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.

Bovine mastitis is a major infectious disease affecting dairy animals resulting in enormous economic losses, prolonged antibiotic treatment, reduced milk yield and death of livestock. Emergence of Methicillin-resistant Staphylococcus aureus (MRSA) among bovine mastitis is matter of concern for animal health and dairy industry. The present study was conducted to detect the distribution of virulence and enterotoxin genes among MRSA isolates from bovine mastitis. Out of 500 milk samples, 126 isolates were identified as Staphylococcus and from these only 56 were S. aureus. S.aureus were resistant to cefoxitin (75%), ceftazidime (75%), amoxicillin (71.4%), cefodaxime (67.8%), cefepime (66.1%), oxacillin (64.3%), norfloxacin (60.7%) and gentamicin (58.9%). Only 42 isolates were identified as MRSA strains among staphylococci isolates. MRSA were harbouring virulence genes; mecA (100%), coa (100%) and nuc (100%). The other virulence factors such as hlg (80.9%, 34/42), pvl (47.6%, 20/42) and spa (92.8%, 39/42) were also reported. Molecular characterisation of enterotoxin genes revealed that out of 42 tested isolates 11 were found negative (26%) for any enterotoxin gene whereas 7 (16.6%), 6 (14.3%), 18 (42.8%), 1 (2.3%), 26 (61.9%),27(64.2%),3 (7.1%) were found positive for sea, seb, sec, sed, seg, sei, and seq enterotoxin respectively.

The aim of present study was to investigate the effect of full mother contact and voluntary colostrum suckling on the health, stress and growth performance of neonatal Murrah buffalo calves. For this, two experiments (exp.) were conducted involving exp.1, 24 mother-calf pairs were divided into three equal groups (eight pairs/each group). In no mother contact (NC) group, weaning was allowed immediately after birth. In restricted mother contact (RC) group, restricted contact was allowed twice daily. In fence-line mother contact (FC) group, calves were in full-time mother contact from birth to day (d) 5 and allowed voluntary colostrum suckling and thereafter fence-line contact. In exp.2, another 24 mother-calf pairs were selected and divided into two groups (12 pairs in each group), with the same conditions as the RC and FC groups in exp.1. Average daily gain, total protein and total Ig concentrations differed between different groups in both experiments. Cortisol levels were lower in FC followed by RC and lowest in NC calves, and similar results were observed in exp.2. It was concluded that the provision of full mother contact along with free choice colostrum suckling from birth to 5 days followed by fenceline mother contact and twice daily suckling at milking time abated the separation and weaning stress and remarkably improved the growth, immune status and health of Murrah buffalo calves as compared to the calves which were allowed only limited contact twice daily or the calves which were weaned at birth.

Bovine tuberculosis (bTB) remains endemic in domestic water buffaloes (Bubalus bubalis) in India and elsewhere, with limited options for control other than testing and slaughter. The prescribed tuberculin skin tests with purified protein derivative (PPD) for diagnosis of bTB preclude the use of Bacille Calmette-Guérin (BCG)-based vaccination because of the antigenic cross-reactivity of vaccine strains with Mycobacterium bovis and related pathogenic members of the M. tuberculosis complex (MTBC). For the diagnosis of bTB in domestic water buffaloes, we here assessed a recently described defined-antigen skin test (DST) that comprises overlapping peptides representing the ESAT-6, CFP-10 and Rv3615c antigens, present in disease-causing members of the MTBC but missing in BCG strains. The performance characteristics of three doses (5, 10 or 20 μg/peptide) of the DST were assessed in natural tuberculin skin test reactor (n = 11) and non-reactor (n = 35) water buffaloes at an organized dairy farm in Hisar, India, and results were compared with the single intradermal skin test (SIT) using standard bovine tuberculin (PPD-B). The results showed a dose-dependent response of DST in natural reactor water buffaloes, although the SIT induced a significantly greater (P < 0.001) skin test response than the highest dose of DST used. However, using a cut-off of 2 mm or greater, the 5, 10, and 20 μg DST cocktail correctly classified eight, 10 and all 11 of the SIT-positive reactors, respectively, suggesting that the 20 μg DST cocktail has a diagnostic sensitivity (Se) of 1.0 (95% CI: 0.72–1.0) identical to that of the SIT. Importantly, none of the tested DST doses induced any measurable skin induration responses in the 35 SIT-negative animals, suggesting a specificity point estimate of 1.0 (95% CI: 0.9–1.0), also identical to that of the SIT and compares favorably with that of the comparative cervical test (Se = 0.85; 95% CI: 0.55–0.98). Overall, the results suggest that similar to tuberculin, the DST enables sensitive and specific diagnosis of bTB in water buffaloes. Future field trials to explore the utility of DST as a defined antigen replacement for tuberculin in routine surveillance programs and to enable BCG vaccination of water buffaloes are warranted.

