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.

Emergence of Mycobacterium orygis-Associated Tuberculosis in Wild Ruminants, India.

Tuberculosis caused by Mycobacterium orygis was detected in 2 spotted deer from a wildlife sanctuary in western India and an Indian bison from a national park in central India. Nationwide surveillance is urgently required to clarify the epidemiology of the Mycobacterium tuberculosis complex at the human-livestock-wildlife interface.

Apoptin NLS2 homodimerization strategy for improved antibacterial activity and bio-stability.

The emergence of antibiotic resistance prompts exploration of viable antimicrobial peptides (AMPs) designs. The present study explores the antimicrobial prospects of Apoptin nuclear localization sequence (NLS2)-derived peptide ANLP (PRPRTAKRRIRL). Further, we examined the utility of the NLS dimerization strategy for improvement in antimicrobial activity and sustained bio-stability of AMPs. Initially, the antimicrobial potential of ANLP using antimicrobial peptide databases was analyzed. Then, ANLP along with its two homodimer variants namely ANLP-K1 and ANLP-K2 were synthesized and evaluated for antimicrobial activity against Escherichia coli and Salmonella. Among three AMPs, ANLP-K2 showed efficient antibacterial activity with 12 µM minimum inhibitory concentration (MIC). Slow degradation of ANLP-K1 (26.48%) and ANLP-K2 (13.21%) compared with linear ANLP (52.33%) at 480 min in serum stability assay indicates improved bio-stability of dimeric peptides. The AMPs presented no cytotoxicity in Vero cells. Dye penetration assays confirmed the membrane interacting nature of AMPs. The zeta potential analysis reveals effective charge neutralization of both lipopolysaccharide (LPS) and bacterial cells by dimeric AMPs. The dimeric AMPs on scanning electron microscopy studies showed multiple pore formations on the bacterial surface. Collectively, proposed Lysine scaffold dimerization of Apoptin NLS2 strategy resulted in enhancing antibacterial activity, bio-stability, and could be effective in neutralizing the off-target effect of LPS. In conclusion, these results suggest that nuclear localization sequence with a modified dimeric approach could represent a rich source of template for designing future antimicrobial peptides.

Coinfection of porcine astrovirus and other porcine viruses in diarrheic pigs in Haryana, India.

In this study, 306 rectal swabs from diarrheal pigs of various ages (0-3 weeks, 3-6 weeks, and >6 weeks) were collected from 54 piggery units in different climatic zones in Haryana state, India. These samples were tested for the presence of porcine astrovirus (PAstV), porcine rotavirus group A (PRV-A), and classical swine fever virus (CSFV) by reverse transcription polymerase chain reaction (RT-PCR), and porcine circovirus 2 (PCV-2) by polymerase chain reaction (PCR). Out of the 306 samples tested, 153 (50%), 108 (35.3%), 32 (10.6%), and three (0.9%) tested positive for PAstV, PCV-2, PRV-A, and CSFV, respectively. A single infection was detected in 135 samples, while mixed infections were found in 77 samples: 70 with two viruses and seven samples with more than two. PAstV was detected most frequently (55.31%) in pigs aged 3-6 weeks. PCV-2 was more predominant in pigs aged 0-3 weeks (36.53%), whereas PRV-A was more common in pigs aged 3-6 weeks (11.3%). CSFV was observed in the age group of 0-3 weeks (1.92%). Phylogenetic analysis revealed the circulation of lineages 2 and 4 of PAstV in this region. Thus, it can be concluded that one or more than one virus is circulating in piggery units in Haryana, India.

Molecular detection of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks.

Respiratory tract infections are of serious concern to the poultry industry. The present study was aimed to delineate the extent of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks. A total of 146 poultry flocks of Haryana and Rajasthan, India, suspected for chronic respiratory disease (CRD) were screened for avian mycoplasmas, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and avian pathogenic Escherichia coli (APEC) by conventional polymerase chain reaction (PCR) assays. A total of 49.31% (72/146) flocks were found positive for Mycoplasma infection. Of the Mycoplasma-positive flocks, 80.55% (58/72) represented pathogenic avian mycoplasmas (MG and/or MS), while 19.44% (14/72) flocks were positive for commensal avian mycoplasmas (other than MG and MS). A correlation was deduced between avian mycoplasmosis and bacterial and/or viral co-infections. The results revealed that 17.24% (10/58) flocks had only avian mycoplasmosis infection. However, in the remaining flocks, the avian mycoplasmosis was associated either with APEC infection [17.24% (10/58)], IBV infection [43.10% (25/58)], or both APEC and IBV infections [22.41% (13/58)], respectively. Further epidemiological studies on respiratory avian mycoplasmosis associated concurrent infections with other pathogens are recommended to assess circulating strains, risk factors, and economic losses.

