Bluetongue Risk Map for Vaccination and Surveillance Strategies in India.

Bluetongue virus (BTV, Sedoreoviridae: Orbivirus) causes an economically important disease, namely, bluetongue (BT), in domestic and wild ruminants worldwide. BTV is endemic to South India and has occurred with varying severity every year since the virus was first reported in 1963. BT can cause high morbidity and mortality to sheep flocks in this region, resulting in serious economic losses to subsistence farmers, with impacts on food security. The epidemiology of BTV in South India is complex, characterized by an unusually wide diversity of susceptible ruminant hosts, multiple vector species biting midges (Culicoides spp., Diptera: Ceratopogonidae), which have been implicated in the transmission of BTV and numerous co-circulating virus serotypes and strains. BT presence data (1997-2011) for South India were obtained from multiple sources to develop a presence/absence model for the disease. A non-linear discriminant analysis (NLDA) was carried out using temporal Fourier transformed variables that were remotely sensed as potential predictors of BT distribution. Predictive performance was then characterized using a range of different accuracy statistics (sensitivity, specificity, and Kappa). The top ten variables selected to explain BT distribution were primarily thermal metrics (land surface temperature, i.e., LST, and middle infrared, i.e., MIR) and a measure of plant photosynthetic activity (the Normalized Difference Vegetation Index, i.e., NDVI). A model that used pseudo-absence points, with three presence and absence clusters each, outperformed the model that used only the recorded absence points and showed high correspondence with past BTV outbreaks. The resulting risk maps may be suitable for informing disease managers concerned with vaccination, prevention, and control of BT in high-risk areas and for planning future state-wide vector and virus surveillance activities.

Comparative analysis of tuberculin and defined antigen skin tests for detection of bovine tuberculosis in buffaloes (Bubalus bubalis) in Haryana state, India.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic disease that results from infection with any member of the Mycobacterium tuberculosis complex. Infected animals are typically diagnosed with tuberculin-based intradermal skin tests according to World Organization of Animal Health which are presently in use. However, tuberculin is not suitable for use in BCG-vaccinated animals due to a high rate of false-positive reactions. Peptide-based defined skin test (DST) antigens have been identified using antigens (ESAT-6, CFP-10 and Rv3615c) which are absent from BCG, but their performance in buffaloes remains unknown. To assess the comparative performance of DST with the tuberculin-based single intradermal test (SIT) and the single intradermal comparative cervical test (SICCT), we screened 543 female buffaloes from 49 organized dairy farms in two districts of Haryana state in India. RESULTS: We found that 37 (7%), 4 (1%) and 18 (3%) buffaloes were reactors with the SIT, SICCT and DST tests, respectively. Of the 37 SIT reactors, four were positive with SICCT and 12 were positive with the DST. The results show that none of the animals tested positive with all three tests, and 6 DST positive animals were SIT negative. Together, a total of 43 animals were reactors with SIT, DST, or both, and the two assays showed moderate agreement (Cohen's Kappa 0.41; 95% Confidence Interval (CI): 0.23, 0.59). In contrast, only slight agreement (Cohen's Kappa 0.18; 95% CI: 0.02, 0.34) was observed between SIT and SICCT. Using a Bayesian latent class model, we estimated test specificities of 96.5% (95% CI, 92-99%), 99.7% (95% CI: 98-100%) and 99.0% (95% CI: 97-100%) for SIT, SICCT and DST, respectively, but considerably lower sensitivities of 58% (95% CI: 35-87%), 9% (95% CI: 3-21%), and 34% (95% CI: 18-55%) albeit with broad and overlapping credible intervals. CONCLUSION: Taken together, our investigation suggests that DST has a test specificity comparable with SICCT, and sensitivity intermediate between SIT and SICCT for the identification of buffaloes suspected of tuberculosis. Our study highlights an urgent need for future well-powered trials with detailed necropsy, with immunological and microbiological profiling of reactor and non-reactor animals to better define the underlying factors for the large observed discrepancies in assay performance, particularly between SIT and SICCT.

