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Objective To determine the prevalence of the different capsular polysaccharide (CP) and major surface-associated non-CP antigen 336 (SP-336) types among Staphylococcus aureus isolated from bovine mastitis cases in Australia and India. Methods A total of 414 strains (154 from Australia, 260 from India) isolated from clinical bovine mastitis were included in the study. Mouse antisera raised against CP types (CP1, CP2, CP5, and CP8) or SP-336 were used in slide agglutination tests and compared with detection of cap1, cap5 and cap8 gene fragments by PCR. Results Serological studies revealed the presence of CP2, CP5, CP8 and SP-336 in 9.1%, 23.4%, 31.8%, and 5.8% of the Australian versus 0.8%, 46.9%, 13.1% and 0% of the Indian isolates, respectively. By PCR, CP1, CP5 and CP8 accounted for 0%, 26.6% and 32.4% of the Australian versus 3.9%, 85% and 8.1% of the Indian isolates, respectively. Conclusions Both PCR and the serological method demonstrated that CP5 and CP8 are the predominant capsular types in Australia, whereas CP5 is the predominant capsular type in India. The study also demonstrated a strong correlation between both methods of typing for CP1, CP5, CP8 and non-typeable S. aureus strains. High-percentage prevalence of non-typeable isolates in both the countries highlights the importance of continued investigations of the identification of unique surface-associated polysaccharide antigens prevalent among S. aureus isolates for the formulation of CP- and SP-based vaccines for bovine mastitis. [ABSTRACT FROM AUTHOR]

We describe the whole-genome sequencing and comparative genomic analysis of 22 mastitis-associated NASM strains isolated from India. The mean genome size of the strains was 2.55 Mbp, with an average GC content of 32.2%. We identified 14 different sequence types (STs) among the 22 NASM strains. Of these, ST1 and ST6 of S. chromogenes were exclusively associated with bovine mastitis. Genome-wide SNP-based minimum spanning tree revealed the intricate phylogenetic relationships among NASM strains from India, categorizing them into five major clades. Interestingly, mastitis-associated strains formed separate subclades in all the NASM species studied, indicating distinct host-specific co-evolution. The study identified 32 antimicrobial resistance (AMR) genes and 53 virulence-associated genes, providing insights into the genetic factors that could contribute to the pathogenicity of NASM species. Some virulence and AMR genes were found in the predicted genomic islands, suggesting possible horizontal transfer events., Competing Interests: Declarations. Competing interests: We declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s).)

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s).)

Interface with animals has been responsible for the occurrence of a major proportion of human diseases for the past several decades. Recent outbreaks of respiratory, haemorrhagic, encephalitic, arthropod-borne and other viral diseases have underlined the role of animals in the transmission of pathogens to humans. The on-going coronavirus disease-2019 (COVID-19) pandemic is one among them and is thought to have originated from bats and jumped to humans through an intermediate animal host. Indeed, the aetiology, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect and cause disease in cats, ferrets and minks, as well as be transmitted from one animal to another. The seriousness of the pandemic along with the zoonotic origin of the virus has been a red alert on the critical need for collaboration and cooperation among human and animal health professionals, as well as stakeholders from various other disciplines that study planetary health parameters and the well-being of the biosphere. It is therefore imminent that One Health principles are applied across the board for human infectious diseases so that we can be better prepared for future zoonotic disease outbreaks and pandemics., Competing Interests: Conflict of interestWe declare that we have no financial or other conflicts of interest., (© Indian Institute of Science 2022.)

