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Canine Leptospirosis pose a public health risk in addition to life threatening disease to dogs. The molecular methods enable faster and sensitive diagnosis of leptospirosis at early stage of infection in contrast to dark field microscopy and culture. The Recombinase Polymerase Amplification (RPA) assay is a versatile alternative to polymerase chain reaction by its simple, fast, inherent enzymatic amplification of nucleic acid at constant temperature. The RPA assay to detect Leptospira DNA was optimized with Leptospira reference strains and its performance characteristic such as analytical, diagnostics and reproducibility were assessed. The limit of detection of RPA assay was estimated as 10 2 copies of genomic DNA was similar to that of PCR assay and it is specific to amplify the pathogenic Leptospira strain rather than non-pathogenic Leptospira strains and other bacterial strains. Out of 150 dog samples screened Leptospira DNA was detected in 42.6% (64/150) by RPA assay and 44.6% (67/150) by PCR. The diagnostic sensitivity and specificity of RPA assay was 92.5% (83.44% to 97.53%, 95% CI) and 97.59% (91.57% to 99.71%, 95% CI) respectively, in comparison with PCR. The RPA assay has a good diagnostic agreement with a kappa value of 0.905 (0.837 to 0.974 at 95% CI). The performance of the assay on different occasion to amplify the DNA with mean concentration of 42.24 ng/µl ± 1.44 with a coefficient of variation of 5.9% showed its good repeatability. The reproducibility assessment with the third-party testing laboratory, revealed better agreement on the performance of the assay with a kappa value of 0.81(0.547 to 1.00 at 95% CI). Since this method is simple, rapid and less expensive enables to perform at resource limited laboratories or point of care testing at field level.

Background: Canine distemper (CD) is a highly fatal disease in dogs. Hence, it is important to document the factors which are significantly correlated with the poor prognosis amongst CD infected dogs to develop a prognostic index. Methods: Forty dogs positive for CD virus by nested reverse transcriptase polymerase chain reaction (nRT-PCR) were included in our study at infectious Disease Unit (IDU), Madras Veterinary College Teaching Hospital, Chennai between March 2021 and August 2022. Two different herbal drugs were tried in two groups of dogs where each group comprised of 20 dogs and clinical responses were observed. Epidemiological and clinical data were collected and analysed between survival and non-survival groups using chi-square test. The statistically significant factors were further analysed by logistic regression to identify their strong association with the event of death. Kaplan-Meier curves for survival were constructed to explore differences in the survival time for different dogs. Result: The overall case fatality rate was found to be 55% (22/40). The event of death due to CD was observed in young dogs having ocular discharge with strong correlation but the chances of survival were found to increase when age advanced.

Objective: This study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions.Methods: A combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5’ labelled with biotin/6-FAM to determine the binding potential and binding pattern.Results: We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between –1.17 to –26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5’-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×106 spermatozoa could trap almost 80% from the suspension.Conclusion: The binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

Heat Shock Protein (HSP) 90 gene is a member of HSPs sub-family that act as molecular chaperons whenever animals come under thermal stress. The genes fulfill essential roles of providing cellular protection, immune response, protein synthesis, protein folding and unfolding, protection from cellular stress, inhibitory apoptosis and adaptation. This study was designed to analyze polymorphisms of HSP 90 and to evaluate their influence on heat tolerance among selected Nigerian zebu. The polymorphisms were also used to determine genetic relationship among the animals. About 450 bp of bovine HSP 90 including part of coding region in exon 3 was sequenced in 90 DNA samples representing four Nigerian zebu namely White Fulani (WF), Sokoto Gudali (SG), Red Bororo (RB) and Ambala (AM). Sequencing was done using an automated ABI-DNA Sequencer. Editing was accomplished using chromatogram analyses on SeqMan Ngen Tool. Rooted phylogenetic tree was constructed using MEGA 5.2 software. In total, 11 genetic variants were determined. Five of these (PRP, RED, ORG, LMN and YLO) were major variants detected in over 70% of the samples. Six (6) were classified as minor variants detected in two breeds or less and in 29.1% of the samples. The GRN and NBL were only detected in RB and SG breeds respectively. We found a shared homology and common ancestral lineage among the breeds. Furthermore, the genetic structure of Nigerian zebu has a common clade architecture to those of goats, sheep, yak, buffalo, camel, horse and other taurines. The gene is conserved among wide range of animals and as such it can serve as one of bio-markers for selection and breeding programmes for thermo-tolerance in wide range of livestock animals under thermal stress. The variant groups could be further interrogated for possible specific effects on thermo-tolerance performance of zebu in hot tropical environments.