This study was carried out with the objective to develop a novel, rapid and cost effective direct PCR-RFLP assay for detection and differentiation of two Anaplasma species (A. ovis and A. marginale). Blood samples were collected randomly from 112 goats. DNA was extracted from blood samples and a direct blood polymerase chain reaction (DT-PCR) for amplifying a fragment of the major surface protein 5 (msp5) gene of A. ovis/A. marginale from whole blood was developed and standardized. Additionally, the 16srRNA and msp4 genes were analysed by nested PCR, semi nested PCR and PCR-RFLP methods. Blood smear examination revealed 33 samples (29.46%) to be positive for Anaplasma inclusion bodies. On the contrary, 54 (48.21%) samples were positive by DT-PCR. The results revealed that DT-PCR was 100% sensitive and 73.41% specific when compared with microscopy based detection (k =0.63). All DT-PCR positive samples were confirmed as A. ovis by RFLP analysis. DT-PCR showed 94.44% sensitivity and 100% specificity compared to conventional PCR results with suspected blood samples (k=0.94). The phylogenetic tree and comparative sequence analysis revealed msp5 gene of Anaplasma species of Indian isolate had maximum distance from A. phagocytophilum followed by A. centrale and A. marginale and 100% sequence identity with A. ovis isolates of Chinese origin. The in-house standardized DT-PCR-RFLP method based on msp5 gene is an easy and reliable diagnostic tool to identify and discriminate between these closely related pathogens in ruminants. This type of study is the first of its kind as it depicts the molecular detection, differentiation and phylogenetic characterization of Anaplasma ovis among goat flocks in India.

Blood samples from suspected crossbred cows (327) were examined microscopically as well as confirmed by in-house standardized duplex PCR assay. Out of 327 samples, 107 (32.72%) and 17 (5.19%) samples were positive for T. annulata and B. bigemina respectively by microscopy. When the samples were screened by duplex PCR, 130 (39.75%) and 27 (8.25%) animals had single infection with T. annulata (Group I) and B. bigemina (Group II), respectively. Duplex PCR was able to detect 11% of mixed infections (Group III) compared to 2.75% by microscopy. Haemato-biochemical profile of infected animals (30 for each group) were studied and compared with each other and normal healthy group (Group IV, n=10). The infected group showed significantly decreased levels of TEC, Hb and PCV, red blood cell indices than healthy control animals indicating microcytic hypochromic anaemia. Marked thrombocytopenia was also observed in affected animals. Serum biochemistry of infected cows revealed significantly higher values of AST and low levels of blood glucose, calcium, total protein, albumin as compared to healthy animals. Group I and Group II were further sub-divided into three sub-groups based on severity of infection as latent, subclinical and clinical. No significant difference among biochemical parameters was observed between subgroups of diseased animals but there was significant decline in hematological parameters, viz. haemaoglobin, PCV and TEC. Haemato-biochemical changes were more severe in B. bigemina infected group in contrast to other groups and anaemia is becoming more severe as the disease progress due to extensive intravascular haemolysis.

The present investigation was designed to develop a novel, rapid and cost effective direct PCR-RFLP assay, as a specific diagnostic tool for detection and differentiation of two Anaplasma species (A. ovis and A. marginale). Blood samples were collected randomly from 83 goats. A direct blood polymerase chain reaction (DT-PCR) for amplifying a fragment of the major surface protein 5 (msp5) gene of A. ovis/A. marginale from whole blood was developed and standardized. Blood smear examination revealed 24 samples (28.91%) positive for Anaplasma inclusion bodies. While, 39 (47%) samples were positive by DT-PCR. The results revealed that DT- PCR was 100% sensitive and 74.57% specific compared to microscopy based detection (k =0.62). Additionally DT-PCR showed 94.44% sensitivity and 100% specificity compared to conventional PCR results with suspected blood samples (k=0.94). All DT-PCR positive samples were confirmed to be A. ovis by restriction fragments length polymorphism (RFLP) analysis. The phylogenetic tree and sequence analysis revealed msp5 gene of Anaplasma species Indian isolate had maximum distance from A. phagocytophilum followed by A. centrale and A. marginale and 100% sequence identity with A. ovis isolates of Chinese origin which further confirmed the sequence identified in native goats to be of A. ovis. The simplified DT-PCR assay as a viable alternative to conventional PCR could be helpful for fast and accurate diagnosis of Anaplasma species and suitable for screening a large number of samples. Furthermore, results revealed that DT-PCR-RFLP of the msp5 gene might be a useful method for simultaneous detection and differentiation of A. ovis and A. marginale in goats.