Background: Bovine ephemeral fever (BEF) is a re-emerging disease caused by bovine ephemeral fever virus (BEFV). Although it poses a huge economic threat to the livestock sector, complete viral genome information from any South Asian country, including India, lacks., Aim: Genome characterization of the first Indian BEFV isolate and to evaluate its genetic diversity by characterizing genomic mutations and their associated protein dynamics., Materials and Methods: Of the nineteen positive blood samples collected from BEF symptomatic animals during the 2018-19 outbreaks in India, one random sample was used to amplify the entire viral genome by RT-PCR. Utilizing Sanger sequencing and NGS technology, a complete genome was determined. Genome characterization, genetic diversity and phylogenetic analyses were explored by comparing the results with available global isolates. Additionally, unique genomic mutations within the Indian isolate were investigated, followed by in-silico assessment of non-synonymous (NS) mutations impacts on corresponding proteins' secondary structure, solvent accessibility and dynamics., Results: The complete genome of Indian BEFV has 14,903 nucleotides with 33% GC with considerable genetic diversity. Its sequence comparison and phylogenetic analysis revealed a close relatedness to the Middle Eastern lineage. Genome-wide scanning elucidated 30 unique mutations, including 10 NS mutations in the P, L and G NS proteins. The mutational impact evaluation confirmed alterations in protein structure and dynamics, with minimal effect on solvent accessibility. Additionally, alteration in the interatomic interactions was compared against the wild type., Conclusion: These findings extend our understanding of the BEFV epidemiological and pathogenic potential, aiding in developing better therapeutic and preventive interventions.

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., (© 2021. The Author(s).)

The dawn of the 20th century saw the formative years of developments in immunology. In particular, immunochemistry, specifically pertaining to antibodies, was extensively studied. These studies laid the foundations for employing antibodies in a variety of ways. Not surprisingly, antibodies have been used for applications ranging from biomedical research to disease diagnostics and therapeutics to evaluation of immune responses during natural infection and those elicited by vaccines. Despite recent advancements in cellular immunology and the excitement of T cell therapy, use of antibodies represents a large proportion of immunotherapeutic approaches as well as clinical interventions. Polyclonal antibodies in the form of plasma or sera continue to be used to treat a number of diseases, including autoimmune disorders, cancers, and infectious diseases. Historically, antisera to toxins have been the longest serving biotherapeutics. In addition, intravenous immunoglobulins (IVIg) have been extensively used to treat not only immunodeficiency conditions but also autoimmune disorders. Beyond the simplistic suppositions of their action, the IVIg have also unraveled the immune regulatory and homeostatic ramifications of their use. The advent of monoclonal antibodies (MAbs), on the other hand, has provided a clear pathway for their development as drug molecules. MAbs have found a clear place in the treatment of cancers and extending lives and have been used in a variety of other conditions. In this review, we capture the important developments in the therapeutic applications of antibodies to alleviate disease, with a focus on some of the recent developments.

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., (Copyright: © Sheela, et al.)

Context: Infections with methicillin-resistant Staphylococcus aureus (MRSA) greatly influence clinical outcome. Molecular characterisation of MRSA can help to predict their spread and to institute treatment and hospital protocols., Aim: The aim of this study is to understand the diversity of MRSA in a tertiary care hospital in Hyderabad, India., Settings and Design: Samples collected at Gandhi Medical College, Hyderabad, and designed to assess hospital-or community-associated MRSA (HA-MRSA or CA-MRSA)., Subjects and Methods: MRSA were subjected to antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and staphylococcal cassette chromosome-mec (SCCmec) typing., Statistical Analysis Used: Discriminatory index and 95% confidence interval., Results: Of the 30 MRSA, (a) 18 and 12 were HA-MRSA and CA-MRSA, respectively, and (b) 23.3% and 6.6% displayed induced clindamycin and intermediate vancomycin resistance, respectively. Genetic diversity was evident from the presence of (a) 20 pulsotypes, (b) eight spa types, with the predominance of t064 (n = 9) and (c) seven sequence types (ST), with the preponderance of ST22 and ST8 (9 each). ST22 and ST8 were the most prevalent among HA-MRSA and CA-MRSA, respectively. SCCmec type IV was the most frequent (n = 8). 44.4% of HA-MRSA belonged to SCCmec IV and V, whereas 33.3% of CA-MRSA belonged to SCCmec I and III; 33.3% (5/15) of the isolates harbouring the pvl gene belonged to SCCmec IVC/H., Conclusions: ST8 was a dominant type along with other previously reported types ST22, ST239, and ST772 from India. The observations highlight the prevalence of genetically diverse clonal populations of MRSA, suggesting potential multiple origins., Competing Interests: None