Molecular investigations on outbreaks of ovine theileriosis among sheep and goats in Haryana, India.

During February 2020 to October 2020, four outbreaks of theileriosis in small ruminants were recorded with overall morbidity, mortality and case fatality rates of 27.95%, 17.46% and 62.5%, respectively. The disease was characterized by high fever (up to 106°F), superficial lymphadenopathy, anaemia, anorexia, lethargy, respiratory distress and death. The presence of pleomorphic intra-erythrocytic piroplasms of Theileria species in Giemsa's stained blood smears was a common finding in all the episodes. Significant haematological alterations including high total leucocyte count and low haemoglobin and packed cell volume were characteristic. Necropsy findings of the icteric liver, enlarged spleen, pulmonary oedema and abomasal ulcerations were observed in three flocks. Smear-positive blood samples from all the episodes were screened by PCR using 18S rRNA gene-specific primer sets for T. lestoquardi, T. luwenshuni, T. uilenbergi and T. ovis. T. lestoquardi which was detected in all four flocks, while there was co-infection of T. ovis in two flocks. Phylogenetic analysis revealed that T. ovis and T. lestoquardi identified in this study had 100% and ~ 99.86% homology, respectively, with the published sequences used for comparison. This is the first confirmed report of outbreaks of malignant ovine theileriosis in the Haryana state of India which caused high morbidity, mortality and case fatality among sheep and goats. Further studies on theilerioses in small ruminants are required to understand epidemiology better.

Epidemiology, clinical features, and molecular detection of orf virus in Haryana (India) and its adjoining areas.

Orf is an acute, highly contagious, and economically important viral disease of small ruminants. In this study, six orf suspected outbreaks among goats and sheep were investigated from Haryana state and adjoining areas of Rajasthan state during the year 2021. The disease was diagnosed on the basis of clinical signs and molecular identification. The causative agent of the disease, orf virus (ORFV), was confirmed using polymerase chain reaction (PCR) targeting immunodominant envelope antigen (B2L) gene and confirmed by sequencing. The morbidity in goats ranged from 8.75 to 100%, whereas in sheep, it ranged from 0 to 8%. The higher mortality was observed among flocks with mixed infections of orf and peste des petits (PPR) or orf and haemonchosis as compared to other outbreaks. The phylogenetic analysis of sequenced PCR products clustered the current study strains in the same clad with Indian as well as strains from other countries with nucleotide identity more than 99%, signifying a close genetic relationship. The study highlighted the circulation of strains of a single cluster among sheep and goats in Haryana and adjoining areas. Prompt diagnosis of the disease is highly important for facilitating the implementation of control measures to minimize the losses suffered by small and marginal farmers in this region. Further detailed studies are required to delineate the molecular details of ORFV for better understanding the dynamics and molecular epidemiology of strains circulating in the country and for designing the effective vaccines against the disease which are currently lacking in the country.

Molecular detection and characterization reveals circulation of multiple genotypes of porcine astrovirus in Haryana, India.

Porcine astrovirus (PAstV) is distributed worldwide and has been reported to cause diarrhea in pigs. PAstV belongs to the family Astroviridae and genus Mamastrovirus. PAstVs are divided into five diverse genotypes (PAstV1-PAstV5) on the basis of phylogenetic analysis of a part of the RNA-dependent RNA polymerase (RdRp) gene and the capsid gene. However, knowledge regarding the clinical significance and molecular characteristics of PAstV in Haryana, India, is limited. In this study, we investigated the presence of PAstV by RT-PCR of the partial RdRp gene in 110 rectal swabs collected from diarrheic pigs in different parts of Haryana, India. Of these, 35 samples (31.8%) tested positive for PAstV, with the highest positivity observed among weaning piglets 3 to 9 weeks of age (47.7%, 21/44), followed by fattening pigs 9 to 24 weeks of age (28.5%, 8/28). Phylogenetic analysis of the partial RdRp gene revealed circulation of four different genotypes (PAstV1, PAstV2, PAstV4, and PAstV5) in Haryana, with PAstV1 being the predominant genotype. To the best of our knowledge, this is the first report of the presence of PAstV1 and PAstV5 in the pig population of India. The PAstV sequences revealed high genetic variability and genetic heterogeneity in a relatively confined area.