The study was conducted to investigate hemato-biochemical and electrolytes changes caused by rumen impaction in 106 buffaloes diagnosed with rumen impaction during the period from July 2016 to June 2017 with the history of mild inappetance to complete anorexia, less water intake, scanty hard mucoid faeces and reduction in milk yield. Clinical examination revealed congested mucous membrane, mild to moderate degree of dehydration, decreased or absence of ruminal motility, slight rise in temperature and hard consistency of rumen on palpation and also on per rectal examination. The affected buffaloes revealed significantly higher values of WBC and neutrophils count indicating neutrophilic leucocytosis. However other haematological parameters were within the normal range. Blood film revealed no haemoprotozoan infection. Coproscopy did not exhibit any parasitic egg/ova/cyst/oocyst. Significant increase in glucose level, indirect bilirubin level, blood urea nitrogen, serum creatinine, ALT and AST enzyme activities was observed in the serum samples of affected buffaloes. A significant decrease was observed in electrolytes viz., sodium, potassium, iCa and chlorides. Present study concludes that marked changes in haematological and serum biochemical profile observed in rumen impaction cases may be useful in understanding the pathogenesis, treatment and prognosis of the disease.

Bovine babesiosis and theileriosis are fatal tick borne haemoparasites of vertebrates imposing serious constraints on health and productivity of livestock. Additionally, the recovered animals become persistent carriers and play a significant role in disease epidemiology. The present investigation describes the development and evaluation of duplex PCR assay for simultaneous detection of Babesia bigemina (B. bigemina) and Theileria annulata (T. annulata) in cattle. Following in silico analysis for candidate target genes representing each of the haemoparasites, an optimised duplex PCR assay was established using two sets of primers, ssurRNA and cytob1 for genomic DNA amplification of B. bigemina and T. annulata encoding product size of 689 and 312 bp, respectively. The results were compared with conventional microscopy and monoplex PCR assay. The sensitivity of each primer pair was checked using serial dilutions of parasite DNA, while specificity was determined by testing for amplification from DNA of different stocks of each pathogen. The duplex PCR detected each parasite species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or with DNA mixture representing the other pathogens. Additionally, single and duplex PCRs could able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of both the pathogen, and nonspecific amplification from non target species was not observed. The developed assay represents an economical, simple, sensitive, specific and reproducible diagnostic tool for simultaneous detection of tropical theileriosis and bovine babesiosis and boosting targeted selective control strategy in endemic areas.

The present study was a undertaken to diagnose Anaplasma marginale in naturally infected crossbred cows and to determine its effect on haemato-biochemical profile. Blood samples were collected from animals (200) for detection of the rickettsial organism by direct smear and direct blood PCR based techniques targeting the major surface protein 5 (MSP-5). Direct blood PCR revealed a 382-bp amplified fragment in positive control samples. When random blood samples were screened under light microscope and direct blood PCR method, 7.5% of samples were positive under microscopic examination whereas PCR analysis revealed 10.5% samples positive for A. marginale. The infected group (25) showed significantly decreased levels of TEC, Hb and PCV than healthy control animals. However, differences in the red blood cell indices were non-significant indicating normocytic normochromic anaemia in affected crossbred cattle. Serum samples (25) of infected cows showed significantly higher values of ALT, AST, BUN, creatinine and TBIL than that of healthy control. A significant decrease of TSP and albumin was also recorded in the infected cows compared to healthy control. The standardized PCR method of the present investigation may be useful for rapid and accurate diagnosis of A. marginale in subclinical/carrier animals as the whole blood could be directly used. Haemato-biochemical studies concluded that anaemia and erythrophagocytosis are considered to be the major components of this disease and adversely affect liver and soft tissue of the affected animals.

