Molecular epidemiology of canine parvovirus in southern India

Aim: The present study was conducted to isolate and characterize canine parvovirus circulating in Southern India by genetic analysis of VP2 capsid protein gene.Materials and Methods: In this study, 128 samples were collected from nine different locations covering five Southern Indian states (Pondicherry, Tamil Nadu, Kerala, Andhra Pradesh and Karnataka) . Out of 128 samples, 69 samples were found to be positive by PCR assay. Out of 69 positive samples, 36 were randomly selected and processed for virus isolation. Twenty viruses could be isolated successfully and 18 randomly selected isolate were subjected to VP2 gene sequence analysis along with 6 random clinical samples.Result: Seventeen isolates and 5 clinical samples were characterized as New CPV-2a (CPV2a with 297-Ser→Ala). But one isolate and one clinical sample had amino acids variations which were characteristics of New CPV-2b. The phylogenetic analysis revealed that one of the field isolates was found to be phylogenetically closely related to New CPV-2b strains of India; rest other sequences was found to share ancestral origins with New CPV-2a reference strains of Japan, China, Thailand and India.Conclusion: The present study revealed that the predominant CPV strain circulating in Southern India is New CPV-2a. There is also enough indication of New CPV-2b strain from different states of Southern India

Full-length VP2 gene analysis of canine parvovirus reveals emergence of newer variants in India

The canine parvovirus (CPV) infection is a highly contagious and serious enteric disease of dogs with high fatality rate. The present study was taken up to characterize the full-length viral polypeptide 2 (VP2) gene of CPV of Indian origin along with the commercially available vaccines. The faecal samples from parvovirus suspected dogs were collected from various states of India for screening by PCR assay and 66.29% of samples were found positive. Six CPV-2a, three CPV-2b, and one CPV-2c types were identified by sequence analysis. Several unique and existing mutations have been noticed in CPV types analyzed indicating emergence of newer variants of CPV in India. The phylogenetic analysis revealed that all the field CPV types were grouped in different subclades within two main clades, but away from the commercial vaccine strains. CPV-2b and CPV-2c types with unique mutations were found to be establishing in India apart from the prevailing CPV-2a type. Mutations and the positive selection of the mutants were found to be the major mechanism of emergence and evolution of parvovirus. Therefore, the incorporation of local strain in the vaccine formulation may be considered for effective control of CPV infections in India

PARTIAL PURIFICATION OF BACTERIOCIN LIKE SUBSTANCE FROM RUMEN LIQUOR OBTAINED FROM THE SLAUGHTERED CATTLE FOR MEDICAL APPLICATIONS

Objective: Rumen is rich source of  anaerobic microbes and these microbes has ability to produce  bacteriocin  like substances ,The present study was aimed at Partial Purification of bacteriocin like substance from Rumen Liquor obtained from the slaughtered cattle. Methods:  Rumen liquor samples were collected from slaughtered cattle   of fifteen samples collected the anti- microbial activity was seen only in those animals which were fed just before slaughter.  The samples were precipitated by 60percent ammonium sulphate saturation these precipitates were further purified using ion-exchange chromatography (SP-Sepharose) and eluted with 1.0 M sodium chloride as a single peak. The purity of protein was confirmed by SDS-PAGE analysis.Results: Antimicrobial activity was obsereved in at 60percent ammonium sulphate saturation and these The protein with anti-microbial activity was eluted with 1.0 M sodium chloride as a single peak using ion exchange chromatography, molecular size  of these protein was around 6.5kDa, . Further the purified protein was subjected for in gel tryptic digestion and nano LC-MS analysis of the peptides released followed by the identification of protein using MASCOT analysis which had shown similarity with the fimbrial protein of Pseudomonas aeruginosa (SO4440) and oligopeptide ABC transporter, ATP binding protein (B72300) ) [23] . Bacteriocin (BViA) falls within the superfamily of ABC transporters and ATP binding proteins demonstrate highest homology matches with the BViA.Conclusion: Thus the observations made in this study clearly demonstrate that the peptide purified from rumen liquor was indeed bacteriocin like substance.     KEY WORDS: Rumen liquor, anti-microbial activity, purification, protein analysi

Immuno-Detection of C3a, a C3 Complement Activated Product in Mastitis Milk, a Potential Diagnostic Marker

The sub-clinical form of mastitis is difficult to detect and causes huge economic loss to the dairy industry. It has become a threat to public health at large, thus there is a need for definite diagnosis of the disease. Therefore, this study was undertaken to identify the novel diagnostic marker for the detection of the sub-clinical form of mastitis. Two-dimensional gel analysis of the whey protein fraction of normal and mastitis milk samples revealed the presence of proteose peptone component 3 precursor, Trypsin precursor, complement component-C3, Ig heavy chain precursors and a C-type lectin domain as differentially expressed protein during the early stage of mastitis. Of these proteins identified, complement component-C3 was tested for its diagnostic potential. Western blot analysis of the milk whey of sub-clinical mastitis cases (M+, M++ & M+++) identified the accumulation of C3a, an activated product of complement component-C3. Further, the hemolytic activity of the above milk whey samples positively correlated with the somatic cell count. As C3a is already reported as an anaphylotoxic agent, it chemo tactically attracts lymphocytes at the site of inflammation, the detection of which in the milk whey can be of diagnostic importance for sub-clinical mastitis

