Incidence of subclinical mastitis and prevalence of major mastitis pathogens in organized farms and unorganized sectors.

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

Salmonella from Farm to Table: Isolation, Characterization, and Antimicrobial Resistance of Salmonella from Commercial Broiler Supply Chain and Its Environment.

Antimicrobial resistance (AMR) in poultry production chain is one of the major food safety concerns due to indiscriminate usage of antibiotics and the presence of pathogens such as Salmonella which causes infections in various stages of production. In the present study, 182 samples were collected from commercial broiler supply chain, viz., three hatcheries (n = 29), three commercial broiler farms (CBF; n = 99), and three retail meat shops (RMS; n = 54), and used for isolation and identification of Salmonella using three different selective agar media and a selective enrichment medium followed by PCR confirmation targeting the hilA gene. The overall prevalence of Salmonella was 47/182 (25.82%), and a significantly higher (P < 0.05) prevalence was observed in retail meat shops (46.29%), CBF (19.19%), and hatcheries (10.34%). Comparison of three agar media for isolation of Salmonella revealed that all the media were equally selective. However, PCR amplification of hilA gene fragment was significantly higher (P < 0.01) in selective enrichment culture tetrathionate brilliant green bile broth (TTB) as compared to all solid (agar-based) media. Susceptibility pattern against most frequently used antibiotics revealed that 100% of the isolates were resistant to at least one antibiotic. High resistance was observed for doxycycline (94.34%), followed by cefpodoxime (84.91%), ciprofloxacin (72.64%), gentamicin (65.09%), enrofloxacin (61.32%), colistin sulphate (40.42%), amikacin (34.91%), ampicillin (33.96%), neomycin (33.02), cefotaxime (30.19%), ceftazidime (29.25%), trimethoprim-sulfamethoxazole (23.58%), amoxicillin+clavulanic acid (21.70%), and chloramphenicol (12.26%); 16.98% of the isolates were ex-tended spectrum ß-lactamase (ESBL) producers, and 76.41% were multidrug resistant (MDR). MDR Salmonella were significantly higher (P < 0.01) in RMS (91.66%) followed by CBF (82.75%), whereas no MDR isolates were present in the isolates from hatcheries. The results indicated a higher prevalence of Salmonella and AMR for commonly used antibiotics in the complete broiler supply chain, especially RMS and CBF. Also, this study idicated that TTB enrichment followed by PCR and colony PCR was found to be rapid, specific and time-saving method.

Randomly amplified polymorphic DNA analysis of Staphylococcus chromogenes isolated from bovine and bubaline mastitis in Karnataka.

BACKGROUND AND AIM: In recent times, non-aureus staphylococci (NAS) have emerged as the major organisms isolated from mastitis cases in dairy animals, with a predominance of Staphylococcus epidermidis and Staphylococcus chromogenes. As compared to Staphylococcus aureus, much less is known about the molecular types or the spatiotemporal epidemiology of these NAS species. In the present study, randomly amplified polymorphic DNA (RAPD) was employed to detect genetic polymorphisms, intraspecies diversity, and epidemiology of S. chromogenes strains (n=37) isolated from bovine and bubaline mastitis cases in the state of Karnataka. MATERIALS AND METHODS: Thirty-seven S. chromogenes isolates (14 from bovines and 23 from bubaline) isolated from subclinical mastitis cases, from organized and unorganized sectors, were subjected to RAPD typing. Further, methicillin resistance was determined by cefoxitin disk diffusion method. RESULTS: The amplified DNA fragments ranged from 150 to 3000 base pairs and yielded several RAPD profiles. Further analysis using Digital Image Correlation Engine correlation coefficient and UPGMA method showed that the 37 isolates could be classified into 12 distinct RAPD types (A to L) at 62% similarity (D=0.889). Four of the most predominant RAPD types, B, A, C, and E, in that order, and together, represented 65% of the isolates. High diversity was observed among the isolates both within farms and between geographic locations. Most of the isolates exhibited methicillin resistance. This is the first such report from India. CONCLUSION: In the absence of defined multilocus sequence type protocols or sufficient sequences available in the public domain, RAPD can be employed to determine genetic diversity of S. chromogenes isolates.

Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin.

6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC50 values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 µM, respectively. Likewise, IC50 values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 µM, respectively. A combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.

