) per mammary gland and euthanized at 6, 12, 24, 48, 72 and 96 h. Mammary gland weight, gross and histopathological changes of mammary gland, liver, kidney, spleen, heart, lung and inguinal lymph node were studied. RESULTS: Mammary gland weight and percentage of body weight increased at 6 and 96 h in S. aureus and S. haemolyticus infected mice. Gross changes were observed in mammary gland but not in other organs. Mammary gland revealed gross changes from 24 to 72 h in three Coagulase negative staphylococcal (CNS) species and persisted up to 96 h in S. aureus infected mice. Histopathological changes in mammary glands was severe in S. aureus and moderate in CNS species. S. aureus infected mice revealed severe damage to alveoli and loss of alveolar architecture at 96 h but three CNS species infection was overcome by host factors which was evident by proliferation of alveolar epithelial cells. No histological changes were observed in kidney, spleen, lung, heart and inguinal lymph nodes. CONCLUSIONS: S. aureus caused severe mastitis in mice when compared to CNS species. Further, it is first report of mice to study CNS mastitis, and in future it can be used as model for CNS mastitis.

Threshold somatic cell count for delineation of subclinical mastitis cases.

AIM: Somatic cell count (SCC) is the most widely used single reliable indicator of udder health. The present study was carried out with an objective to find the exact threshold of SCC. MATERIALS AND METHODS: Milk samples collected from a total of 214 Holstein Friesian crossbred dairy animals were subjected to bacterial DNA extraction and SCC estimation by digital PortaCheck. California Mastitis Test and polymerase chain reaction based on amplification of organism using reported primers were performed to diagnose subclinical mastitis. Receiver's operating characteristic (ROC) curve analysis and discriminate function analyses were performed using SPSS 18 software. RESULTS: ROC curve analysis represented that the area under the curve was 0.930 with the standard error of 0.02. Results indicated that 93% of the case could be correctly predicted as mastitis infected using SCC as a marker (p<0.001). At cut score level of 282 000 cells/ml, 285,000 cells/ml and 288,000 cells/ml, sensitivity remained 92.6% and specificity augmented as 86.3%, 87.2%, and 88%, respectively. At SCC value of 310,000 cells/ml of milk, sensitivity and specificity were optimal, namely, 92.6% and 91.5%, respectively. The function fitted demonstrated 89.2% accuracy with p<0.001. The functions at group centroids were -0.982 and 1.209, respectively, for normal and mastitis-infected animals and log_SCC value was the most important factor contributing 38.30% of the total distance measured. CONCLUSION: Our study supports that the threshold value to delineate subclinical mastitis case from the normal is 310,000 somatic cells/ml of milk and a model so fitted using the variable SCC can be successfully used in field for the diagnosis of subclinical cases of mastitis which otherwise would be difficult to differentiate based on clinical signs.

Cytokine gene expression and pathology in mice experimentally infected with different isolates of Trypanosoma evansi.

Aim of the present study was to assess the cytokine gene expression in liver, kidney and spleen and histopathological changes in mice infected with buffalo and dog isolates of Trypanosoma evansi. Forty-four Swiss albino mice was divided into eleven groups of four mice each and injected subcutaneously with 1 × 105 trypanosomes of buffalo and dog isolate to twenty mice each, four mice served as control. Mice were examined for clinical signs, blood smear for trypanosome counts. Blood for PCR, liver, kidney, spleen, heart, lung, testis and abdominal muscle for histopathology and liver, kidney, spleen for cytokine gene expression studies, were collected. Mice showed dullness, lethargy, hunched back, sluggish movements on D4 and D5 in buffalo and dog isolate, respectively. Parasite count in blood varied between the two isolates of T. evansi. By PCR, trypanosome DNA was detected on D1 and D2 for buffalo and dog isolate, respectively. Splenomegaly was observed in mice infected with buffalo isolate but not with dog isolate. Histopathological changes were observed in liver, kidney, spleen and heart of mice but no changes in testis and abdominal muscles. Blood vessels of liver, heart, lung showed presence of trypanosomes in mice infected with buffalo isolate but not for dog isolate. Cytokine gene expression of IL-2, IL-4, IL-6, IL-12, TNF-α and IFN-γ increased in liver, kidney and spleen in both these isolates. However, the buffalo isolate exhibited pronounced increase in cytokine gene expression when compare to dog isolate of T. evansi. Anti-inflammatory cytokine gene IL-10 showed 50-60 and 10-20 folds increment in buffalo and dog isolates, respectively. This is the first report of IL-4, IL-6, IL-10 and IL-12 cytokine changes in mice infected with T. evansi. A variation in pathogenicity between buffalo and dog isolates was recorded indicating buffalo isolate of T. evansi remained more pathogenic in mice.