The present study was undertaken to evaluate the effect of Picrorhiza kurroa (Kutki) feeding on immunological response and protection in chickens vaccinated and challenged with Salmonella enterica serovar gallinarum (SG) vaccine. Day-old broiler chicks (n=220) were randomly divided into six groups A, B, C, D, E and F. The chicks in groups A, B, C and D were immunized with 2x1010 cfu per 0.4 ml-1 SG formalin killed aluminum hydroxide adjuvant vaccine (SGFKV) subcutaneously at day zero of age. Chicks in group B were fed with P. kurroa @ 5 g per kg of feed, group C with aflatoxin B1 @ 2 ppm per kg feed and group D with P. kurroa and aflatoxin B1 with the same dose as in groups B and C from day-one of age. Group E chicks were fed P. kurroa @ 5 g per kg feed and group F were kept as control (no aflatoxin, no P. kurroa, no vaccine). At four weeks post immunization, chicks of all the six groups were challenged with 2x1010 cfu/0.5 ml/bird of SG organisms. The maximum lymphocyte transformation responses (LTR’s) with mean stimulation indices of 9.60, 9.94, 9.04, 9.36 and 2.43 were observed at 4 weeks post immunization against sonicated bacterial cell protein antigen (sbcp) antigen and 20.41, 21.85, 18.51, 19.49 and 11.51 against concanavalin A (Con A) in groups A, B, C, D and E, respectively. Maximum ELISA titers of 3.42, 3.70, 2.82, 3.04 and 1.78 were observed in groups A, B, C, D and E, respectively at 3 weeks PI. It was concluded that feeding of aflatoxin B1 to broiler chicken lowered the cell mediated immunity and humoral immune response. P. kurroa feeding to chicks immunized with SGFKV showed an increase in HI and CMI responses. However, the immunoprotective efficacy of the SGFKV was reduced in groups C and D chickens due to immunosuppression caused by feeding of aflatoxin B1.

Swinepox virus (SWPV), a member of the genus Suipoxvirus causes generalized pock-like lesions on the body of domestic and wild pigs. Although outbreak has been reported in India since 1987, virus isolation and genetic characterization remained elusive. In September 2013, an outbreak of acute skin infection occurred in piglets in a commercial piggery unit at Rohtak district in Haryana, India. The presence of SWPV in scab samples collected from piglets succumbed to infection was confirmed by virus isolation, PCR amplification of SWPV-specific gene segments and nucleotide sequencing. Phylogenetic analysis of host-range genes of the SWPV revealed that the Indian isolate is genetically closely related to reference isolate SWPV/pig/U.S.A/1999/Nebraska. To the best of our knowledge this is the first report on isolation and genetic characterization of SWPV from pigs in India.

Aim: The present study was investigated to ascertain the epidemiological status of fowl typhoid (FT) in broilers in some parts of Haryana during January 2011 to December 2013. Materials and Methods: To elucidate the epidemiological status of FT in broiler chickens for the 3 years (2011-2013) and to study the prevalence of various Salmonella serovars in poultry on the basis of culture characteristics, biochemical features, serotyping, and their antibiogram profile from some parts of Haryana (India). Results: A total of 309 outbreaks of FT were recorded in chickens during this period. Overall percent morbidity, mortality, case-fatality rate (CFR) in broiler chicks due to FT during this period was 9.45, 6.77, and 71.55. The yearly observations were divided into quarters A (January-March), B (April-June), C (July-September) and D (October-December). Maximum number of outbreaks - 106 (34.3%) was recorded in quarter D followed by quarters B - 84 (27.3%), C - 64 (20.7%), and A - 55 (17.7%). Salmonella isolates (253) were recovered from disease outbreaks in broilers from different parts of Haryana. Typical morphology and colony characters on MacConkeys Lactose Agar and Brilliant Green agar, biochemical reactions, serotyping along with antibiogram profiles were able to group these isolates into 3 groups namely Salmonella Gallinarum (183), Salmonella Enteritidis (41) and Salmonella Typhimurium (29). The antibiogram pattern of 183 isolates of S. Gallinarum revealed that most of the isolates were sensitive to gentamicin (76%) followed by amikacin (72%), kanamycin (71%). Conclusion: FT is prevalent in commercial broiler flocks in different parts of Haryana and is responsible for considerably high morbidity and mortality in affected flocks. Isolation of S. Gallinarum (9, 12:183) from FT cases suggest it to be the primary pathogen, however, isolation of S. Typhimurium and S. Enteritidis from these cases is a major concern. The detection of S. Enteritidis and S. Typhimurium from FT cases assumes significance from public health point of view.

bacterium widely distributed in the soil and water. It is considered as an important enterotoxigenic food borne pathogen causing diarrhoeal and emetic food poisoning (Johnson, 1984; Kramer and Gilbert, 1989). Apart from gastroenteritis, B. cereus has a role in causing variety of non-gastro intestinal tract infections such as meningitis, endopthalmitis, endocarditis, periodontitis, osteomyelitis, wound infections and septicaemia in humans (Schoeni et al., 2005). The presence of B. cereus in pasteurized milk affects its shelf life and poses other defects such as off flavors, sweet curdling and bitty cream. This organism is also responsible for spoilage of different food products (Meer et al., 1991). Public health significance of its presence in pasteurized milk is due to its heat resistance and potential pathogenic character.