For segmented viruses, rapid genomic and phenotypic changes can occur through the process of reassortment, whereby co-infecting strains exchange entire segments creating novel progeny virus genotypes. However, for many viruses with segmented genomes, this process and its effect on transmission dynamics remain poorly understood. Here, we assessed the consequences of reassortment for selection on viral diversity through time using bluetongue virus (BTV), a segmented arbovirus that is the causative agent of a major disease of ruminants. We analysed ninety-two BTV genomes isolated across four decades from India, where BTV diversity, and thus opportunities for reassortment, are among the highest in the world. Our results point to frequent reassortment and segment turnover, some of which appear to be driven by selective sweeps and serial hitchhiking. Particularly, we found evidence for a recent selective sweep affecting segment 5 and its encoded NS1 protein that has allowed a single variant to essentially invade the full range of BTV genomic backgrounds and serotypes currently circulating in India. In contrast, diversifying selection was found to play an important role in maintaining genetic diversity in genes encoding outer surface proteins involved in virus interactions (VP2 and VP5, encoded by segments 2 and 6, respectively). Our results support the role of reassortment in driving rapid phenotypic change in segmented viruses and generate testable hypotheses for in vitro experiments aiming at understanding the specific mechanisms underlying differences in fitness and selection across viral genomes.

Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.

The Indian subcontinent with its population density, climatic conditions, means of subsistence, socioeconomic factors as well as travel and tourism presents a fertile ground for thriving of RNA viruses. Despite being pathogens of huge significance, there is very little focus on research into the biology and pathogenesis of RNA viruses in India. Studies on epidemiology and disease burden, risk factors, the immune response to RNA viruses, circulating virus strains and virus evolution, animal models of disease, antivirals and vaccines are strikingly absent. Emerging RNA viruses such as Zika virus, Nipah virus and Crimean-Congo haemorrhagic fever virus are a matter of grave concern to India. Here we summarize the outcome of the India|EMBO symposium on "RNA viruses: immunology, pathogenesis and translational opportunities" organized at Faridabad, National Capital Region, India, on March 28-30, 2018. The meeting focused on RNA viruses (non-HIV), and both national and international experts on RNA viruses covered topics ranging from epidemiology, immune response, virus evolution and vaccine trials concerning RNA viruses. The aim of the symposium was to create a road map for RNA virus research in India. Both concrete and tentative ideas pointing towards short-term and long-term goals were presented with recommendations for follow-up at government level.

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population., (© 2017 Blackwell Verlag GmbH.)

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., Competing Interests: The authors declare no conflicts of interest.

Introduction: Vaccinology has evolved from a sub-discipline focussed on simplistic vaccine development based on antibody-mediated protection to a separate discipline involving epidemiology, host and pathogen biology, immunology, genomics, proteomics, structure biology, protein engineering, chemical biology, and delivery systems. Data mining in combination with bioinformatics has provided a scaffold linking all these disciplines to the design of vaccines and vaccine adjuvants. Areas covered: This review provides background knowledge on immunological aspects which have been exploited with informatics for the in silico analysis of immune responses and the design of vaccine antigens. Furthermore, the article presents various databases and bioinformatics tools, and discusses B and T cell epitope predictions, antigen design, adjuvant research and systems immunology, highlighting some important examples, and challenges for the future. Expert opinion: Informatics and data mining have not only reduced the time required for experimental immunology, but also contributed to the identification and design of novel vaccine candidates and the determination of biomarkers and pathways of vaccine response. However, more experimental data is required for benchmarking immunoinformatic tools. Nevertheless, developments in immunoinformatics and reverse vaccinology, which are nascent fields, are likely to hasten vaccine discovery, although the path to regulatory approval is likely to remain a necessary impediment.