Background: Campylobacter species are a leading cause of most important food-borne diarrhoeal illness worldwide while, poultry has been identified as a significant cause of Campylobacter infection in humans. C. jejuni is highly effective in colonizing chicken intestinal mucosa without causing any clinical manifestations and the consumption of poultry meat is the major source of transmission of bacteria to humans. Methods: The total of 19 chicken meat samples collected from retail markets in Chennai were screened by cultural examination, further subjected to phenotypic characterization using biochemical test and genotypic characterization using polymerase chain reaction assay targeting hip O and map A genes. Result: All the isolates showed growth on modified blood free charcoal cefoperazone deoxycholate agar media (mCCDA) and 18 (94.73%) samples showed typical morphological characteristics. The 12 (63.15%) isolates showed biochemical reactions positive. The results from polymerase chain reaction showed that 10 (83.33%) isolates were positive for C. jejuni. This study suggested that, it is essential to investigate the incidence of Campylobacter jejuni infection in poultry and the risk factors at all production stages of meat production to help reducing the disease in humans in terms of food safety.

Background: Campylobacter has emerged as an important zoonotic food borne pathogen of human and animals worldwide. Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. There are very few cases reported from mastitis therefore this study was aimed to determine the incidence of Campylobacter jejuni from from mastitis milk and raw milk samples. Methods: Total of 72 milk samples comprising mastitis milk (20) and raw milk (52) were collected. The samples were subjected to cultural examination, biochemical as well as molecular identification. The isolates were further subjected to phenotypic characterization by biochemical test and genotypic characterization by Polymerase Chain Reaction. The isolates were subjected to PCR targeting hip O and MAP A genes. Result: The 52 samples showed growth on modified Blood Free Charcoal Cefoperazone Deoxycholate agar media and 18 (34.61%) samples showed typical morphological characteristics. The result revealed that 10 (19.23%) isolates were positive by phenotypic characteristic and 7(70%) by Polymerase chain reaction for C. jejuni. The outcome result showed that importance of Campylobacter jejuni in cattle, especially raw milk and milk from mastitis cows, as a potential source for transmission of Campylobacteriosis in human and dairy farm environment. This can cause acute bacterial gastroenteritis in humans and associated with foodborn infection, food safety and a serious public health threat.

Background: Infectious virus antigen is not recommended for disease monitoring in global Peste des petits ruminants eradication strategies. Native virus antigens are gradually being replaced with recombinant or synthetic peptide antigens. The focus of the present study is to optimize and develop peptide-based immunoassay for the detection of antibodies to PPRV N and H proteins. Methods: Epitopes of PPRV H and N proteins were selected based on prediction with bioinformatics tools and from previous studies. Two peptides each were synthesized for N and H proteins and peptide ELISA developed. The peptide ELISA’s sensitivity and specificity were tested with sera samples collected at different time intervals of vaccination (goat =73, sheep= 62) and 88 random serum samples (goat =47, sheep=41). The collected sera were screened using cELISA before proceeding to peptide ELISA. Result: In competitive ELISA, 106 goat serum samples and 96 sheep serum samples were found to be positive. Fourteen goat serum samples and seven sheep serum sample were shown to be negative. Among120 goat serum samples tested, 114 were found to be positive by peptide ELISA. Similarly, out of 103 sheep serum samples analyzed, 96 were found to be positive with peptide ELISA. The peptide ELISA based on the highly conserved and antigenic N and H epitope detected antibodies to PPRV in precise manner. This study demonstrated the effective use of synthetic peptides as an antigen in the detection of antibodies to PPRV.

An attempt was made to assess the effects of aging, regional differences, and draught load applied on draught potential of Umblachery cattle, an important draught breed of South India. Age had a highly significant effect (p less than 0.01) on all morphometric traits, stride length, and significant effect (p less than 0.05) on pulse rate after work. The middle age group (5.0 to 7.5 years) with more substantial stride length was identified as the critical productive age group for draught ability. Regional differences had a highly significant (p less than 0.01) influence on stride length, horsepower, and a significant effect (p less than 0.05) on pulse rate after work. The optimum draught load with which Umblachery breed could give uniform and maximum power output was found to be around 75 to 78 kg.