A total of 87 samples (40 samples from enteritis-affected birds and 47 samples were from healthy birds) of intestinal contents from commercial broiler chickens were processed for isolation of Clostridium perfringens. Of these, 32 (68.08%) samples from healthy birds and 34 (85%) samples from diseased birds yielded C. perfringens. Thirty C. perfringens isolates each from healthy birds and enteritis-affected birds were subjected to the PCR for the detection of α toxin (cpa), β-2 toxin (cpb2) and NetB (NetB) toxin gene using gene specific primers. Of the 30 isolates from healthy birds; 10, 2 and 7 isolates were positive for α toxin alone, β-2 toxin alone and both α and β toxins, respectively. In case of enteritis-affected birds, this number was 16, 2 and 10, respectively. None of the isolates was found positive for NetB toxin. The study indicates that α and β-2 toxin producing strains of C. perfringens are prevalent in broiler chickens in Haryana.

The present study was conducted to prepare dietary fiber rich chicken meat rolls and patties. The combination of two different sources of dietary fiber has been used viz. rice bran and psyllium husk. Three different levels of rice bran and psyllium husk viz. 10% rice bran and 2% psyllium husk, 10% rice bran and 4% psyllium husk and 10% rice bran and 6% psyllium husk were tried. The sensory characteristics such as colour, flavour, tenderness, juiciness, texture and overall acceptability of fiber added chicken meat rolls and patties were studied. The sensory scores showed a decreasing trend with increasing levels of incorporation but 10% rice bran and 4% psyllium husk combination was found to be suitable organoleptically.

This investigation was carried out to develop high fibre chicken meat patties using psyllium husk. Three different levels (4, 6 and 8 percent) of psyllium husk were tried. On the basis of results of physico-chemical properties, proximate composition and sensory evaluation, its incorporation at 4% level was found suitable. The patties with 4% psyllium husk had significantly higher total dietary fibre content than control. The cholesterol content was found to be significantly lower in psyllium husk incorporated patties than control. During storage at 4±1°C, the psyllium husk added patties were found to be microbiologically safe and organoleptically acceptable up to 15 days. It can be concluded that addition of psyllium husk at 4% level increases dietary fibre content of patties without deteriorating quality parameters.

In the present study, 69 pork samples including 23 each of pig tongue, lymphoid tonsils and ground pork were procured from local market and processed for isolation of Yersinia enterocolitica using both direct broth enrichment and cold broth enrichment techniques. In both enrichment procedures, before inoculation on selective agar medium, inoculum was subjected to alkali treatment with 0.5% KOH in saline to inhibit the non-yersinia organisms. Of the 69 samples, twelve (17.69%) were positive by the culture methods including three (13.0%) of pig tongue, eight (34.78%) of lymphoid tonsils and one (4.35%) of ground pork. The positive samples included six (8.69%) by direct broth enrichment and one (1.44%) by cold broth enrichment method alone and five (7.25%) were positive by both the methods. The number of pork samples positive for Y. enterocolitica by direct broth enrichment was more (11; 15.94%) as compared to cold broth enrichment (6; 8.69%). The present investigation, thus, revealed that isolation rate of Y. enterocolitica from pork samples was better with direct broth enrichment than with cold broth enrichment.

The present study was envisaged to prepare dietary fiber rich chicken meat rolls and patties. The combination of two different sources of dietary fiber viz. black gram hull and psyllium husk has been used. They were added in three different levels viz. 5% black gram hull and 2% psyllium husk, 5% black gram hull and 4% psyllium husk and 5% black gram hull and 6% psyllium husk. The sensory characteristics such as colour, flavour, tenderness, juiciness, texture and overall acceptability of fiber added chicken meat rolls and patties were studied. In general, the sensory scores showed a decreasing trend with increasing levels of incorporation but 5% black gram hull and 4% psyllium husk combination was found to be suitable organoleptically.