Multiple antigenic peptide-based flow through dot-blot assay for simultaneous antibody detection of infectious bronchitis virus and Newcastle disease virus.

The present study describes the development of a novel affordable and rapid visual dot-blot assay using synthetic multiple antigenic peptides (MAP) for simultaneous detection of antibodies to infectious bronchitis virus (IBV) and Newcastle disease virus (NDV). Antibody detection efficiencies of MAP peptides namely, NP1 MAP (Nucleoprotein IBV) and HN MAP (Haemagglutinin-neuraminidase NDV) were studied in solid-phase indirect peptide ELISA. In comparison with the commercial kit, the NP1 MAP showed 89.20% diagnostic sensitivity (DSn) and 85.90% diagnostic specificity (DSp) at 19.45% ROC cut-off. Similarly, HN MAP was evaluated and showed 89.70% DSn and 92.90% DSp at 19.90 % ROC cut-off. The peptides after evaluating their ELISA performance were further used to device a flow-through dot-blot assay (FT-DBA) for simultaneous detection of IBV and NDV antibodies. The kappa value for IBV by FT-DBA in comparison to commercial ELISA was 0.64 whereas for NDV, FT-DBA gave a kappa value of 0.68 in comparison to commercial ELISA indicating substantial agreement between the assays. In essence, the divergent MAP based diagnostic design could provide an alternative for antibody detection of IBV and NDV. Further, the FT-DBA approach could be used for low cost, rapid and pen-side detection of IBV and NDV antibodies simultaneously.

Detection and molecular characterization of astroviruses in turkeys.

This study was conducted to determine the prevalence and molecular characteristics of turkey astrovirus 1 (TAstV-1) and avian nephritis virus (ANV) in turkeys with light turkey syndrome (LTS), which is characterized by lower body weight in market-age turkeys than their standard breed character. We collected pools of fecal samples from four LTS and two non-LTS turkey flocks in Minnesota at 2, 3, 5 and 8 weeks of age. Of the 80 LTS pools tested, 16 (20.0 %) and 11 (13.8 %) were positive for TAstV-1 and ANV, respectively. For non-LTS flocks, these numbers were 8 (20.0 %) and 5 (12.5 %), respectively. The maximum number of birds was positive at five weeks of age. We also tested 130 fecal samples of poult enteritis syndrome (PES) cases submitted to the Minnesota Veterinary Diagnostic Laboratory and found 19 and 11 positive for TAstV-1 and ANV, respectively. RdRp gene sequences were determined for a total of 29 TAstV-1 and 22 ANV samples. Phylogenetic analysis of the RdRp gene revealed 92-100 % and 88-100 % nucleotide sequence identity among TAstV-1 and ANV sequences, respectively. A large number of nucleotide and amino acid substitutions were observed in LTS and PES flocks than in non-LTS flocks. One of the PES sequences grouped with ANV-like sequences detected in chickens, indicating that regular screening of birds should be continued. Further, complete genome analysis should be conducted to determine whether this virus is a novel divergent strain or a recombinant of chicken and turkey ANV-like viruses. The detection of TAstV-1 and ANV in a considerable number of non-LTS cases emphasizes the need for further studies on the transmission pattern and pathogenesis of these viruses to determine their role as pathogens of turkeys.

Molecular characterization of turkey enteric reovirus S3 gene.

The molecular diversity in S3 gene sequences of turkey reovirus (TRV) was determined in poult enteritis syndrome (PES)-affected and apparently healthy turkey poults. Twenty-nine TRV-positive samples (15 from PES-affected flocks and 14 from apparently healthy flocks) were tested using self-designed primers for the S3 gene. Phylogenetic analysis revealed that the TRV S3 sequences of this study clustered in clade III and formed two different groups in this clade. The avian reoviruses from duck and goose formed clade I and those from chickens formed clade II. The clade III TRV sequences had a nucleotide percent identity of 88.9 to 100% among themselves but only of 59.5 to 63.5% and 69.2 to 72.6% with clades I and II, respectively. More amino acid substitutions were present in TRVs from PES-affected flocks than in those from apparently healthy flocks using ATCC VR-818 (AY444912) as a benchmark. All TRVs of this study showed substitutions at positions 244 and 285. The impact of these changes on the virulence of the virus, if any, needs to be studied.