Poliovirus (PV) contains a single-stranded positive-sense RNA genome, which is translated into a single polyprotein. Viral proteases process this polyprotein to produce several individual as well as fused proteins. The major viral protease 3C cleaves at nine of the eleven cleavage sites. During the process of expressing PV 3ABC protein in Escherichia coli, we identified a 3C mutant (L70P), which lost its protease activity. This loss of function was confirmed by generating recombinant adenoviruses expressing mutant and wild-type 3C. Further, infectious PV could not be recovered from PV full-length cDNA containing the L70P mutation. However, 3C L70P mutant cDNA could complement a PV cDNA containing a 1AB deletion, producing a viable virus population containing defective complementing genomes. Structural analysis of the mutant protein indicated that the L70P mutation resulted in the loss of a hydrogen bond between two residues located within a loop between two β-sheets, potentially leading to strain on the catalytic site. We conclude that L70P inactivates 3C protease because of its close proximity to the 3C catalytic site.

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks., (© 2016 Blackwell Verlag GmbH.)

Biotinylation has been extensively used for antibody tagging, affinity-based purification, and in protein/DNA-protein interaction studies. Here we describe the use of biotinylation to study the turn-over of proteins in cells. We use the prokaryotic biotin ligase (BirA) to biotinylate the human leukocyte antigen (HLA)-A2 (A2) heavy chain (HC), which was engineered to contain a biotin acceptor peptide (BAP). Controlled availability of biotin in combination with visualization using streptavidin-conjugated peroxidase made it possible to detect biotinylated BAP-A2. Further, we exploited the effects of human cytomegalovirus (HCMV) unique short (US) proteins US2 and US11 on the turn-over of BAP-A2 HC. The full-length BAP-A2 HC and its mutants lacking either the cytosolic tail (tail-less) or both the transmembrane and cytosolic regions (soluble) were expressed via recombinant adenoviruses (rAd). The effect of US2, US11 and a control HCMV protein US9, also expressed via rAd, on each of the BAP- A2 forms was assessed. Experiments using this system showed that US2 and US11 cause proteasome-mediated degradation of full-length BAP-A2 HC but only US2 could cause degradation of tail-less BAP-A2. The results demonstrate that the technique of biotinylation can be used to study protein turn-over in cells., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Japanese encephalitis (JE) is a serious public health concern in most of Asia. The disease is caused by JE virus (JEV), a flavivirus transmitted by Culex mosquitoes. Several vaccines have been developed to control JE in endemic areas as well as to protect travelers and military personnel who visit or are commissioned from non-endemic to endemic areas. The vaccines include inactivated vaccines produced in mouse brain or cell cultures, live attenuated vaccines, and a chimeric vaccine based on the live attenuated yellow fever virus 17D vaccine strain. All the marketed vaccines belong to the JEV genotype III, but have been shown to be efficacious against other genotypes and strains, with varying degrees of cross-neutralization, albeit at levels deemed to be protective. The protective responses have been shown to last three or more years, depending on the type of vaccine and the number of doses. This review presents a brief account of the different JE vaccines, their immunogenicity and protective ability, and the impact of JE vaccines in reducing the burden of disease in endemic countries.