Infectious laryngotracheitis (ILT), caused by Gallid herpesvirus1, is one among the respiratory diseases of poultry gradually spreading worldwide, including Indian subcontinent. Present study was carried out to identify the pathogen from the suspected cases of the disease. The tracheal tissue samples (pooled) were collected from the birds suspected to have died of ILT from 26 commercial poultry farms located in and around Namakkal district of Tamil Nadu state of India. On post-mortem examination, haemorrhagic caseous exudate and fibrinous pseudo-membrane adhered to tracheal mucosa were noticed. A total of 22 farms were found positive through PCRs targeted against ICP4 gene and the thymidine kinase (TK) gene followed by confirmation through sequencing. Histopathology examination revealed decilliation, hyperplasia, degeneration and/or loss of tracheal epithelium with severe submucosal edema. There was infiltration with numerous lymphocytes, macrophages and plasma cells in milder infections, whereas presence of fibrinous exudates admixed with numerous erythrocytes and inflammatory cells like heterophils and lymphocytes were seen indicating the severe acute form of the disease. A fibrinous pseudomembrane was seen firmly attached to the inflamed and necrotic mucosa in subacute cases. Further, virus was isolated from randomly selected 5 PCR positive tracheal tissue samples in embryonated chicken eggs through chorioallantoic membrane (CAM) route. The typical pock lesions were observed on CAM along with engorged blood vessels and thickening of the membrane. Present study has reported the disease ILT among poultry flocks in the Namakkal district of Tamil Nadu and raises the concern for thorough investigation of the nature of prevailing pathogen in the region.

Vector borne pancytopenia is emerging as a life threatening entity in animals. In India babesiosis is one among the most prevalent tick-borne parasitic diseases of dogs caused by either Babesia gibsoni or Babesia canis. Bleeding due to thrombocytopenia and the concurrent anaemia and leukopenia were difficult to manage. This study assessed the efficacy of Filgrastim in pancytopenia associated with Babesia gibsoni in dogs presented to the Small Animal Medicine Referral Clinic, Madras Veterinary College. The therapeutic practices included Injection Filgrastim @ 10 μg/kg, SC, SID in combination with the standard triple therapy to manage the pancytopenia and the infection. Twenty numbers of PCR positive Babesia gibsoni dogs were used for this study. The animals were divided in to two groups based on therapeutic practices. First group consisted of dogs treated with triple therapy and the second group consisted of dogs treated and evaluated with Filgrastim along with triple therapy. The study showed that there was a significant increase in leukocyte count in Filgrastim treated group when compared to the other group. Integration of G-CSF along with standard triple therapy helped in better survival in pancytopenic dogs with Babesia gibsoni.

Newcastle disease (ND) is a major risk to the poultry industry which results in severe economic loss throughout the world even with vaccination. The vaccine viruses that are used in many countries include the LaSota and other live viruses that were isolated in the early and late 1950s. Reports from several laboratories including ours indicate a greater variance of the circulating strains and recent classification indicates the existence of XVIII different genotypes of NDV strains. The efficiency of the LaSota vaccination in inducing protective immunity to different heterologous strains has been a question and its efficacy upon exposure to a virulent genotype IV strain has not been reported after 1989 world-wide except for India. Serum antibody negative (SAN) chicks of either sex obtained by hatching specific-pathogen-free (SPF) eggs were vaccinated with increasing doses of the vaccine virus from 101 to 107 EID50 per bird delivered through occulo-nasal route and challenged 20 days later with NDV-2K3 (genotype IV) strain of virus isolated in the year 2000 from pigeon in India. The birds were monitored for serum antibody titers and following challenge for morbidity, mortality, viral load in the cloacal swab and different tissues. We could clearly show that a minimum vaccine titre of 104 EID50 could establish protective antibody levels and also prevent viral replication post challenge upon exposure to the virulent genotype IV strain. We conclude based on our results and previous observation that there do exist differences in the levels of the antibody that could limit viral replication and shedding upon exposure to different heterologous genotype of NDV. Developing a strain matched vaccine might less potential to result in better protection by limiting the viral shedding.