Seroprevalence and risk factors associated with Toxoplasma gondii in pigs in Haryana, India.

AIMS: Toxoplasmosis is one of the most common food-borne parasitic zoonosis, caused by Toxoplasma gondii, an obligate intracellular protozoan parasite. A cross-sectional study was carried out to determine the seroprevalence of T. gondii and associated risk factors in pigs in Haryana, India. METHODS AND RESULTS: Serum samples were collected from 429 pigs from three agroclimatic zones (I-III) of Haryana and analysed for the presence of antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Anti-T. gondii antibodies were detected in 106 animals (24.7%), with the highest seropositivity in zone II (31.3%) followed by zone III (24.4%) and zone I (18.3%). Risk factors associated with higher seropositivity in pigs were farm size (higher in large-sized farms), age (higher in pigs >1 year of age), sex (higher in males), type of feeding (higher in combination of homemade and hotel waste) and housing (higher in free-ranging pigs). CONCLUSIONS: The findings of the study testify to the exposure of pigs (of all agro-climatic zones) to T. gondii. Hence, the observations are of significant medical and veterinary importance for devising and implementing control measures to check the dissemination of toxoplasmosis to pigs and eventually to humans.

First Molecular Confirmed Outbreak of Malignant Ovine Theileriosis in Sheep from North India.

CONTEXT: Malignant ovine theileriosis is a tick-borne disease of sheep and goats, caused by protozoan Theileria lestoquardi. The disease has serious economic implications for small ruminant production around the world. METHODS: An outbreak of malignant ovine theileriosis in a sheep flock was investigated from Hisar district of Haryana, India, in March 2022. The etiological agent was identified using polymerase chain reaction assay with genus specific primers targeting 18S rRNA gene and subsequently confirmed by sequencing. RESULTS: The morbidity, mortality and case fatality rate reported in the outbreak were 22.2, 18.8 and 85%, respectively. The phylogenetic analysis clustered the present study T. lestoquardi isolate in the same clade with T. lestoquardi from Iraq, Iran and Pakistan with maximum nucleotide identity of 99.37% with strains from Iraq. The tick vector Hyalomma anatolicum recovered from dead animals was implicated in the disease's transmission. CONCLUSIONS: Malignant ovine theileriosis resulted in high case fatality rate. This study presents the first molecularly confirmed outbreak of malignant ovine theileriosis outbreak in the North Indian region, with characteristic post-mortem findings.

uspA gene-based phylogenetic analysis and antigenic epitope prediction for Escherichia coli strains of avian origin.

Pathogenic Escherichia coli (E. coli) is responsible for various local and systemic infections in animal and human populations. Conventional methods for the detection and identification of E. coli are time-consuming and less reliable for atypical strains. The uspA gene has been widely used as a target for the detection of E. coli. The present study was aimed at phylogenetic analysis of the uspA gene sequences to determine the evolutionary relationships between the strains and other members of the Enterobacteriaceae family. In addition, the unique differences in the sequences of the current study with Salmonella and Shigella species were tested using Tajima's molecular clock test. Antigenic epitope prediction was performed to locate the B-cell epitope region of the UspA protein. Two E. coli isolates of avian origin and strains from the National Center for Biotechnology Information (NCBI) database were used for prediction. The Immune Epitope Database (IEDB) server, Bepitope, ABCpred, SVMTrip, and ElliPro server were used to identify B-cell epitopes. The 3D structure was predicted using SWISS-MODEL. Phylogenetic analysis of the isolates from the current study revealed that both OM837340 and OM837341 sequences from the current study had maximum nucleotide homology (nt) of 99.87%-100% with E. coli isolates and minimum nt homology of 84.08% with Salmonella enteritidis and S. Hissar. The isolates in the current study had a homology of 98.87%, while the homology with Shigella species was 99.25%. Seven silent mutations were observed in the coding region of the UspA protein of ECO9LTBW (current study). Modeling of the UspA protein revealed a maximum homology of 67.86% with the Protein Data Bank in Europe (PDBe), also validated by the Ramachandran plot. No significant differences were found in the coding regions of uspA of Salmonella, Shigella, and E. coli with Tajima's test. For the E. coli isolates, a total of 24 linear B-cell and seven discontinuous epitopes were predicted using in-silico analysis. When the results of the predicted peptides were compared, two peptides, namely ARPYNA and YSDLYTGLIDVNLGDMQKRISEE, were found suitable candidates. In conclusion, the uspA gene appears to be conserved among E. coli isolates and can be used for molecular detection.

