Genome sequencing and comparative genomic analysis of bovine mastitis-associated Staphylococcus aureus strains from India.

BACKGROUND: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. RESULTS: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. CONCLUSION: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

Simultaneous detection of velogenic Newcastle disease virus of genotype XIII 2.2 from spot-billed pelican and backyard chicken: implications to the viral maintenance and spread.

Despite the widespread occurence of Newcastle disease virus (NDV) in different avian species, there has been scanty reports on genetic characterization of NDV strains from wild birds in India. During 2017-18, a total of forty eight cloacal swab samples were collected from apparently healthy migratory birds (painted storks, n = 32 and spot-billed pelicans, n = 16) at the Telineelapuram bird sanctuary of Andhra Pradesh, India. NDV was isolated from a spot-billed pelican (NDV/Pelican/Telineelapuram/2018) which is genetically identical to that isolated from a naturally infected backyard chicken flock (NDV/Chicken/SKLM-1/2018). The isolates are found to be velogenic based on mean death time, intracerebral pathogenicity index and the putative fusion protein cleavage site (112R-R-R-K-R-F117). Phylogenetic analysis based on full-length fusion gene classified the isolates into genotype XIII, sub-genotype 2.2, however these isolates demonstrated multiple amino acid substitutions in the critical domains of F and HN proteins. The pelican strain (MIG-9) was tested for its pathogenic and transmission potential in three-weeks-old broiler chickens and the isolate proved to be highly virulent to chickens. To the best of our knowledge, this is the first evidence for the role of spot-billed pelicans in the maintenance of virulent NDV and its transmission to chickens in India. This study further highlights the role of wild birds in NDV transmission and the need for enhanced biosecurity in commercial poultry operations. Keywords: Newcastle disease virus; Pelecanus philippensis; chicken; transmission; pathogenicity; India.

Assessing rabies-free status of Andaman, Nicobar, and Lakshadweep Islands, India.

The Indian Islands of Andaman, Nicobar, and Lakshadweep have been historically rabies-free. However, reliable laboratory evidence to substantiate rabies-free status was lacking. In this background, the study was conducted as a component of the World Health Organization-Association for Prevention and Control of Rabies in India, Indian Multi-Centric Rabies Survey; to assess the rabies-free status of the two Islands and to examine the feasibility of initiating laboratory surveillance for rabies in dogs in Andaman, Nicobar, and Cats in Lakshadweep Islands. A team of medical and veterinary investigators visited these Islands in 2017. A review of 10 years records (2007-2017) in medical and veterinary institutes and interviews with different stakeholders were conducted. Based on the review of records, there was no evidence of human/animal rabies in the Islands. Eight dog brain samples from Andaman, Nicobar Islands, and ten cat brain samples from Lakshadweep Islands were tested negative for rabies by fluorescent antibody test at two rabies diagnostic laboratories at Bengaluru.

Health-seeking behavior and compliance to post exposure prophylaxis among animal bite victims in India.

BACKGROUND: In rabies endemic countries, where every animal bite is potentially a suspected rabid exposure, the exposed individuals should seek early and proper health care. It is also essential to complete the full course of postexposure vaccination to protect against rabies. OBJECTIVES: The study aimed at determining the health-seeking behavior of animal bite victims; assessing the perceived risk of rabies transmission from different animals and knowledge on its prevention and finding out the compliance to complete course of rabies vaccination and associated factors. METHODS: A multi-centric, health facility-based survey was conducted during May 2017 to January 2018 in six regional-representative states involving 18 health facilities. Study participants were animal bite victims attending the health facilities. The data from all the study participants across the country were compiled and analyzed using descriptive statistics and Chi-square test to find out the association of factors influencing compliance. RESULTS: Among a total of 529 animal bite victims, 83.6% sought postexposure prophylaxis coming directly to health facility; others visited nonallopathic/traditional healers/veterinarians/Auxiliary Nursing Midwifery before coming to health facility. The perceived risk of disease transmission and knowledge on the prevention of rabies was insufficient among the exposed victims. All participants were started with anti-rabies vaccination; the compliance rate for the full course of intramuscular rabies vaccination was 65.9% and for intra-dermal rabies vaccination, it was 85.1%. Among Category III exposures, only 46.2% received rabies immunoglobulin. CONCLUSIONS: Health-seeking behavior and compliance to complete course of anti-rabies vaccination is unsatisfactory, which has to be improved to prevent rabies.

Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens.

The development of persistent antibiotic resistance by human methicillin-sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly-N-acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm-forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm-associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface-associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype.

Genetic characterization of pigeon- origin avian avulavirus-1 reveals unique substitutions in F and HN proteins.

Since decades, Newcastle disease (ND) has become endemic in the poultry population of the Indian subcontinent. ND is a highly contagious disease of poultry and other avian species. However, the genetic nature of ND viruses circulating in the rock pigeons is unraveled. The present investigation is a part of Newcastle disease virus (NDV) surveillance in wild birds. Two velogenic NDV strains could be isolated from apparently healthy rock pigeons, thus establishing the status of carrier/reservoir host. The fusion protein cleavage site in the fusion protein has multiple basic amino acid (RRRKRF) motifs similar to velogenic isolates. Phylogenetic analysis based on complete fusion gene sequences confirmed that the isolates belong to NDV sub genotype XIII 2.2. Further analysis revealed several amino acid substitutions in the hypervariable region, heptad repeat regions and neutralizing epitopes of the fusion protein and heptad repeat regions and antigenic sites of the hemagglutinin-neuraminidase (HN) protein that are critical for fusion. A unique D170A substitution in the neutralizing epitope is identified that is critical for structure and function of the fusion protein. Mutations within the virulence determinants including fusion (F) and HN, elucidate continuous evolution of the viruses among the rock pigeons. Accidental spillover of these mutated viruses into commercial poultry operations may result in disease outbreaks with economic breakdown.

Studies on the sequential pathology of Kyasanur Forest Disease (KFD) in Mouse brain: KFD virus induces apoptosis of neurons in cerebrum and hippocampus.

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

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.

Developments in Rabies Vaccines: The Path Traversed from Pasteur to the Modern Era of Immunization.

Rabies is a disease of antiquity and has a history spanning millennia ever since the first interactions between humans and dogs. The alarming fatalities caused by this disease have triggered rabies prevention strategies since the first century BC. There have been numerous attempts over the past 100 years to develop rabies vaccineswith the goal of preventing rabies in both humans and animals. Thepre-Pasteurian vaccinologists, paved the way for the actual history of rabies vaccines with the development of first generation vaccines. Further improvements for less reactive and more immunogenic vaccines have led to the expansion of embryo vaccines, tissue culture vaccines, cell culture vaccines, modified live vaccines, inactivated vaccines, and adjuvanted vaccines. The adventof recombinant technology and reverse genetics have given insight into the rabies viral genome and facilitated genome manipulations, which in turn led to the emergence of next-generation rabies vaccines, such as recombinant vaccines, viral vector vaccines, genetically modified vaccines, and nucleic acid vaccines. These vaccines were very helpful in overcoming the drawbacks of conventional rabies vaccines with increased immunogenicity and clinical efficacies. The path traversed in the development of rabies vaccines from Pasteur to the modern era vaccines, though, faced numerous challenges;these pioneering works have formed the cornerstone for the generation of thecurrent successful vaccines to prevent rabies. In the future, advancements in the scientific technologies and research focus will definitely lay the path for much more sophisticated vaccine candidates for rabies elimination.

Comparative Evaluation of Intradermal vis-à-vis Intramuscular Pre-Exposure Prophylactic Vaccination against Rabies in Cattle.

Rabies is a progressively fatal viral disease affecting a wide variety of warm-blooded animals and human beings. With cattle being major part of Indian livestock population, rabies can result in significant financial losses. Immunization of livestock vulnerable to exposure is the best way to control rabies. The present study was undertaken to investigate the efficacy of a rabies pre-exposure prophylactic vaccine administered through different routes and to sequentially monitor the levels of rabies virus-neutralizing antibody (RVNA) titers in cattle. Thirty cattle were divided into five groups of six animals each. Group I and III animals were immunized with 1 mL and 0.2 mL of rabies vaccine through intramuscular (IM) and intradermal (ID) routes, respectively, on day 0, with a booster dose on day 21; Group II and IV animals were immunized with 1 mL and 0.2 mL of rabies vaccine, respectively, without the booster dose; unvaccinated animals served as a control (Group V). Serum samples were collected on days 0, 14, 28, and 90 to estimate RVNA titers using the rapid fluorescent focus inhibition test (RFFIT). The titers were above an adequate level (≥0.5 IU/mL) on day 14 and maintained up to 90 days in all animals administered the rabies vaccine through the IM and ID route with or without a booster dose. The study indicated that both routes of vaccination are safe and effective in providing protection against rabies. Hence, both routes can be considered for pre-exposure prophylaxis. However, the ID route proved to be more economical due to its dose-sparing effect.