Genetic clustering of recent classical swine fever virus isolates from Karnataka, India revealed the emergence of subtype 2.2 replacing subtype 1.1.

The phylogenetic analysis of 11 CSFV isolates from Karnataka, India obtained during the year 2012-13 was undertaken to obtain the most reliable genetic typing of the CSFV isolates based on E2, NS5B and 5'UTR genomic regions. The study indicated that all the 11 CSFV isolates belonged to subgroup 2.2. The most reliable classification was obtained with sequence data from the NS5B region which separated all the isolates based on the history of outbreak and geographic origin. Analysis of full length E2 amino acid sequences revealed different genetic makeup of Indian 2.2 isolates compared to 2.2 isolates from different countries. The group 2.2 viruses are gradually spreading as confirmed by frequent detection/ isolation of group 2.2 viruses in the recent years and replacing the subgroup 1.1 viruses, which were hitherto predominantly involved in CSF outbreaks in India.

Prevalence of classical swine fever in Karnataka, India.

AIM: The present study was conducted to know the current scenario of classical swine fever (CSF) in Bengaluru Urban, Bengaluru Rural, Chikkaballapur, Madikeri, Mandya, Bagalkot, Gadag, Yadgir, Koppal, and Bidar districts of Karnataka with the using of both antigen and antibody ELISA. MATERIALS AND METHODS: We collected 218 sera and 121 blood samples from pigs from 10 different districts of Karnataka. Screening of sera for CSF IgG antibody and whole blood for CSF virus antigen were carried out using the CSF virus (CSFV) antibody and antigen ELISA kits, respectively. RESULTS: The mean seroprevalence was 41% (89/218) and prevalence of CSFV antigen in blood samples was 32% (39/121) for the 10 districts of Karnataka. Seroprevalence of 61%, 29%, 20%, and 21%; and antigen prevalence of 40%, 50%, 13%, and 12% were recorded for Bangalore, Mysore, Belgaum, and Gulbarga divisions of Karnataka, respectively. CONCLUSIONS: The study revealed an alarmingly high prevalence of CSF, both for the antigen (32%) and antibody (41%) in Karnataka. Southern Karnataka has the highest seroprevalence (61% in Bangalore and 29% in Mysore divisions), which confirms the endemicity of the disease in that region. This could be attributed to the intensive pig farming practices in the region as compared to Northern Karnataka (Seroprevalence of 20% in Belgaum and 21% in Gulbarga divisions), where the commercial pig farming is still in infantile stages.

Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis.

Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and ß-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis.

Isolation, Biochemical and Molecular Identification, and In-Vitro Antimicrobial Resistance Patterns of Bacteria Isolated from Bubaline Subclinical Mastitis in South India.

Buffaloes are the second largest source of milk. Mastitis is a major impediment for milk production, but not much information is available about bubaline mastitis, especially subclinical mastitis. The aim of this study was to (a) investigate the application of various tests for the diagnosis of bubaline subclinical mastitis, (b) identify the major bacteria associated with it, and (c) evaluate the antibiotic resistance pattern of the bacteria. To this end, 190 quarter milk samples were collected from 57 domesticated dairy buffaloes from organized (64 samples) and unorganized (126 samples) sectors. Of these, 48.4%, 40.0%, 45.8%, 61.1%, and 61.6% were positive for subclinical mastitis by somatic cell count, electrical conductivity, California mastitis test, bromothymol blue test, and N-acetyl glucosaminidase test, respectively. As compared to the gold standard of somatic cell count, California mastitis test performed the best. However, a combination of the two methods was found to be the best option. Microbiological evaluation, both by biochemical methods as well as by monoplex and multiplex polymerase chain reaction, revealed that coagulase-negative staphylococci were the most predominant (64.8%) bacteria, followed by streptococci (18.1%), Escherichia coli (9.8%) and Staphylococcus aureus (7.3%). Most of the pathogens were resistant to multiple antibiotics, especially to ß-lactam antibiotics. We propose that California mastitis test be combined with somatic cell count for diagnosis of subclinical mastitis in domestic dairy buffaloes. Further, our results reveal high resistance of the associated bacteria to the ß-lactam class of antibiotics, and a possible major role of coagulase-negative staphylococci in causing the disease in India.

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.