Molecular epidemiology, antibiogram profile and risk factor analysis of pathogenic Escherichia coli associated with pre-weaning diarrhoea in piglets from Haryana, India.

BACKGROUND: Piglet diarrhoea is a multifactorial disease with serious implications for the swine industry worldwide, including India. The Escherichia coli (E. coli) pathotypes, i.e., enterotoxigenic E. coli (ETEC) and Shiga toxin-producing E. coli (STEC) are among the major bacterial agents attributed as causative agent for piglet diarrhoea, but studies related to genetic diversity, antibiogram profile and their correlation with risk factors of these pathogens are sparse. MATERIAL AND METHODS: A total of 104 faecal swab samples were collected from 32 different piggery units of Haryana, India and confirmed as E. coli by standard microbiological methods. The identified E. coli were characterized as ETEC and/or STEC using PCR assays and were studied for their genetic diversity by phylogenetic analysis of the sequences. All the isolates were subjected to antimicrobial susceptibility testing. Further, the correlation of variables with presence or absence of ETEC and/or STEC was also investigated by using Fisher's exact test. RESULTS: Microbiological isolation led to identification of 208 E. coli isolates. A total of 17.3% (31/208) isolates were characterized as ETEC and 4.8% (10/208) isolates as STEC, whereas 2.4% (5/208) isolates exhibited both ETEC and STEC pathotype. Of the total studied piggery units (n = 32), ETEC were isolated from fourteen and both ETEC and STEC from eight farms. The phylogenetic analysis of Stx2 gene revealed 100% homology with Stx2eA variant from Germany, while analysis of STII gene revealed a distinct nucleotide and amino acid substitution when compared with standard strains. The antibiotic susceptibility testing revealed maximum resistance to moxifloxacin (71.9%) followed by tetracycline (58.1%) and amoxicillin with a total of 41.8% (87/208) E. coli isolates designated as multi-drug resistant (MDR). The multiple antibiotic resistance index varied from 0.05 to 0.75. The statistical analysis suggested three factors viz., number of farm worker(s), frequency of using disinfectant for floor cleaning and use of antibiotic in feed as risk factors significantly associated (p < 0.05) with ETEC associated diarrhoea at piggeries under study. CONCLUSION: Current study warrants a need for systematic studies on the ETEC/STEC associated diarrhea and antibiotic resistance among these isolates to understand the mechanisms of origin and dissemination of drug resistant pathogens and to design suitable prevention and control measures to curb emergence of antibiotic resistance in the farm settings.

A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys.

This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting "poult enteritis syndrome" were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys.

Sub-lineages of Taenia solium Asian Genotype Recorded in North India.

PURPOSE: Porcine cysticercosis is a neglected zoonotic disease of significant veterinary and medical importance owing to its economic impact and public health significance. The present study aimed at genetic characterization of Taenia solium metacestodes in slaughtered pigs of Haryana (North India). METHODS: A total of 213 (160 and 53 from Chandigarh and Hisar, respectively) slaughtered pigs intended for human consumption were screened for the presence of T. solium metacestodes. The retrieved metacestodes were confirmed molecularly based on the partial amplification of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Evolutionary divergence, haplotype and nucleotide diversities and neutrality indices of the retrieved isolates were also assessed. RESULTS: Out of the 213 pigs, 2 (0.94%) revealed the presence of metacestodes involving 1 pig each from Chandigarh (0.62%) and Hisar (1.9%). The sequences obtained after custom sequencing were submitted to GenBank under the accession numbers LC661682-83. The present study haplotype clustered with haplotypes of Asian origin and showed variation from other haplotypes by 1-23 mutational steps. However, the present study isolates also showed nucleotide polymorphisms (A198T, A199G, A201T, G204A, T206A, C210T, T212G, T213A, T216G/A, T217C, T221C, C524T, G994A) at different positions, which indicated the presence of sub-lineages. Low nucleotide diversity (π = 0.020) and negative value of Tajima's D (- 1.304) observed for the haplotypes under consideration was indicative of purifying selection and recent population expansion. CONCLUSIONS: Our study confirms the circulation of T. solium Asian genotype (with distinct sub-lineages) in study area and recommends strict control measures to contain the zoonotic disease.