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine.

We evaluated the safety and potency of the Kyasanur Forest disease (KFD) vaccine inactivated with different formalin concentrations in mice, since the side effects due to higher formalin concentrations have been a major reason for vaccine refusal. Furthermore, with an objective to reduce the use of mice in vaccine testing, we performed quantification of the KFD virus by real-time PCR and compared it with in vivo titration in mice. The KFD vaccine prepared in chicken embryo fibroblast cells was inactivated with 0.04%, 0.06%, and 0.08% concentrations of formalin. The vaccine inactivated with 0.04% and 0.06% formalin failed the safety test, whereas the KFD vaccine inactivated with 0.08% formalin was safe and potent with a log protective index of 5678 in mice. This reduced formalin content may induce no/lesser side-effects of pain/swelling which may increase the vaccine acceptance. The real-time PCR on individual KFD vaccine harvests interpreted that when the CT value of each harvest is <20, the vaccine will have sufficient viral particles to pass the potency test. Comparison of the real-time PCR on tenfold dilutions of the pooled harvests with in vivo mice inoculation test revealed that the 1MLD50 of the vaccine lies in the tenfold dilution that yields CT values between 31 and 34.

Assessment of Immune Responses to Rabies Vaccination in Free-Ranging Dogs in Bengaluru, India.

Rabies is a fatal encephalomyelitis mainly transmitted to humans and other animals by rabid dog bites. Hence, vaccination programs are being instituted for the control of rabies in dogs. Though stray dogs have been vaccinated for years under various programs initiated for control of the disease, the effectiveness of these programs can be ascertained only by assessing the immunity of these dogs. With this in view, a study was conducted to assess the effectiveness of the ongoing mass dog vaccination (MDV) program by the Bengaluru City Municipal Corporation, Bengaluru, India. Whole blood and serum samples (n = 260) from vaccinated stray dogs in 26 wards of 8 corporation zones were tested by rapid fluorescent focus inhibition test (RFFIT) as well as an in-house quantitative indirect enzyme-linked immunosorbent assay (iELISA) for a humoral response and by interferon-gamma (IFN-γ) ELISA for a cellular response. As determined by the cut-off value of 0.5 IU/mL of serum, 71% and 87% of the samples from vaccinated dogs revealed adequate levels of antibodies presumed to confer protection by RFFIT and iELISA, respectively. The sensitivity and specificity of the iELISA were 100% and 63.3%, respectively. The IFN-γ ELISA revealed adequate cellular response in 50% of the samples. The quantitative iELISA was found to be useful in large-scale seromonitoring of MDV programs to aid in the elimination of dog-mediated rabies.

Diagnosis of rabies using reverse-transcription polymerase chain reaction on post-mortem skin tissue specimens of the nasolabial plate in a rabies suspected cow: a case study.

In India, rabies in cattle is under-reported. Religious sentiments hamper its diagnosis, discouraging post-mortem examination, particularly opening the cranium. Specimens of peripheral tissue innervated by the cranial nerves could potentially be used as alternative diagnostic specimens to the brain. Herein, we present a case study of a novel approach for diagnosing rabies in a cow suspected of having rabies, using skin tissue specimens of the nasolabial plate obtained post-mortem. Brain and nasolabial tissue specimens tested positive for rabies using conventional reverse-transcription polymerase chain reaction. This approach has been previously shown to have a high diagnostic sensitivity in animals. We encourage further studies with more nasolabial plate skin specimens for both post- and antemortem diagnosis of rabies in cattle.

Molecular typing of Brucella species isolates from livestock and human.

Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus-melitensis-ovis-suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.

Review of Oral Rabies Vaccination of Dogs and Its Application in India.