A cross sectional study was conducted to determine the dynamic prevalence of bovine tuberculosis amongst bovine using single intra dermal cervical comparable test (SICCT or CIDT) in North-Eastern agro-climatic zone of Tamil Nadu. In the present study, Out of 1119 blood samples tested from four selected districts of North-Eastern agro-climatic zone of Tamil Nadu, the overall prevalence of M. bovis infection in North-Eastern agro-climatic zones of Tamil Nadu was found to be 8.46 per cent by CIDT and highest prevalence was noticed in Vellore (10.86%) district followed by Kancheepuram (8.20%), Thiruvallur (7.92%) and Chennai (6.86%) districts, which shows the potential to increase the incidence as well as prevalence of the disease since the “test and slaughter” or “culling policy” are yet to be strengthened intensively or warranting the calf-hood immunization against tuberculosis.

The NOD-like receptors (NLRs) are a group of intracellular germline encoded innate immune receptors that recognize conserved microbial structures in various invasive pathogens. Atleast 23 different NLRs have been identified with CARD15 being the major NLR playing an important role in the co-ordination of adaptive immune response induced by the CARD15 agonist Muramyl dipeptide (MDP). CARD15 has been reported to confer resistance or susceptibility to Crohn's disease in humans and Johne's disease in animals. We amplified and sequenced the major region of the CARD15 gene (3042 bp) from three native goat breeds (Barbari, Tellicherry and Kanni), screened for polymorphisms and correlated the expression levels of MDP induced CARD15 mRNA and downstream cytokines with that of the polymorphisms. The different exons that contribute to the LRR domain of the CARD15 gene were screened from a 3 breed panel with twenty un-related animals each. Four polymorphisms were located in the coding region of the gene (one each in exon 5 and 10 and two in exon 6) and one in the non-coding region of the gene (Intron 5–6). Two nonsynonymous substitutions R2441H (in Barbari and Tellicherry) and A2492G (in Tellicherry and Kanni) were observed in exons 5 and 6 respectively. The major genotype in exon 5 was GG across Barbari, Tellicherry and Kanni. The polymorphism at position 2492 with respect to mRNA in exon 6 revealed the major genotypes GG, CC and GC in Barbari, Tellicherry and Kanni respectively. Upon MDP stimulation, there was no significant change in the expression levels of CARD15 and TNF-α mRNA across the three breeds while the IL1-β mRNA levels were significantly higher in Kanni breed of goat. The results provide us some basic data on the genotypes and the polymorphisms in the CARD15 gene in native breeds of goat. Future studies with an increased sample number, screening for paratuberculosis and understanding the host genetic background would help in disease association.

Peste des petits ruminants virus (PPRV) is known to replicate in a wide variety of ruminants causing very species-specific clinical symptoms. Small ruminants (goats and sheep) are susceptible to disease while domesticated cattle and buffalo are dead-end hosts and do not display clinical symptoms. Understanding the host factors that influence differential pathogenesis and disease susceptibility could help the development of better diagnostics and control measures. To study this, we generated transcriptome data from goat and cattle peripheral blood mononuclear cells (PBMC) experimentally infected with PPRV in-vitro. After identifying differentially expressed genes, we further analyzed these immune related pathway genes using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and selected candidate genes were validated using in-vitro experiments. Upon PPRV infection, we identified 12 and 22 immune related genes that were differentially expressed in goat and cattle respectively. In both species, this included the interferon stimulated genes (ISGs) IFI44, IFI6, IFIT1, IFIT2, IFIT3, ISG15, Mx1, Mx2, OAS1X, RSAD2, IRF7, DDX58 and DHX58 that were transcribed significantly higher in cattle. PPRV replication in goat PBMCs significantly increased the expression of phosphodiesterase 12 (PDE12), a 2&prime, 5&prime, oligoadenylate degrading enzyme that contributes to the reduced modulation of interferon-regulated gene targets. Finally, a model is proposed for the differential susceptibility between large and small ruminants based on the expression levels of type-I interferons, ISGs and effector molecules.