Characterization and antimicrobial susceptibility of biofilm-producing Avian Pathogenic Escherichia coli from broiler chickens and their environment in India.

Avian pathogenic Escherichia coli (APEC) is responsible for colibacillosis in poultry. APEC remains a constant problem for the poultry industry, despite the use of antimicrobials and disinfectants in farms. The endemicity of APEC in poultry farms is associated with its biofilm-forming ability, which is further aggravated by various virulence factors and resistance to multiple drugs that help bacteria to thrive under different environmental conditions. To characterize APEC from affected broiler chickens and their environments, samples (n=114) from dead birds (heart, liver, lungs, and cloacal swab) and surrounding environments such as feeder, drinker, litter, PVC pipe, water tank wall, feed, and water were collected. The collected samples were subjected to microbial isolation using MacConkey Lactose agar (MLA) and Eosin Methylene Blue agar (EMB), which led to the isolation of 62 E. coli isolates. This was confirmed by uspA gene amplification and Vitek 2 Compact. These isolates were characterized using a set of five virulence genes (hlyF, ompT, iroN, iss, iutA), which yielded 47 (75.80%) isolates as APEC and the remaining as non-APEC. Furthermore, all the 62 isolates were subjected to microtiter plate assay for biofilm detection and the result showed that 36 (58.06%) isolates were able to form moderate to strong biofilms in Trypticase soy broth (TSB) at 72h of incubation. Of the 36 biofilm-producing isolates, 30 were APEC. Biofilm-related genes (crl, csgA, fimH, luxS, and papC) were also detected with higher prevalence among APEC isolates. Antimicrobial susceptibility test using Vitek 2 Compact revealed 43 (91.48%) of 47 APEC isolates as multiple drug resistant (MDR) and 8 (17.02%) as ESBL positive. This study reveals that APEC with biofilm formation ability is present in poultry farms. Further studies are needed to understand the role of biofilms in the pathogenesis and antimicrobial resistance of APEC.

Immunosorbent assay for detection of Trypanosoma evansi infection in multiple host species using chimeric protein A/G conjugate.

Surra caused by an extracellular hemoflagellate, Trypanosoma evansi, leads to severe economic loss to livestock productivity in India. Among the various mammalian pathogenic trypanosomes, T. evansi has the widest host range.Usually, species specific conjugates are used in conventional indirect immunosorbent assay (ELISA) for diagnosis of T. evansi infection in different animal species. The aim of the study was to explore the use of non-species specific conjugates viz., protein A, G and chimeric protein A/G instead of species specific conjugates for development of indirect ELISAs. These assays were used for detection of antibodies against T. evansi infection in multiple animal species viz., equine, cattle, buffalo, dog, pig and camel. The functional affinities of serum immunoglobulins of six different animal species with different conjugates were determined by estimation of relative avidity index (RAI). The species specific conjugate based whole cell lysate- T. evansi antigen ELISA was considered as reference assay for comparison of sensitivity and specificity of non-species specific conjugates based ELISAs optimized in the present study. Data showed that serodiagnosis of T. evansi can be carried out by using chimeric protein A/G conjugate in multiple hosts viz., equine, buffalo, camel, pig and dog; protein G conjugate in equine and buffalo and protein A conjugate in camel, pig and dog. The relative diagnostic sensitivity and specificity for chimeric protein A/G conjugate varied from 60 to 100% and 79-100%, respectively for different livestock species. This approach might be helpful in monitoring and surveillance of T. evansi infection in multiple hosts in particular when host specific secondary antibody conjugates are not available. Investigations should be made in wild animals and other warm-blooded vertebrates to validate this hypothesis.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays.

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a 'perfect' agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.