Parasitic pneumonia caused by Paragonimus spp. in a wild Royal Bengal Tiger, Mysuru, South India.

Parasitic pneumonia induced by genus Paragonimus involves many species, which affects both humans and animals and it is a food borne zoonotic disease. In this report, we have described the gross and histopathological findings of Paragonimus fluke infection in lungs of tiger. The postmortem examination of sub adult male wild tiger (Panthera tigris tigris) died in captivity was conducted, earlier which was rescued by Forest Department, Mysuru, Karnataka, India. External examination of carcass revealed pale oral and conjunctival mucous membranes with sunken eye balls. During necropsy, moderate congestion, consolidation and numerous transparent to dark encysted lesions were found in the parenchyma of all lobes of lungs visible grossly on pleural surface. Lungs were hemorrhagic with necrotic foci around the cysts. The incision of encysted lesions revealed the presence of flukes (2-3 in numbers) in each cyst with brownish exudate. The lung tissues with lesions were collected in 10% formalin and haematoxylin and eosin staining was done for histopathological evaluation. The flukes were identified as Paragonimus spp. based on the morphology and micrometry. The histopathological examination revealed presence of longitudinal sections of flukes in bronchial lumen (in pair) with tegument and tegumental spines surrounded by connective tissue capsule as cystic encapsulation and numerous eggs in adjacent lung parenchyma. Necrosis and moderate fibrosis of lung parenchyma with infiltration of polymorphonuclear and mononuclear inflammatory cells were observed around fluke as well as eggs. The squamous cell metaplasia of lining bronchial epithelium and atelectasis of alveoli were also prominently seen.

Survivin expression in canine spontaneous cutaneous and subcutaneous tumors and its prognostic importance.

AIM: The present study was carried out to know the expression level of survivin, an inhibitor of apoptosis protein with an objective to determine its prognostic importance in cutaneous and subcutaneous tissue tumors of dogs. MATERIALS AND METHODS: Forty cases of canine cutaneous and subcutaneous tissue tumors on histopathological examination revealed various round cell, epithelial, and mesenchymal cell tumors. Survivin gene expression was detected in all tumors tested by TaqMan real-time polymerase chain reaction assay by comparative cycle threshold method. RESULTS: The mean survivin gene expression value of benign tumors was 0.94±0.63 folds and that of malignant tumors was 18.87±5.30 folds. Postsurgical follow up of 30 malignant tumor cases revealed death in 8, recurrence in 7, and neoplastic free alive status in 15 dogs with mean survivin fold difference values of 48.49±12.39, 14.63±6.37, and 5.034±2.27, respectively. The mean survivin gene expression value was significantly higher in malignant (30 cases, 18.87±5.30) compared to benign tumors (10 cases, 0.94±0.63), and it varied between various postsurgical follow-up groups (p<0.05). Survival analysis, using survivin gene expression median cutoff value of 3.74 in 30 malignant tumors, was performed to predict probable survival period in malignant cutaneous and subcutaneous tumors of dogs. CONCLUSIONS: Results of the present study indicated that the expression of survivin in canine cutaneous and subcutaneous tumors has prognostic value, and survivin expression greater than median cutoff value of 3.74 has a poor prognosis.

Studies on occurrence, characterisation and decontamination of emerging pathogenic Escherichia coli (STEC, ETEC and EIEC) in table eggs.