Oral rabies vaccines (ORVs) have been in use to successfully control rabies in wildlife since 1978 across Europe and the USA. This review focuses on the potential and need for the use of ORVs in free-roaming dogs to control dog-transmitted rabies in India. Iterative work to improve ORVs over the past four decades has resulted in vaccines that have high safety profiles whilst generating a consistent protective immune response to the rabies virus. The available evidence for safety and efficacy of modern ORVs in dogs and the broad and outspoken support from prominent global public health institutions for their use provides confidence to national authorities considering their use in rabies-endemic regions. India is estimated to have the largest rabies burden of any country and, whilst considerable progress has been made to increase access to human rabies prophylaxis, examples of high-output mass dog vaccination campaigns to eliminate the virus at the source remain limited. Efficiently accessing a large proportion of the dog population through parenteral methods is a considerable challenge due to the large, evasive stray dog population in many settings. Existing parenteral approaches require large skilled dog-catching teams to reach these dogs, which present financial, operational and logistical limitations to achieve 70% dog vaccination coverage in urban settings in a short duration. ORV presents the potential to accelerate the development of approaches to eliminate rabies across large areas of the South Asia region. Here we review the use of ORVs in wildlife and dogs, with specific consideration of the India setting. We also present the results of a risk analysis for a hypothetical campaign using ORV for the vaccination of dogs in an Indian state.

A Comparative Evaluation of the Estimation of Rabies Virus Antibodies among Free-Roaming, Vaccinated Dogs in Bengaluru, India.

Vaccination is the practical solution for the prevention of rabies in dogs. Assessment of the immunogenicity of vaccination includes estimation of specific rabies virus neutralizing antibodies (VNA) in the target species. We undertook a study to estimate the levels of VNA in free-roaming dogs with a history of rabies vaccination in Bengaluru city, India. We compared the rapid fluorescent focus inhibition test (RFFIT) and an in-house quantitative indirect ELISA (iELISA). The study area comprised the jurisdiction of Bruhat Bengaluru Mahanagara Palike (BBMP), the Bengaluru civic body. The BBMP, along with several non-government organizations (NGO), were conducting a trap- neuter- vaccinate- release program for the prevention of dog rabies. Serum samples were collected from 250 free-roaming dogs from representative regions of BBMP, of which 125 had a VNA titre of 0.5 IU or more by the RFFIT. Furthermore, 126 dogs showed percent positivity values (PP values) more than the cut off PP value of 57.1 by the iELISA, accounting for 50.4% of satisfactory post-vaccinal serum conversion. The sensitivity and specificity of the iELISA was 94.4% and 95.2%, respectively. Based on these data, a quantitative iELISA may be a complementary tool for sero-monitoring immune responses of free-ranging animals after rabies vaccination.

Randomly amplified polymorphic DNA analysis of Staphylococcus chromogenes isolated from bovine and bubaline mastitis in Karnataka.

BACKGROUND AND AIM: In recent times, non-aureus staphylococci (NAS) have emerged as the major organisms isolated from mastitis cases in dairy animals, with a predominance of Staphylococcus epidermidis and Staphylococcus chromogenes. As compared to Staphylococcus aureus, much less is known about the molecular types or the spatiotemporal epidemiology of these NAS species. In the present study, randomly amplified polymorphic DNA (RAPD) was employed to detect genetic polymorphisms, intraspecies diversity, and epidemiology of S. chromogenes strains (n=37) isolated from bovine and bubaline mastitis cases in the state of Karnataka. MATERIALS AND METHODS: Thirty-seven S. chromogenes isolates (14 from bovines and 23 from bubaline) isolated from subclinical mastitis cases, from organized and unorganized sectors, were subjected to RAPD typing. Further, methicillin resistance was determined by cefoxitin disk diffusion method. RESULTS: The amplified DNA fragments ranged from 150 to 3000 base pairs and yielded several RAPD profiles. Further analysis using Digital Image Correlation Engine correlation coefficient and UPGMA method showed that the 37 isolates could be classified into 12 distinct RAPD types (A to L) at 62% similarity (D=0.889). Four of the most predominant RAPD types, B, A, C, and E, in that order, and together, represented 65% of the isolates. High diversity was observed among the isolates both within farms and between geographic locations. Most of the isolates exhibited methicillin resistance. This is the first such report from India. CONCLUSION: In the absence of defined multilocus sequence type protocols or sufficient sequences available in the public domain, RAPD can be employed to determine genetic diversity of S. chromogenes isolates.