Background The caecal microbiota plays a key role in chicken health and performance, influencing digestion and absorption of nutrients, and contributing to defence against colonisation by invading pathogens. Measures of productivity and resistance to pathogen colonisation are directly influenced by chicken genotype, but host driven variation in microbiome structure is also likely to exert a considerable indirect influence. Methods Here, we define the caecal microbiome of indigenous Indian Aseel and Kadaknath chicken breeds and compare them with the global commercial broiler Cobb400 and Ross 308 lines using 16S rDNA V3-V4 hypervariable amplicon sequencing. Results Each caecal microbiome was dominated by the genera Bacteroides, unclassified bacteria, unclassified Clostridiales, Clostridium, Alistipes, Faecalibacterium, Eubacterium and Blautia. Geographic location (a measure recognised to include variation in environmental and climatic factors, but also likely to feature varied management practices) and chicken line/breed were both found to exert significant impacts (p

The incidence of resistant mastitis in dairy cows in Tamil Nadu, India was 56.l %. The predominant resistant causative pathogen was Escherichia coli (50.64 %) followed by Staphylococcus aureus (44.25 %) and Methicillin resistant Staphylococcus aureus (5.11 %). Incidence of resistant mastitis was high in Holstein Friesian cross breed followed by Jersey cross breed and non descript. Highest incidence was observed in early stage of third lactation. In vitro antibiotic sensitivity test revealed the E. coli, S. aureus and MRSA organisms showed more sensitivity to enrofloxacin, amoxicillin+sulbactam, gentamicin and ceftriaxone and had highest resistant to penicillin followed by amoxicillin, oxytetracycline and methicillin. The study highlights the need for preventing the indiscriminate use of antibiotics.

Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages. Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem in dairy animals suffering from mastitis. The study was carried out to evaluate the incidence of Methicillin resistant S. aureus from clinical mastitis milk samples and their antibiotic resistance profile and characterised with respect to the molecular features that contributed to the resistance in these pathogens. Isolation and identification of Methicillin resistant S. aureus were performed from acute clinical mastitis samples. The isolates were tested using agar disc diffusion method for their antimicrobial susceptibility and modified resazurin assay micro dilution technique for MIC to 8 different antimicrobial drugs. A total of 235 clinical mastitis milk samples from dairy cows were cultured for incidence of S. aureus. Methicillin resistant S. aureus was isolated from a total of 12 (44.25%) of the 116 S. aureus samples. Based on the antimicrobial sensitivity and MIC results, MRSA isolates were found sensitive to gentamicin, enrofloxcain, amoxicillin+sulbactam, ceftriaxone and resistant to amoxicillin, oxytetracycline, penicillin G and oxacillin. Most of MRSA isolates were found to be multi-drug resistant. MRSA alert kit test and mecA and blaZ target gene PCR were found to be useful in the confirmation of MRSA.

The study was undertaken to determine the prevalence and treatment of antibiotic resistant mastitis in dairy cows. The predominant resistant causative pathogen was Escherichia coli (50.64 %) followed by S. aureus (44.25 %) and Methicillin resistant Staphylococcal aureus (5.11%).These isolates were found sensitive to gentamicin, enrofloxcain, amoxicillin+sulbactam, ceftriaxone and resistant to amoxicillin, oxytetracycline, penicillin G and oxacillin. In all the treatment groups of E. coli, S. aureus and MRSA mastitis, the post treatment pH, SCC was significantly (P < 0.01) decreased when compared to pre treatment pH, SCC values and the post treatment electrical conductivity was significantly (P < 0.01) increased when compared to pre treatment electrical conductivity value. In E. coli mastitis, treated with amoxicillin+sulbactam, ceftriaxone, enrofloxacin and gentamicin showed 74.1%, 67.75 %, 76.67 % and 64.52 % clinical recovery and in S. aureus mastitis, showed 65.25 %, 65.25 %, 72.43 % and 68.98 % clinical recovery. In MRSA mastitis, enrofloxacin was found to be highly effective in comparison to amoxicillin+sulbcactam.