Poliomyelitis, caused by poliovirus, is on the verge of eradication, and the world is preparing to shift from live to inactivated polio vaccine. In view of the requirement of non-infectious reagents, especially protein antigens, for surveillance during the final phase of poliovirus eradication, we have attempted to generate reagents that may be of use for the development of diagnostic tests. Polioviral proteins VP0, VP3, VP1, and 3AB were expressed in Escherichia coli using the autoinduction system, purified, and the proteins were used to raise antisera in rabbits. All antisera detected all three serotypes of PV from infected cell lysates in enzyme-linked immunosorbent assay, immunofluorescence and western blotting., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen that can cause a wide variety of infections. Comparative genetic analyses have led to the discovery that despite the existence of a vast number of genotypes, outbreak strains of MRSA appear to be limited to certain genotypes, some of which are further restricted to certain geographical locations. Whereas extensive literature is available in several countries, the complexity of the clonal distribution both of healthcare-associated (HA) and community-associated (CA) MRSA in India is only now beginning to be understood. Studies have revealed that MRSA in India is distributed among all of the major staphylococcal cassette chromosome mec (SCCmec) types. The majority of HA-MRSA isolates belong to SCCmec type III and sequence type (ST) 239. By contrast, CA-MRSA mostly belong to ST22 (SCCmec IV), ST772 (SCCmec V) and ST672 (SCCmec V) genotypes. Similar to the global scenario, CA-MRSA is becoming more invasive and transmissible and is increasingly becoming difficult to be differentiated from HA-MRSA. In addition, it is disturbing that some of the HA-MRSA isolates have been reported to be vancomycin-resistant. On the other hand, almost no information is available on the genotypes of livestock-associated MRSA or their potential impact on human infections in India. Concerted efforts are needed to further understand the genetic epidemiology of MRSA in India., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Bluetongue (BT) is an arthropod-borne viral disease mostly of sheep. Bluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of family Reoviridae and is transmitted by midges belonging to Culicoides spp. The disease is endemic in the tropics and subtropics, and the incidence is high in southern India. Twenty-six serotypes of BTV have been reported worldwide. Although most of the serotypes have been reported in India, information regarding currently circulating serotypes is essential to develop control programs. Both serological assays and nucleic acid-based assays have been used for typing BTV. Segment 2, which codes for the outer capsid protein VP2, is the target for PCR-based typing; however, the VP2 sequence diversity among viruses belonging to the same serotype but isolated from different geographical areas makes it essential to develop geographical based reagents. In this study, reverse transcription PCR was developed based on sequences of Indian isolates of BTV (serotypes 1, 2, 9, 10, 12, 16, 21 and 23), and this was applied to type 52 isolates obtained during the last decade. It was found that multiple serotypes circulate, with involvement of more than one serotype infecting individual animals and herds over a period in a given area. Detection of circulating serotypes and estimation of herd immunity against different serotypes of BTV may provide important information for predicting the distribution of these serotypes and inclusion of serotypes in vaccines., (© 2015 Blackwell Verlag GmbH.)

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern., (© 2016 Blackwell Verlag GmbH.)

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations., (© 2014 Blackwell Verlag GmbH.)

Buffaloes are the second largest source of milk. Mastitis is a major impediment for milk production, but not much information is available about bubaline mastitis, especially subclinical mastitis. The aim of this study was to (a) investigate the application of various tests for the diagnosis of bubaline subclinical mastitis, (b) identify the major bacteria associated with it, and (c) evaluate the antibiotic resistance pattern of the bacteria. To this end, 190 quarter milk samples were collected from 57 domesticated dairy buffaloes from organized (64 samples) and unorganized (126 samples) sectors. Of these, 48.4%, 40.0%, 45.8%, 61.1%, and 61.6% were positive for subclinical mastitis by somatic cell count, electrical conductivity, California mastitis test, bromothymol blue test, and N-acetyl glucosaminidase test, respectively. As compared to the gold standard of somatic cell count, California mastitis test performed the best. However, a combination of the two methods was found to be the best option. Microbiological evaluation, both by biochemical methods as well as by monoplex and multiplex polymerase chain reaction, revealed that coagulase-negative staphylococci were the most predominant (64.8%) bacteria, followed by streptococci (18.1%), Escherichia coli (9.8%) and Staphylococcus aureus (7.3%). Most of the pathogens were resistant to multiple antibiotics, especially to β-lactam antibiotics. We propose that California mastitis test be combined with somatic cell count for diagnosis of subclinical mastitis in domestic dairy buffaloes. Further, our results reveal high resistance of the associated bacteria to the β-lactam class of antibiotics, and a possible major role of coagulase-negative staphylococci in causing the disease in India.

Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and β-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis.