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.

Staphylococcus aureus spa type t267, clonal ancestor of bovine subclinical mastitis in India.

AIM: To evaluate the virulence determinants and genetic diversity of Staphylococcus aureus from bovine subclinical mastitis milk. METHODS AND RESULTS: PCR detection of virulence genes was performed for 173 Staph. aureus from bovine subclinical mastitis milk. Further, genetic diversity was analysed by agr and spa typing followed by pulsed field gel electrophoresis (PFGE) of selected isolates. Screening of virulence genes (n = 19) showed the adherence genes viz. fnbA, clfA, fnbB and cna in 98·8, 97·1, 68·8 and 28·3 percentage of isolates, respectively, and 80 strains (46·24%) positive for enterotoxin genes were distributed as 23 toxinotypes, of which, 5 genotypes contained a single gene and the rest comprised of multiple toxin genes. Out of agr type-1 (87·3%), 74·2 per cent belonged to the three predominant spa types. Of 27 spa types, 11 were identified for the first time. The predominant spa types were t267 (N =44), t359 (N = 42) and t6877 (N =29), which together accounts to 66·5 per cent of isolates. PFGE analysis of isolates (N = 45) covering all the spa types revealed mostly similar or closely related pulsotypes. Local emergence of spa type t6877 in herd-dependant manner was observed. spa sequence-based phylogenetic analysis suggested t267 as the ancestral clone of t359, t6877 and other spa types except two. CONCLUSION: Heterogenous virulence profile of the isolates had no significant association with the genotype. High prevalence of agr group I reaffirms their association with persistent subclinical mastitis. The spa type t267 appears to be the ancestral clone endemic in the region causing subclinical mastitis. In addition, few new spa types have emerged in the geographic region. SIGNIFICANCE AND IMPACT OF STUDY: Gives an insight into the genetic and evolutionary behaviour of Staph. aureus associated with bovine subclinical mastitis in India. The study would pave the way for devising effective control strategy for bovine mastitis in Indian context.

Incidence of subclinical mastitis and prevalence of major mastitis pathogens in organized farms and unorganized sectors.

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

Multiplex PCR assay for species identification of bovine mastitis pathogens.

AIM: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. METHODS AND RESULTS: A two-tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10(3) CFU ml(-1). A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. CONCLUSION: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.

PCR-based diagnosis of surra-targeting VSG gene: experimental studies in small laboratory rodents and buffalo.

Trypanosoma evansi, the causative organism of 'surra' expresses its variable surface glycoprotein (VSG) at early, middle and late stages of infection in animals. The variable antigenic nature of VSG caused by switching its expression type favours evasion from the host immune response and leads to chronic and persistent infection. Developing a polymerase chain reaction (PCR)-based diagnostic tool targeting the VSG gene is expected to be highly specific and sensitive for diagnosis of surra. Hence, in the present study, we have designed EXP3F/4R primer pair and amplified the 1.4 kb of VSG gene of T. evansi and studied the phylogenetic relationship by in silico analysis. The PCR method was standardised using another set of primer, DITRYF/R, and 400 bp was amplified from blood and tissue samples of experimentally infected animals. Applying the PCR method, we were able to detect as low as 0.15 trypanosomeml(-1). Considering the number of parasite-to-DNA concentration, the PCR method has a sensitivity of 0.015 pg ml(-1). The PCR could detect the presence of the parasite as early as 24h post-infection (p.i.) and 72 h p.i., respectively, in experimentally infected rats and buffalo. No amplification was observed with DNA of Babesia bigemina and Theileria annulata, indicating the primers are specific for T. evansi. The PCR method could detect the dog, lion and leopard isolates of T. evansi. Similarly, amplifying the DNA from the experimentally infected tissues was also found to be sensitive. Thus, the findings of this study favour the application of PCR over the parasitological methods for the detection of the early and/or chronic stage of surra in domestic and wild animals.

Investigation of haemorrhagic enteritis in pygmy hogs (Sus salvanius) from India.

The pygmy hog is a representative of the smallest and rarest wild species of known living Suidae. This paper reports the investigation of haemorrhagic enteritis encountered amongst the pygmy hogs at the Research and Breeding Centre of the Pygmy Hog Conservation Programme, Guwahati, Assam, India. Three out of 68 pygmy hogs died of enteric infection. Post-mortem examination and bacteriological investigation of two out of the three animals that died revealed clostridial infection. The isolates harboured two plasmids of molecular weight 42.8 kilobases (kb) and 51.9 kb. Clostridium perfringens Type A positive for the beta2 toxin (cpb2) gene was detected by polymerase chain reaction. Sequence analysis of the partial alpha toxin (cpa) gene showed 98% to 100% homology with isolates from different geographical locations.