Molecular fingerprinting of bovine mastitis-associated Staphylococcus aureus isolates from India.

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. Owing to the mostly backyard dairy practices, we hypothesized that genetic diversity among mastitis-associated S. aureus from India would be high, and investigated 166 isolates obtained mostly from the Southern State of Karnataka, but also from a few other states. The results revealed (a) 8 to 13 fragments in pulsed-field gel electrophoresis (PFGE), forming 31 distinct patterns, and (b) 34 spa types, of which three (t17680, t18314, and t18320) were newly identified. Multi-locus sequencing typing (MLST) identified 39 sequence types (STs), with ST2454 (34.4%) and ST2459 (24%) being the most commonly represented, which clustered to clonal complexes (CC) CC9 and CC97, respectively; 12 STs were newly identified. Thirty-four (20.5%) of the 166 isolates displayed oxacillin resistance. On the other hand, whereas none were mecC+, 44 (26.5%) isolates were mecA+, with a predominance of SCCmecIVb (26/32 isolates, others being untypeable); 24 isolates (14.46%) were oxacillin-susceptible methicillin-resistant S. aureus (OS-MRSA; mecA+ but OS). Integrated analysis revealed that CC9-ST2454- and CC97-ST2459-SCCmecIVb were the predominant MRSA, although the distribution of CC9 and CC97 was similar between methicillin-resistant and -susceptible isolates. By PCR, 56.25%, 28.75% and 47.5% of the 166 isolates were positive for hlg, tsst and pvl genes, respectively. Our results, for the first time describe the application of a combination of various molecular methods to bovine mastitis-associated S. aureus isolates from India, corroborate the worldwide distribution of CC97 and CC9, and suggest pathogenic potential of the isolates.

Reviewing Solutions of Scale for Canine Rabies Elimination in India.

Canine rabies elimination can be achieved through mass vaccination of the dog population, as advocated by the WHO, OIE and FAO under the 'United Against Rabies' initiative. Many countries in which canine rabies is endemic are exploring methods to access dogs for vaccination, campaign structures and approaches to resource mobilization. Reviewing aspects that fostered success in rabies elimination campaigns elsewhere, as well as examples of largescale resource mobilization, such as that seen in the global initiative to eliminate poliomyelitis, may help to guide the planning of sustainable, scalable methods for mass dog vaccination. Elimination of rabies from the majority of Latin America took over 30 years, with years of operational trial and error before a particular approach gained the broad support of decision makers, governments and funders to enable widespread implementation. The endeavour to eliminate polio now enters its final stages; however, there are many transferrable lessons to adopt from the past 32 years of global scale-up. Additionally, there is a need to support operational research, which explores the practicalities of mass dog vaccination roll-out and what are likely to be feasible solutions at scale. This article reviews the processes that supported the scale-up of these interventions, discusses pragmatic considerations of campaign duration and work-force size and finally provides an examples hypothetical resource requirements for implementing mass dog vaccination at scale in Indian cities, with a view to supporting the planning of pilot campaigns from which expanded efforts can grow.

Application and Comparative Evaluation of Fluorescent Antibody, Immunohistochemistry and Reverse Transcription Polymerase Chain Reaction Tests for the Detection of Rabies Virus Antigen or Nucleic Acid in Brain Samples of Animals Suspected of Rabies in India.

Accurate and early diagnosis of animal rabies is critical for undertaking public health measures. Whereas the direct fluorescent antibody (DFA) technique is the recommended test, the more convenient, direct rapid immunochemistry test (dRIT), as well as the more sensitive, reverse transcription polymerase chain reaction (RT-PCR), have recently been employed for the laboratory diagnosis of rabies. We compared the three methods on brain samples from domestic (dog, cat, cattle, buffalo, horse, pig and goat) and wild (leopard, wolf and jackal) animals from various parts of India. Of the 257 samples tested, 167 were positive by all the three tests; in addition, 35 of the 36 decomposed samples were positive by RT-PCR. This is the first study in which such large number of animal samples have been subjected to the three tests simultaneously. The results confirm 100% corroboration between DFA and dRIT, buttress the applicability of dRIT in the simple and rapid diagnosis of rabies in animals, and reaffirm the suitability of RT-PCR for samples unfit for testing either by DFA or dRIT.