Bluetongue virus (BTV) causes disease mainly in sheep, but can be transmitted via other domestic and wild ruminants, resulting in pecuniary burden and trade restrictions. Segmented genome with the possibility of reassortment, existence of 26 serotypes, geographical restriction in the distribution of many of the serotypes, use of live attenuated vaccines and the lack of complete sequences of viruses isolated from several parts of the globe have complicated our understanding of the origin, movement and distribution of BTV. Recent efforts in genome sequencing of several strains have helped in better comprehending BTV epidemiology. In an effort to contribute to the genetic epidemiology of BTV in India, we report the isolation and complete genome sequencing of a BTV serotype 12 virus (designated NMO1). This is the first BTV-12 isolated from India and the second BTV-12 to be sequenced worldwide. The analysis of sequences of this virus suggests that NMO1 derived its segments from viruses belonging to western topotype viruses, as well as those from South-East Asia and India. The results have implications for understanding the origin, emergence/re-emergence and movement of BTV as well as for the development of vaccines and diagnostics based on robust epidemiological data., (© 2013 Blackwell Verlag GmbH.)

Background: Japanese encephalitis (JE) is a vaccine-preventable acute disease. We report the results of a phase 2/3 trial of JENVAC, a Vero cell-derived vaccine developed using an Indian strain of JE virus (JEV)., Methods: JENVAC was administered in 2 doses 28 days apart, and immunogenicity was compared to that from a single dose of SA-14-14-2, the only approved JE vaccine and regimen at the time in India., Results: After both the doses, seroconversion and seroprotection were >90% for JENVAC. For SA-14-14-2, seroconversion and seroprotection were 57.69% and 77.56%, respectively, on day 28 and 39.74% and 60.26%, respectively, on day 56. The geometric mean titers at day 28 and day 56 were 145.04 and 460.53, respectively, for JENVAC and 38.56 and 25.29, respectively, for SA-14-14-2. With a single dose of JENVAC, seroprotection titers lasted at least 12 months in >80% of the subjects. Following receipt of 2 doses, 61.17% of subjects retained seroprotection titers at 24 months, and immunogenicity criteria were higher than that for SA-14-14-2 at 12, 18, and 24 months each. Sera from JENVAC subjects neutralized JEV genotypes I, II, III, and IV equally well. Adverse events were not significantly different between the 2 vaccines., Conclusions: JENVAC elicits long-lasting, broadly protective immunity., Clinical Trials Registration: CTRI/2011/07/001855., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Health monitoring is an integral part of laboratory animal quality standards. However, current or past prevalence data as well as regulatory requirements dictate the frequency, type and the expanse of health monitoring. In an effort to understand the prevalence of rodent pathogens in India, a preliminary study was carried out by sero-epidemiology. Sera samples obtained from 26 public and private animal facilities were analyzed for the presence of antibodies against minute virus of mice (MVM), ectromelia virus (ECTV), lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), Sendai virus (SeV), and Mycoplasma pulmonis in mice, and SeV, rat parvo virus (RPV), Kilham's rat virus (KRV) and sialodacryoadenitis virus (SDAV) in rats, by sandwich ELISA. It was observed that MHV was the most prevalent agent followed by Mycoplasma pulmonis and MVM in mice, and SDAV followed by RPV were prevalent in rats. On the other hand, none of the samples were positive for ECTV in mice, or SeV or KRV in rats. Multiple infections were common in both mice and rats. The incidence of MHV and Mycoplasma pulmonis was higher in facilities maintained by public organizations than in vivaria of private organizations, although the difference was not statistically different. On the other hand the prevalence of rodent pathogens was significantly higher in the northern part of India than in the South. These studies form the groundwork for detailed sero-prevalence studies which should further lay the foundations for country-specific guidelines for health monitoring of laboratory animals.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV 'topotype'. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of 'live' South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively., (Copyright © 2015 Maan et al.)

The traditional platform of using embryonated chicken eggs for the production of influenza vaccines has several drawbacks including the inability to meet the volume of required doses in the case of widespread epidemics and pandemics. Cell culture platforms have therefore been explored in the last 2 decades, and have attracted further attention following the H1N1 pandemic outbreak. This platform, while not the most economical for large-scale production, has several advantages, and can supplement the vaccine requirement when needed. Recent developments in production technologies have contributed greatly to fine-tuning this platform. In combination with other technologies such as live attenuated and recombinant protein or virus-like particle vaccines, and different adjuvants and delivery systems, cell culture-based influenza vaccine platform can be used both for production of seasonal vaccine, and to mitigate vaccine shortages in pandemic situations.