Aim: To study the prevalence of drug resistant mastitis and their pattern of antibiotic resistance in dairy cows from Tamil Nadu. Materials and Methods: Isolation and identification of resistant pathogens were performed from acute clinical mastitis samples. Based on culture, isolation and sensitivity tests, cows with resistant mastitis were grouped as; Group I: Escherichia coli (n=119), Group II: Staphylococcus aureus (n=104) and Group III: Methicillin-resistant Staphylococcal aureus (MRSA) (n=12). The isolates were tested using agar disc diffusion method for their antimicrobial susceptibility and modified resazurin assay microdilution technique for minimum inhibitory concentration (MIC) to 8 antimicrobial drugs. The organisms were also confirmed for their identity by performing PCR on the bacterial pellet targeting the specific genes such as 16s-23s rRNA, mecA and blaZ respectively for the resistant pathogens and also confirmed by sequencing. Results: Antibiotic resistant mastitis was detected in 235 out of 401 cows accounting to 56.1%. The predominant resistant causative pathogen was E. coli (50.64%) followed by S. aureus (44.25%) and MRSA (5.11%). In vitro antibiotic sensitivity test and MIC breakpoints, E. coli, S. aureus and MRSA organisms showed more sensitivity to enrofloxacin, amoxicillin + sulbactam, gentamicin and ceftriaxone and had highest resistant to penicillin followed by amoxicillin, oxytetracycline and methicillin. E. coli and S. aureus isolates were found to be resistant to 1 or 2 antimicrobials, whereas most of the MRSA isolates were found to be multi-drug resistant i.e resistance to 3 or more of antimicrobials. Out of 235 milk samples, the specific target gene 16s-23s rRNA (E. coli ), 16s-23s rRNA (S. aureus) and MRSA (mecA and blaZ) could be amplified from 119, 104 and 12 isolates with a percentage positivity of 50.64 (119/235), 89.64 (104/116) and 10.34 (12/116) respectively. Conclusion: Prevalence of antimicrobial resistance (AMR) in bovine mastitis pathogens was high. Most MRSA pathogens were multidrug resistant. E. coli and S. aureus isolates were resistant to few antimicrobials.

The polymorphism of A781G locus of the growth hormone (GH) gene was analyzed in 112 Vembur sheep. HaeIII – RFLP of the GH gene (422 bp) revealed the existence of two genotypes viz., AA (366 and 56 bp) and AB (422, 366 and 56 bp), and two alleles viz., A and B. Genotypic frequencies for AA and AB were 0.43 and 0.57, and allelic frequencies were 0.71 and 0.29 for A and B respectively. BB genotype was absent. Highly significant Chi-square value (P

Bluetongue is an infectious disease caused by bluetongue virus (BTV), which affects sheep, goat, cattle and certain wild ruminants. However severe clinical signs are usually seen with significant mortality in sheep than cattle and goat. To date, comparative studies on innate immune responses of sheep and goat infected with BTV is lacking. In this study, we compared the innate immune response of sheep and goat by infecting the peripheral blood mononuclear cells (PBMCs) with BTV serotype 23. In our study, we observed that sheep PBMCs supports higher virus replication than goat PBMCs. To delineate the role of innate immune response in differential viral replication observed in this study, we examined TLR3 (Receptor for dsRNA virus) mRNA expression and cytokine profiles (IL-1β, Il-6, IL-8, Il-10, IL-12p40, TNF-α, IFN-γ and IFN-α) following Poly I:C (TLR3 ligand) stimulation and BTV 23 infection. In our present study, sheep PBMCs had significantly higher TLR3 mRNA levels, TLR3 specific ligand (Poly I:C) stimulation resulted in increased levels of IFN-γ at transcriptional and translational levels along with IL-8 and IL-10 at transcriptional levels. Whereas, the levels of TNF-α was higher in goat PBMCs at transcriptional levels. BTV infected sheep PBMCs expressed significantly higher levels of IFN-γ at transcriptional and translational levels along with IL-6, IL-8 and IL-10 at transcriptional levels. Whereas the expression levels of TNF-α and IFN-α at transcriptional and translational levels were significantly high in goat PBMCs. To examine the potential factor for consistent increase in the expression of TNF-α, we sequenced the promoter region of TNF-α and identified a total of five single nucleotide polymorphisms (SNP) and one indel in goat TNF-α promoter region. Luciferase assay for transcriptional activity of the promoter showed that goat TNF-α has significantly enhanced transcriptional activity in comparison with sheep TNF-α promoter. Altogether, our data suggests that the expression levels of TNF-α and IFN-α and/or IL-10 plays crucial role in replication of BTV 23.