1. Escherichia coli is one of the most common facultative anaerobic species present in the gastrointestinal tract of animals and human beings. Usually they occur as commensals, but some serotypes can cause significant illnesses in humans as well as mammals and birds. 2. The occurrence of E. coli in different categories of table eggs collected from markets was evaluated. Isolates were analysed for the presence of virulence genes, antibiotic susceptibility pattern and efficacy of peracetic acid and chlorine for the purpose of decontaminating table eggs. 3. Significant differences were observed in the occurrence of E. coli between different groups viz. processed (cleaned, washed, sanitised and packed eggs), unprocessed (un-cleaned, un-sanitised and loose eggs) and free range (eggs obtained from backyard poultry) table eggs. Overall, E. coli occurred in table eggs at 28.6% with 22.9, 29.2 and 50.0% occurrence in processed, unprocessed and free-range table eggs, respectively. 4. A total of 24 isolates of E. coli were obtained and screened for virulence genes viz. STH, SLT1/2 and INVE genes. Of the 24 isolates recovered, 10 typeable isolates belonged to O141, O119, O9, O120 and O101 serotypes, while the remaining 14 were untypeable. Antibiograms of the isolates showed multiple antimicrobial resistance (MAR) index in the range of 0.13-0.40. 5. Peracetic acid (PAA) and chlorine (CL) were studied for their sanitisation efficacy; concentrations of 100 mg/kg of PAA and 200 mg/kg of CL completely inactivated E. coli over the egg surface and also resulted in 2.58 and 2.38 log reduction in total viable counts (TVC), respectively. 6. The presence of virulence-associated shiga-like toxin (SLT1/2) and invasion E (INVE) genes and antimicrobial resistance among the emerging serotypes of pathogenic E. coli isolated from table eggs has public health implications. It underscores the need to implement better management practices across the production systems and marketing channels to produce E. coli-free wholesome eggs for consumers.

Seasonal prevalence of blood parasitic diseases in crossbred cattle of Mysore and its surrounding districts of Karnataka.

The present study report the seasonal prevalence of blood parasitic diseases in cross bred cattle in Mysore and its surrounding districts of Karnataka such as Mandya, Chamaraj Nagar and Kodagu. The study was undertaken for a period of 2 years from August 2013 to July 2015. A total of 1655 blood samples were collected from clinically suspected cattle for blood parasitic diseases with clinical symptoms of anorexia, high fever, anaemia, salivation, enlargement of superficial lymphnodes, haemoglobinuria and sudden drop in milk yield. The blood samples were examined by giemsa's staining technique. Of the 1655 blood samples screened, 673 (40.22%) blood samples were found positive for blood parasites. Amid 673 positive samples, 609 (90.49%), 19 (2.82%) and 45 (6.68%) were found positive for Theileria annulata, Babesia bigemina and Anaplasma marginale respectively. The season wise prevalence revealed that, the highest prevalence was observed in summer months (March-June) (43.17%) followed by rainy (July-October) (39.53%) and winter season (November-February) (39.35%). Further, the month wise prevalence showed highest in August (77.64%) (Rainy month) followed by November (38.23%) and January (35.93%). During August-2013 to July 2014 and between August-2014 and July 2015, the highest was found in the month of May (85%) followed by July (70%) and April (69.81%). Theileriosis was most prevalent in summer (92.73%) followed by rainy (90.95%) and winter season (87.61%). Babesiosis was most prevalent in winter season (5.04%) followed by rainy (1.8%) and summer season (1.7%) whereas, Anaplasmosis was most prevalent in rainy season (7.23%) followed by winter (6.88%) and summer season (5.55%) during two years of study period.

Prevalence of different species of Culicoides in Bangalore rural and urban districts of South India.

A study was conducted to observe the prevalence of Culicoides a biting midge, important pest and prime vector for various viruses, protozoa and filarid worms. In the vicinity of 11 different farms of cattle, buffalo, sheep and goats in Bangalore rural and urban districts the flies were collected by using UV traps (Onderstepoort Veterinary Institute. ARC. LNR) connected with suction fan for the period of 1 year (2012-2013). Around 83,629 Culicoides were collected of which 77,906 (93.16 %) were female and 5,723 (6.84 %) were males and 40,120 (47.97 %) of C. imicola, 39,366 (47.07 %) C. oxystoma, 2,504 (2.99 %) C. actoni, 1,145 (1.37 %) C. peregrinus, 145 (0.17 %) C. huffi, 120 (0.16 %) C. innoxius, 90 (0.11 %) C. palpifer, 67 (0.08 %) C. anopheles, 37 (0.04 %) C. circumscriptus and 25 (0.03 %) were C. arakawae. It was observed that C. imicola and C. oxystoma were the most predominant species prevalent in Bangalore rural and urban districts of Karnataka.