The clinical evaluation of the MDCK-based H1N1 pandemic influenza vaccine HNVAC in adults aged 18-65 years is reported. In the Phase I randomized, double-blind, placebo-controlled, single-centre study, 160 subjects were parallelly assigned 3:1 to vaccine:placebo groups (n=60:20) with both the aluminium hydroxide adjuvanted and non-adjuvanted vaccine formulations. A single dose of both the formulations containing 15 μg of haemagglutinin protein showed minimal adverse reactions, the most common of which were pain at injection site (11.67%) and fever (10.00%). Both formulations produced 74-81% seroprotection (SRP: titre of ≥40), 67-70% seroconversion (SRC: four-fold increase in titres between days 0 and 21), and a four-fold increase in geometric mean titres (GMT). Aluminium hydroxide did not have a significant effect either on immunogenicity or on reactogenicity. Nevertheless, based on its recognized positive effects on the stability and immunogenicity of many vaccines, and its marginal benefit in both pre-clinical and Phase I studies of HNVAC, alum adjuvanted HNVAC was further tested in a staggered Phase II/III randomized, double-blind, placebo-controlled, multi-centre study of 200 and 195 subjects, respectively, parallelly assigned 4:1 to adjuvanted vaccine and placebo groups. In these studies, the most common adverse reactions were pain at injection site (6.88% and 5.77% in Stage 1 and Stage 2, respectively) and fever (7.50% and 7.05%, respectively), and a single dose resulted in 87-90% SRP, 85-86% SRC, and a nearly six-fold increase in GMT, meeting or exceeding licensing criteria. It is concluded that HNVAC is safe and immunogenic to adults of 18-65 years., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Several limitations of the use of embryonated eggs and the threat of pandemics have highlighted the need for other platforms for the production of influenza vaccines. We report the indigenous development and pre-clinical testing of an MDCK-based H1N1 pandemic influenza vaccine HNVAC from India. The cell bank and virus seed were characterized extensively. The cells were characterized by PCR, electron microscopy, and karyotyping, and found to be of female canine epithelial origin. The virus was confirmed by neutralization, haemagglutination inhibition, neuraminidase inhibition, and PCR and nucleotide sequencing. Adventitious agent testing was performed by both in vitro and in vivo studies. The in vitro studies included culturing, haemadsorption, haemagglutination, PCR and RT-PCR, whereas in vivo studies included passage in embryonated eggs and in laboratory animals. Both cell bank and virus seed were free of adventitious agents. MDCK cell lysates as well as cellular DNA did not produce tumours in newborn or adult laboratory animals. The bioprocess parameters were standardized to recover antigen with minimal levels of process-related impurities. The vaccine bulk was tested for the presence of specific antigen, and quantified by single radial immunodiffusion. Finally, non-adjuvanted and aluminium hydroxide adjuvanted vaccine formulations were found to be safe in preclinical toxicity studies in mice, rats, guinea pigs and rabbits, and immunogenic in mice and rabbits. This is the first and only cell culture-based influenza vaccine platform developed in any developing country., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Brucella spp. are facultative intracellular bacterial pathogens causing the zoonotic disease brucellosis. Here, we report the draft genome sequence of the Brucella melitensis strain from India designated Bm IND1, isolated from stomach contents of an aborted goat fetus., (Copyright © 2014 Rao et al.)

Bluetongue (BT) is an infectious, arthropod-borne viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), which is a double-stranded segmented RNA virus. Of the 26 confirmed BTV serotypes, 23 were reported in India based on the detection of antibodies or virus. In order to assess the prevalence of different serotypes in Andhra Pradesh, serum samples which were positive for BTV by group-specific antibody ELISA were subjected to type-specific neutralization of BTV serotypes 1, 2, 9, 10, 21 and 23. Of the 52 samples tested, 50.0, 44.23, 21.15, 26.92, 0, and 15.38 % neutralized BTV serotypes 1, 2, 9, 10, 21 and 23, respectively. However, 32.69 % of the ELISA positive sera could not neutralize any of these serotypes, indicating that there could be other serotype viruses (e.g., BTV-3 and -16) circulating in the State. This method can be used for surveillance of the circulating serotypes as well as for assessing the level of herd immunity, and assist in determining the vaccine strains to be used in multivalent vaccines.