Toll-like receptors (TLRs) are transmembrane receptors composed of extra cellular leucine rich repeats (LRRs) that identify specific pathogen associated molecular patterns triggering a innate immune cascade. The LRR regions of TLR 1–10 proteins of goat (Capra hircus), sheep (Ovis aries), buffalo (Bubalus bubalis) and bovine (Bos taurus) were modeled using MODELLER 9v7 tool and validated. The similarities and variations of these 10 TLRs extracellular regions of each species were compared using online servers like FATCAT, SSM and SSAP. It was evident that the LRRs of TLRs like 1, 2, 3 and 6 showed structural convergence with

The objective of this study was to assess cytokine production upon activation of pattern recognition receptors responsible for sensing bacterial and viral pathogen associated molecular patterns in two genetically diverse buffalo breeds, Toda and Murrah. A very limited molecular-epidemiological analysis showed a higher prevalence of Anaplasma and Theileria in Murrah than Toda buffaloes. Toda buffalo peripheral blood mononuclear cells (PBMC) produced significantly higher levels of IFN γ and/or TNF α mRNAs in response to peptidoglycan, poly I:C, lipopolysaccharide, imiquimod and CpG. Flagellin stimulation did not result in any significant differences in the expression levels of the cytokines tested between these breeds. The levels of ligand induced IFN γ and TNF α mRNA and proteins also correlated except when induced with CpG. The proximal promoter region of TNF α across these two breeds were also sequenced to detect SNPs and promoter assay performed to determine their role in altering the transcriptional activity. Two polymorphisms were identified at −737 (T/A) and −1092 (G/T) positions in Toda buffalo TNF α promoter and promoter assay revealed higher transcription activity in Toda buffalos than in Murrah. This suggests that disease tolerance of these buffalo breeds could be due to the differences in their cytokine transcription levels in response to the respective PAMPs that may be at least in part determined by polymorphisms in the cytokine promoter regions.

Sharks are a species of delight for immunologists from the evolutionary perspective since it is considered as the first species to have evolved the adaptive immune responses in addition to the innate immune system. One of the components of the highly conserved innate immune system is the toll-like receptors (TLR) which has a conserved overall protein structure throughout deuterostome evolution. There is no report that demonstrates the expression of these receptors in sharks. In this study we successfully amplified a 270 bp amplicon using a degenerate primer design strategy that corresponded to the Toll/IL-1 receptor (TIR) domain of TLR2 (GenBank ID: JF792813 ). BLAST analysis revealed a maximum nucleotide identity of 87% and 76% with the TLR2 of higher mammals and teleost fishes respectively. Domain prediction revealed a TIR structure between 1 and 87 amino acids that had a maximum identity of 58% and 76% with TLR2 – TIR protein of teleost fishes and higher mammals respectively. Phylogenetic analysis revealed a closer clustering of the shark TIR sequence with those from human, cattle, goat, sheep and chicken than with other fish species. Basal expression levels of the TLR2-TIR mRNA were found to be significantly higher in kidneys followed by fins, spleen and intestinal spiral valve (ISV). In tissues such as spleen and kidney the expression of the TLR2-TIR mRNA could be localized to lymphoid and macrophages like cells and tubular epithelial cells respectively. In-vivo exposure of sharks to peptidoglycan (TLR 2 ligand) resulted in 9 folds higher expression of TLR2-TIR mRNA in gills followed by 5 folds in the fins. However, when inoculated with a TLR ligand pool, the expression levels significantly increased to 12 fold in skin followed by epigonal, kidneys and ISV. These findings not only support the presence of the TLRs in sharks but also their induction upon exposure to specific ligands. Further studies are needed to identify their numbers, their ligand specificity and downstream cytokine responses.

The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.

Newcastle disease virus (NDV) has been a threat to poultry industry in most of the developing countries with a wide variety of avian species being susceptible, coupled with the presence of mobile wild bird reservoirs contributing not only to the vast genomic diversity of this virus but also to the diagnostic failures. NDV of multiple genotypes (I–XI) is known to be prevalent and reported worldwide. However, there is a paucity of information on the circulating genotypes of NDV in India. This study utilized the fusion protein cleavage site (FPCS) sequence to determine the different genotypes of NDV circulating in India. Our results indicate that majority of NDV isolates from southern states of India namely, Tamil Nadu, Kerala and Karnataka were found to belong to genotype II. However, some of the strains from Tamil Nadu and most from Uttar Pradesh belong to genotype groups VI and VII. Interestingly, three isolates recovered from Tamil Nadu grouped with genotype IV viruses (namely Herts/33) which had not been hitherto reported to GenBank since 1989. This preliminary information points to the existence of multiple genotypes and also the need for efficacy studies with vaccines incorporating multiple genotypes in controlling virulent NDV (vNDV) outbreaks in India.