Serosurveillance of foot and mouth disease in Karnataka state, India: a 3 years study.

A prospective serological investigation was conducted to determine the prevalence and distribution of foot-and-mouth disease (FMD), as well as to monitor the effectiveness of the FMD control programme (FMD-CP) through vaccination in Karnataka, India. Random serum samples were collected every year between May and August before the start of vaccination in 2011, and subsequently following two phases of vaccination in 2012 and 2013. Infection status (seroprevalence) was inferred by subjecting the sera to indirect r3AB3 non-structural protein-ELISA, using kits developed by the Project Directorate on FMD, India. The seromonitoring of FMD-CP was carried out by detecting antibodies deemed to be protective in the pre- and post-vaccinal sera, using liquid-phase blocking-ELISA for structural proteins. The results revealed significant decrease in seroprevalence from 58 to 21 %, providing more definitive data supporting our earlier findings obtained through clinical observations (Hegde et al. in Virusdisease 25:504-509, 2014), and detecting active infection in some of the populations which were considered to be free based on passive surveillance. On the other hand, after four rounds of vaccination, a gradual and significant increase from 4.5 to 59 % of animals carrying antibody levels deemed to be protective was observed against all the three serotypes. The findings of this study could be useful for further strategizing to strengthen the ongoing FMD-CP in Karnataka State, India.

Microsatellite based genetic diversity study in indigenous chicken ecotypes of Karnataka.

AIM: The current study was the first of its kind taken upon indigenous ecotypes of the Karnataka in order to unravel the diversity details at 20 chicken microsatellite regions. MATERIALS AND METHODS: 210 indigenous chicken belonging to six districts of Bangalore and Mysore division formed the target sample for the present study. The genomic deoxyribonucleic acid was isolated by phenol chloroform isoamyl alcohol method. A panel of 20 microsatellite regions, including 14 recommended by FAO and six identified from published scientific literature became the targeted chicken genomic region. 27-33 samples were successfully genotyped in each of the six ecotypes through simplex or multiplex polymerase chain reactions, polyacrylamide gel electrophoresis and silver staining for the selected microsatellite panel. RESULTS: The chickens of Ramanagara and Chamrajnagara were most distant with a Nei's genetic distance value of 0.22. The chickens of Bangalore rural and Mysore were least distant with a value of 0.056. The Ramanagara and Chamrajnagara pair had Nei's genetic identity value of 0.802, which is least among all pairs of ecotypes. There were five main nodes from which the six ecotypes evolved on the basis 20 microsatellite markers used in this study. This study indicates that the four ecotypes Ramnagara, Bangalore Rural, Chickaballapura and Mysore are genetically identical due to their common ancestral evolution while, Mandya and Chamrajnagara ecotypes formed a relatively different cluster due to a separate common ancestral chicken population and less number of generations since drifting from bifurcation node. CONCLUSION: Twenty microsatellite markers based genetic diversity study on six indigenous ecotypes indicated lower genetic distances as well as lower FST values compared to the distinguished breeds reported. There were two main clusters, which differentiated into six ecotypes. They may differentiate into more distinct varieties if bred in isolation for a longer number of generations.

Comparative immunogenicity of two peste des petitis ruminants (PPR) vaccines in South Indian sheep and goats under field conditions.

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.

Synthesis of 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine derivatives as antimicrobial agents.

A series of novel substituted 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine benzamides 9(a-h) were synthesized to determine their antibacterial and antifungal activities as well as possible structure-activity relationships (SARs) to improve therapeutic efficacy. The pyrazol-5-amine benzamides were screened for their antibacterial activity against standard strains of Gram-positive (Streptococcus pyogenes NCIM 2608, Staphylococcus aureus ATCC 29737, Bacillus subtilis NCIM 2010) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 20852, Klebsiella pneumoniae MTCC 618) bacteria by using streptomycin as positive control. They were also tested for their antifungal activities against mycotoxic strains of Fusarium verticillioides, Aspergillus ochraceous, Aspergillus flavus, Alternaria alternata, and Penicillium chrysogenum using nystatin as positive control. Among the synthesized compounds, 9d, 9g, and 9h showed potent antimicrobial activities.