Peste des petitis ruminants (PPR) is an economically important endemic viral disease of sheep and goats in India, where several different homologous PPR vaccine candidates have been developed. We evaluated the serological response to two vaccine strains, Arasur/87 and Sungri/96, in South Indian cross-bred and native sheep and goats reared under organized and unorganized settings. Animals seronegative (percent inhibition or PI <40) by competitive enzyme-linked immunosorbent assay (c-ELISA) were immunized with either of the vaccine strains or placebo. Sera collected on 21, 60 and 90 days post-vaccination were subjected to c-ELISA and serum neutralization test (SNT). Seropositivity (PI >40), seroconversion (fourfold increase in SNT titres) and seroprotection (SNT titre of ≥8 deemed to be protective) ranged from 66.7 to 84.0 %, 56.0 to 69.2 %, and 60.0 to 76.0 %, respectively. However, no significant difference was observed between responses to the two vaccine strains. These results support the premise that the two vaccine strains are equally efficacious.

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The second complete genome of bluetongue virus serotype 9 (BTV-9) is presented in this report. The sequence analysis points to continued circulation in India of a mixed topotype virus apparently belonging to the BTV-9 serotype, and it raises questions about approaches for serotyping bluetongue viruses.

Bluetongue virus serotype 21 (BTV-21) was originally isolated from Australia, but has now been reported from India, Indonesia, China and Japan. We report the isolation, and sequencing of BTV-21 from India. The complete ORF sequence of VP2 gene of this isolate showed that it is closely related to recent BTV-21 isolates from Japan (93-94% identity), and distantly related to BTV-21 reference strain (86% identity). Our results, along with the available sequences of Japanese isolates, suggest that the currently circulating BTV-21 strains from India and Japan are divergent from the original strain(s) from Australia and shed light on designing molecular diagnostics for the detection of BTV.

Bluetongue, a transboundary disease, is endemic in several tropical countries and is caused by bluetongue virus (BTV). The origin and movement of BTV can be predicted by comparing nucleotide sequences of its segmented RNA genome. Such analyses have been useful in evaluating the source of the virus responsible for recent incursion of BTV into previously unreported areas. Besides several serotypes, genetically related BTV strains circulate in each endemic area, but such clusters of strains have been reported to be distinct from one geographical region to another. We obtained partial or complete sequences of the open reading frames encoded by VP2, VP6, VP7, NS1 and NS2 genes of a BTV-10 isolate of India and compared them with other BTV-10 sequences available in public database. Sequences of all the five genes showed >99% identity to BTV-10 prototype, vaccine strain and vaccine-like virus isolates from the USA. Our results suggest that Indian BTV-10 virus analysed in this study possibly originated from the United States., (© 2011 Blackwell Verlag GmbH.)

Recent incursions of bluetongue virus (BTV) into previously naive geographical areas have emphasised the need to better understand virus movement and epidemiology. Several bluetongue virus (BTV) serotypes are known to exist in India, and some serotype viruses have been isolated. However, the complete genome of not a single isolate is available to date. We report the complete genome sequence of one, and partial sequences of three other Indian isolates of BTV-9. Evolutionary relationships with segment-2 and -6 sequences of BTV isolates around the world, deduced using four different phylogenetic analyses and a similarity programme, show that BTV-9 (Eastern), BTV-9 (Western), and BTV-5 form a triad of equidistant, genetically distinct groups of viruses. The Indian BTV-9 isolates were closely related to Mediterranean and European BTV-9 isolates (Eastern topotype) based on segment-2 and -6 sequences. By contrast, segment-5 analyses clustered the Indian BTV-9 isolates with South African BTV-3 reference strain (98% identity), which belongs to one of the Western types. These results have implications on BTV origin and movement, genotyping, serotyping, and vaccine design.