Purpose The present study was carried out to identify and assess the expression levels of toll-like receptors (TLRs) 1–10 mRNA in corneal epithelial cells of buffalo, goat, sheep and bull. Materials and methods The globes from the respective species were collected and the corneal epithelium was denuded. The expression levels of the different TLR mRNAs were assessed by densitometry of the band intensities following reverse transcriptase polymerase chain reaction (RT-PCR). Results In all species TLR5 and TLR7 were highly expressed with lower levels of TLR10. The TLR6 mRNA levels were the lowest in the cornea of bull. The TLR2 expression was high in sheep and bulls, while TLR4 mRNA was higher in sheep alone. The level of the other TLRs varied across other species. Conclusion Based on these observations it may be inferred that these animals would be resistant to flagellin (TLR5) and single-stranded RNA viruses (TLR7), while sheep alone may show more resistance to lipo-polysaccharide (TLR4) and peptidoglycan (TLR2)-mediated injury of the cornea. This work reports for the first time the expression of TLR mRNAs in the corneal epithelial cells of farm animals. The relationship of the TLR mRNA levels to corneal disease susceptibilities and pathogenicity remains to be explored further.

This chapter introduces the development of the polymerase chain reaction (PCR) and its modifications that enabled the development of in-situ PCR and its successful application in localizing RNA and DNA targets. We provide detailed descriptions of methods and materials for successful demonstration of rabies virus (RABV) nucleic acid by in-situ PCR. The essential components of this topic include sample preparation, including care during pre-treatment, in-situ reverse transcription (RT) PCR amplification, and detection of target genes. The parameters pertaining to critical steps in the procedure are discussed.

Genetic variation in a part of growth hormone (GH) gene was studied in Vembur and Kilakarsal breeds of sheep. Two regions of GH gene viz., 214 bp fragment (includes part of intron 3, exon 4 and part of intron 4) and 365 bp fragment (includes part of intron 4, exon 5 and part of 3'UTR) were studied. DNAsequencing of GH gene revealed absence of SNPs in exons 4 and 5. It showed a T→C transition in Vembur breed of sheep in the 3 ’untranslated region of growth hormone gene. Genotyping of T→C transition mutation (T1965C) was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique using EcoP15I endonuclease and found to be polymorphic in Vembur sheep. Further studies are required to investigate the relationship between GH gene polymorphisms and the performance traits in these breeds.

The genome of a Newcastle disease virus isolated from a Japanese quail in 2003 is reported here. The genome is 15,192 nucleotides (nt) long, as found in the recent genotypes, and grouped as genotype VIIb, with a 6-nt insertion. This is the first report on the sequence of a genotype VII Newcastle disease virus (NDV) from India.

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3′ leader and 5′ trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have “died out” after the first panzootic (1926–1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India. Published version

This study involved cloning and sequencing of the coding regions of all 10 Toll-like receptor (TLR) genes of goat. Goat TLR 1-10 gene sequences revealed a high degree of nucleotide identity with sheep and cattle sequences (>90%) and 75-85% with pig, mouse and human sequences. At the amino acid level, 85-99% similarity was observed with sheep and cattle and 60-85% with pig, mouse and human. TLR9c DNA of goat showed the highest amino acid identity to that of sheep (99%) while TLR8 cDNA showed the lowest identity of 88.7% to that of sheep. Variations were seen in the number of leucine rich repeats (LRRs) of goat TLRs as compared to other ruminant species with maximum differences in the TLR3 gene. Phylogenetic analysis through molecular evolution and genetic analysis (MEGA) software and multi dimensional scaling revealed a high degree of conservation of goat TLRs with those from other species. However when the TIR domain of all the TLRs were compared, goat TLR7 TIR alone showed a high divergence of 19.3 as compared to sheep sequences. This is the first report of the full-length cDNA sequences of all the 10 TLR genes of goats which would be a useful tool for the study of evolutionary lineages and for phylogenetic analysis.

This report describes Newcastle disease in peacock and the isolation and characterization of the virus. The virus had an intracerbral pathogenicity index of 1.71 and mean death time of 47 h. The isolate had multiple basic amino acids at the fusion protein cleavage site sequence ((110)GGRRQRRFIG(119)) with a phenylalanine at residue 117. Biological and molecular characterization revealed that the virus is velogenic. Phylogenetic analysis placed the isolate in genotype II.

Background CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene. Methods We cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies. Results TNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to some of the putative cd38 GREs by dexamethasone. Conclusion The EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-κB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.