Characterization of VP2 gene of an Indian Bluetongue virus serotype 2 and its close phylogenetic relationship to the Taiwan isolate.

In this study we present the first report on partial amplification, sequencing and phylogenetic relationship of VP2 of the Indian isolate BTV-2. A PCR product of 1135 bp was amplified, cloned and sequenced. About 1063 bp of partial VP2 gene (1792-2854 bp region) of the Indian isolate was subjected to sequence analysis with already published sequences available in the genome database. The percent similarity of 85.2 was observed with Taiwan isolate and 59% with other isolates of BTV-2. However, 46.2% similarity with Australian BTV-1 and no significant similarity were noted with other serotypes. In-silico analysis and restriction enzyme digestion confirmed the presence of conserved SalI site at 2380 bp position in both Indian and Taiwan isolates. Phylogenetic analysis showed that all BTV-2 isolates formed one distinct group in which BTV-2 Indian and Taiwan isolate is more closely related and further demonstrated that BTV's of the same serotype from different geographical regions were closely related at nucleotide and amino acid level, respectively.

A short note on peste des petits ruminants in Karnataka, India.

Retrospective quantitative analysis of epidemiological data on peste des petits ruminants (PPR) from the Department of Animal Husbandry and Veterinary Sciences in Karnataka, southern India, revealed significant information about the disease in this area. In the nine years between April 1998 and March 2007 a total of 624 outbreaks were reported in the state. With the exception of the 12-month period between April 2001 and March 2002 the disease occurred every year. The study shows clearly that incidences were highest during the rainy season and in the dry agro-climatic zones. The density of the PPR-susceptible population in different districts of the state played a major role in disease incidences. Environmental factors also influenced disease occurrence. Vaccination programmes are slowly being taken up in the state. The disease data documented in this study provide information about the endemicity of the disease that can help to formulate an effective strategy for a PPR-control programme in the state.

Standardization of large scale production of homologous live attenuated PPR vaccine in India.

Live attenuated homologous vaccine against peste des petits ruminants of sheep and goats was produced on a large scale basis in roller culture bottles using seed virus developed at the Indian Veterinary Research Institute, Muktheswar, India. Vero cells between 130-150 passages with six percent foetal calf serum were used for the production of vaccine. The cells were infected with 0.01 multiplicity of infection and harvested when the cytopathic effect was 80%. The vaccine was freezedried in order to maintain the stability of the vaccine. Identity test and titration was performed and the vaccine titre was monitored to be minimum of 10(5)/100 doses. In-house sterility tests and quality control tests using experimental animals and small ruminants were performed. The vacuum and moisture content of the vaccine were also regulated to be within the normal limits.

Nucleotide sequence analysis of variable region of VP2 gene of two infectious bursal disease virus isolates from commercial poultry farms.

Two infectious bursal disease virus (IBDV) isolates were obtained from commercial poultry farms with a history of severe outbreaks. A 474-bp product encompassing hypervariable region of IBDV VP2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequences of two isolates, VMB1 and VMB2, were determined and compared with those of twenty IBDV strains, including seven very virulent, four classical virulent, four classical attenuated, three antigenic variants and two avirulent serotype 2 strains. The two isolates showed a similarity of 96.5-98.4% with very virulent strains, 84.6-94.6% with classical virulent strains, 90.0-91.4% with classical attenuated strains, 83.0-91.9% with antigenic variants and 65.8-68.7% with avirulent strains. The deduced amino acid sequences of the two isolates showed amino acid substitutions of V256I, N279D, L294I and N299S, specific for very virulent strains. Phylogenetic analysis showed that the two isolates, along with a reported very virulent Indian strain, were closely related to European, Japanese and Chinese very virulent strains indicating their evolutionary origin.