Prabhakar Xavier Antony

Background: Mastitis is an economically important disease of bovine species. Among the causative agents of mastitis, Staphylococcus species are the leading cause of contagious mastitis and are also responsible for milk-borne intoxication in consumers. The study aims to assess the distribution of enterotoxins in various species of coagulase-positive and coagulase-negative Staphylococci isolated from bovine mastitis milk samples. Methods: A total of 135 mastitis bovine milk samples collected were subjected to cultural isolation followed by biochemical and molecular identification. The distribution of nine enterotoxins among coagulase-positive and coagulase-negative Staphylococcus species was done by PCR. Result: About 74.07% (100 isolates) of mastitis samples were found positive for Staphylococci of which 44% were coagulase-positive and 56% coagulase-negative. Totally sixteen different species were isolated which included two species of coagulase-positive and fourteen species of coagulase-negative staphylococci. Out of nine Staphylococcal Enterotoxin (SE) genes screened, seven SE encoding genes namely sea, seb, sec, sed, seg, sei and sej were detected among the isolates with a range of one to six SE genes per isolate; however, see and seh could not be detected in any of these isolates. About 84.09% and 96.42% of CPS and CNS isolates were shown to have SE genes respectively and a maximum of six SE genes (sea, seb, sed, seg, sei and sej) were detected in a CNS isolate belonging to S. caprae. The high prevalence of SE encoding genes in both CPS and CNS isolated from bovine mastitis cases highlights the possible risk of staphylococcal food poisoning.

Background and Aim: Diagnosis of query fever (QF) is mostly done on the basis of serological/molecular tests, due to the stringent requirement of biosafety level-3 containment facilities for isolating Coxiella burnetii in culture. QF is an important zoonosis and is considered to be an occupational hazard to livestock handlers. This report describes our study on the serological as well as molecular evidence of QF in animal handlers from Puducherry and surrounding Tamil Nadu, from where, to the best of our knowledge, no such reports are available so far. Materials and Methods: Seventy-five animal handlers were recruited, comprising veterinarians, slaughterhouse workers, butchers, and animal attendants of various government veterinary clinics from Puducherry and surrounding areas of Tamil Nadu state. QF serology was performed to identify Phase I and Phase II immunoglobulin G antibodies to C. burnetii. Nested polymerase chain reaction (N-PCR) was carried out to detect C. burnetii DNA in buffy coat samples by targeting IS1111 gene element. N-PCR-positive samples were sequenced and phylogenetic analysis was performed using MEGA software version 10.0. Results: A total of 21 animal handlers (28.1%) were positive for either serology or PCR. PCR alone was positive in 10 (13.4%), only serology was positive in 8 (10.7%), and both serology and PCR were positive in three samples (4.0%). GenBank accession numbers were obtained for 13 N-PCR-positive samples (MG548608-MG548620). Six of our study sequences showed close similarity with the reference isolates from Bengaluru, Colombia, Brazil, France, and Iran. Conclusion: A significant percentage of QF positivity in animal handlers of this part of South India, Puducherry, warrants a prospective study with follow-up of a large number of this occupational group.

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.

In the course of our Indian Council of Medical Research project on coxiellosis in Puducherry and Tamil Nadu, 5.64% goat, 1.85% sheep, 1.06% buffaloes, and 0.97% cattle were positive for Coxiella burnetii antibodies by enzyme linked immunosorbent assay kit (IDEXX, Liebefeld, Switzerland)

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.

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.

The present study was undertaken to characterize the mutation in gyrA (DNA gyrase) and parC (topoisomerase IV) genes responsible for fluoroquinolone resistance in Escherichia coli isolates associated with the bovine mastitis. A total of 92 milk samples from bovine mastitis cases were sampled in and around Puducherry (Southern India). Among these samples, 30 isolates were bacteriologically characterized as E. coli. Minimum inhibitory concentrations (MIC) of fluoroquinolones of these 30 E. coli isolates were evaluated by resazurin microtiter assay. Then, the quinolone resistance determining region (QRDR) (gyrA and parC genes) of these E. coli isolates was genetically analyzed for determining the chromosomal mutation causing fluoroquinolone resistance. E. coli isolates showed a resistance rate of 63.33%, 23.33% and 30.03% to nalidixic acid, ciprofloxacin and enrofloxacin, respectively. Mutations were found at 83(rd) and 87(th) amino acid position of gyrA gene, and at 80(th) and 108(th) amino acid position of parC gene in our study isolates. Among these five isolates, one had a single mutation at 83 amino acid position of gyrA with reduced susceptibility (0.5 µg/ml) to ciprofloxacin. Then, in remaining four isolates, three isolates showed triple mutation (at gyrA: S83⟶L and D87⟶N; at parC: S80⟶I) and the fifth isolate showed an additional mutation at codon 108 of parC (A108⟶T) with the increased ciprofloxacin MIC of 16-128 µg/ml. The most common mutation noticed were at S83⟶L and D87⟶N of gyrA and S80⟶I of ParC. The study confirms the presence of mutation/s responsible for fluoroquinolone resistance in QRDR of gyrA and parC genes of E. coli isolates of animal origin, and there is increased rate of fluoroquinolone resistance with high-level of MIC. The mutations observed in this study were similar to that of human isolates.