Characterization of Haptoglobin Isotype in Milk of Mastitis-Affected Cows

Haptoglobin is a major acute phase protein in bovines and reportedly increases in serum and milk whey during mastitis, highlighting its potential as a diagnostic biomarker. Since haptoglobin is known to undergo tissue specific glycosylation resulting in different isoforms, this study was undertaken to characterize the isoforms of haptoglobin. Milk whey fraction and serum obtained from animals with or without clinical mastitis in Puducherry, India, were subjected to SDS-PAGE followed by western blot and immuno-detection of haptoglobin protein. All subunits (β, α1 and α2) of haptoglobin protein were detected in serum sample obtained from clinical cases. However, only the β-subunit was detected in milk whey fraction obtained from the respective animals. Similar results were observed with milk whey fractions from subclinical cases indicating difference in isoform of haptoglobin detected in milk whey from serum. This was further supported by RT-PCR (Reverse Transcription Polymerase Chain Reaction) analysis of haptoglobin gene (Hp) confirming the tissue specific origin of haptoglobin

Bacterial DNA Induced TNF-alpha Expression in Buffaloes (Murrah) in Comparison to that of Cross Breed Cattle

Abstract: Buffaloes are generally considered to be disease resistance. But systematic studies to understand the underlying mechanism of disease resistance in buffaloes in comparison to that of cattle are scanty. Therefore, the present study was undertaken to study the immune response in terms of TNF-alpha expression in PBMCS isolated from buffaloes in comparison to that of cattle. PBMCs were isolated from blood collected from healthy buffaloes and cross breed cattle and incubated with bacterial (E. coli) DNA at different concentration for a different period of time. Total RNA was isolated and mRNA expression of TLR9 and TNF-alpha was studied. Expression of actin gen was studied as positive control. Incubation of PBMCs with bacterial DNA resulted in the expression of TLR9 in both, buffaloes and cattle. But, the expression of TNF-alpha was seen only in the case of buffaloes and the level was found to increase with the increase in bacterial DNA concentration and time. Thus this study reports the inherent difference in the immune response of buffaloes in comparison to that of cattle

Comparative immune responses of pups following modified live virus vaccinations against canine parvovirus

Background and Aim: Canine parvovirus (CPV) is the most important viral cause of enteritis and mortality in pups. Evaluation and monitoring of pre- and post-vaccine immune responses may help to determine the efficacy of the current vaccination schedule being followed in pups in India. This study aimed to evaluate and monitor the pre- and post-vaccine immune responses of CPV vaccinated pups using hemagglutination inhibition (HI) assay. The neutralizing antibody titer levels were also detected using serum neutralization test (SNT). Materials and Methods: The pups were categorized into two groups, the double booster and the single booster groups. In this study, serum samples were subjected to HI and SNT for measuring the CPV antibody titer at frequent intervals for up to 6 months from 27 healthy pups following primary and booster CPV vaccinations. Results: The antibody titers in double booster pups reached their peaks at the 21st day after the second booster vaccination with a geometric mean (GM) of 3.57. The antibody titers in single booster pups reached their peaks at the 21st day after the first booster vaccination with a lower GM of 3.18. Conclusion: The double booster pups maintained a higher immune response throughout the period of the study compared to single booster pups though the difference in titers was not statistically significant. SNT results indicated that the raised antibody titer was also able to yield virus-neutralizing antibodies. No interfering maternally derived antibodies were found in the pups at the age of primary vaccination (45th day) in our study. Therefore, the second booster vaccination may be useful in maintaining the protective titer for a prolonged period

Methicillin resistant staphylococci associated with bovine mastitis and their zoonotic importance

Aim: The present study was conducted to determine the zoonotic importance of methicillin resistant staphylococci associated with bovine mastitis and their potential role in transmission to animal handlers. Materials and Methods: A total of 158 milk samples from bovine mastitis cases and 126 nasal swabs from the animal handlers were sampled in and around Pondicherry (Southern India). The Presence of Staphylococcal organism was confirmed by PCR amplification using the genus specific primers and among the isolated Staphylococci; methicillin resistance was identified by genetic amplification of mec A methicillin resistant gene. Then the amplified gene from the bacteria expressing the mecA gene (PBP2a) (~2kb fragment) was further sequenced using four sets of primer pairs and aligned for determining their genetic relatedness between the sequences. Both phenotypic and genotypic analysis was carried out for the six MRS isolates (three bovine and three human) in this study. Results: Out of 158 mastitis milk samples; 96 and 19 bovine isolates were found to be positive for Staphylococcal genus specific PCR and methicillin resistant (mecA) gene PCR, respectively. Similarly, Out of 126 human nasal swabs, 64 and 13 human isolates were found to be positive for Staphylococcal genus specific PCR and mec A gene PCR, respectively. Among the 160 staphylococcal isolates (Bovine and Human origin); 51 were identified as coagulase-positive staphylococci (CPS) and remaining as coagulase-negative staphylococci (CONS). The results obtained in this study revealed the presence of many species of Staphylococci but the predominant species were Staphylococcus aureus and S. epidermidis. The Sequence analysis of the mec A gene of human isolates obtained in this study had a maximum identity (99% -100%) with the bovine isolates. Conclusion: The phenotypic and genotypic analysis carried out for the six MRS (Methicillin Resistant Staphylococci) isolates in this study were indistinguishable and epidemiologically related, which may indicate the transmission of MRS between bovine and humans. The occurrence of methicillin resistance among staphylococci isolated from cases of bovine mastitis is increasing, necessitating the periodic surveillance for antimicrobial resistance patterns of Staphylococci in order to control the spread of MRS

Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.6/Nov-2013/20.pdf RESEARCH ARTICLE Open Access Seroepidemiology of canine leptospirosis by iELISA and MAT

Leptospirosis is a widespread zoonotic disease and is a real public health concern around the world. The disease is caused by spirochetes of the genus Leptospira which comprises of more than 260 serovar