The Prevalence and Molecular Characterization of Bovine Babesia Species and the First Report of B. bovis from Kashmir Himalayas.

Background: Bovine babesiosis, a global disease, has not been studied so far in Kashmir valley, which is having temperate type of climate as compared to rest of India having tropical to sub-tropical climate, so we felt the need to investigate it. Methods: To diagnose the babesiosis in clinically suspected cattle (n=450), peripheral blood film examination and PCR tests using generic and species-specific primers targeting Babesia/Theleria genera and B. bigemina, B. bovis as well as B. divergens, respectively were conducted. Four PCR products were sequenced and subjected to BLASTn analysis. Ticks were collected from the clinically suspected animals and identified as per the standard morphological keys. Results: The prevalence of babesiosis among suspected cattle in central Kashmir by peripheral blood film examination and PCR technique was 11.11% and 33.62%, respectively. The 18S rRNA gene of Isolate B1 of Babesia spp. showed 99.0 to 100% nucleotide sequence homology with 18S rRNA gene of different isolates of B. bigemina registered in the GenBank, while as 18S rRNA gene of Isolate Z showed 98.5 to 99.2% and 93.1 to 93.9% nucleotide sequence homology with 18S rRNA gene of different isolates of Babesia spp. and B. bigemina, respectively, registered in the GenBank. Rhipicephalus spp. and Haemaphysalis spp. were the two major tick genera identified in the present study. Conclusion: Bovine Babesiosis in Kashmir is attributed to B. bovis, B. bigemina and some other Babesia spp. or strains which needs further investigation. To our knowledge, this is the first report of Babesia bovis from northern India in cattle.

Bio-typing of Mycobacterium avium subspecies paratuberculosis isolates recovered from the Himalayan sheep and goats.

Information on bio-type profile of Mycobacterium avium subspecies paratubeculosis (MAP) in sheep flocks and goat herds of Himalayan region is not reported earlier. The aim of our study was to determine the bio-type of MAP infecting livestock of this region. A total of 71 faecal samples (sheep-57, goats-14) were screened by Ziehl-Neelsen (ZN) staining and IS900 PCR, and then processed for culture on Herrold's egg yolk medium (HEYM) having mycobactin J (MJ). Out of 71 faecal samples, MAP colonies were seen only in four samples (sheep-3 and goat-1). Isolates were confirmed as MAP on the basis of slow growth, acid fastness, MJ dependency, IS900 and IS1311 PCR. All the IS900 and IS1311 PCR positive samples were bio-typed by IS1311 PCR-REA (restriction endonuclease analysis), which confirmed all four isolates as 'bison type.' In IS1311 based phylogeny of MAP isolates by ClustalW method of the MegAlign program of DNASTAR Lasergene software, the four sequences of MAP isolates (NCBI sequence nos. MH988763, MH988765, MH988766 and MH988764) did not show any distinct clustering/grouping pattern. However, these four isolates showed a bit of closeness to the MAP sequences (KC990353.1 and KC990352.1) of 'bison type' isolated from wood bison in Canada. In conclusion, this is the first report on isolation and bio-type profile of MAP infecting sheep and goats of Himalayan region. Study will help in devising prevention and control strategies against spread of MAP infection in livestock population of Himalayan region.

Novel Candidates for Vaccine Development Against Mycoplasma Capricolum Subspecies Capripneumoniae (Mccp)-Current Knowledge and Future Prospects.

Exploration of novel candidates for vaccine development against Mycoplasma capricolum subspecies capripneumoniae (Mccp), the causative agent of contagious caprine pleuropneumonia (CCPP), has recently gained immense importance due to both the increased number of outbreaks and the alarming risk of transboundary spread of disease. Treatment by antibiotics as the only therapeutic strategy is not a viable option due to pathogen persistence, economic issues, and concerns of antibiotic resistance. Therefore, prophylactics or vaccines are becoming important under the current scenario. For quite some time inactivated, killed, or attenuated vaccines proved to be beneficial and provided good immunity up to a year. However, their adverse effects and requirement for larger doses led to the need for production of large quantities of Mccp. This is challenging because the required culture medium is costly and Mycoplasma growth is fastidious and slow. Furthermore, quality control is always an issue with such vaccines. Currently, novel candidate antigens including capsular polysaccharides (CPS), proteins, enzymes, and genes are being evaluated for potential use as vaccines. These have shown potential immunogenicity with promising results in eliciting protective immune responses. Being easy to produce, specific, effective and free from side effects, these novel vaccine candidates can revolutionize vaccination against CCPP. Use of novel proteomic approaches, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, immunoblotting, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, tandem mass spectroscopy, fast protein liquid chromatography (FPLC), bioinformatics, computerized simulation and genomic approaches, including multilocus sequence analysis, next-generation sequencing, basic local alignment search tool (BLAST), gene expression, and recombinant expression, will further enable recognition of ideal antigenic proteins and virulence genes with vaccination potential.

Comparative evaluation of different therapeutic protocols for contagious caprine pleuropneumonia in Himalayan Pashmina goats.

Therapeutic management of contagious caprine pleuroneumonia (CCPP) involves mostly the use of oxytetracycline followed by enrofloxacin and rarely tylosin. In many parts of the world including India, the former antibiotics are commonly available than the latter. Therefore, prolonged use of the same leads to the development of antibiotic resistance and decreased efficacy of drug. Besides, inflammatory and allergic pathogenesis of CCPP envisages combination therapy. In this study, we evaluated the effectiveness of the combination therapy using different antibiotics (oxytetracycyline @ 10: group I, enrofloxacin @ 5 group II, and tylosin: group III, @ 10 mg/kg body weight), along with anti-inflammatory (meloxicam @ 0.5 mg/kg) and anti-allergic (pheneramine maleate @ 1.0 mg/kg) drugs. These drugs were given intramuscularly at the interval of 48 h for four times in three test groups (n = 10) of Pashmina goats, viz. groups I, II, and III, respectively, affected with CCPP. Group IV (n = 10) was kept as healthy control when group V (n = 10) treated with oxytetracycline @ 10 mg/kg alone was used as positive control. Clinical signs, clinical parameters, pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α)), and oxidative stress indices (total oxidant status (TOS), total antioxidant status (TAS)) were evaluated at hours 0, 48, 96, and 144 of experimental trial. Tylosin-based combination therapy resulted in a rapid and favorable recovery resulting in restoration of normal body temperature (102.46 ± 0.31 °F), respiration rate (16.30 ± 0.79 per minute), and heart rate (89.50 ± 2.63 per minute) compared to the oxytetracycline (102.95 ± 0.13, 21.30 ± 1.12, 86.00 ± 2.33, respectively) and enrofloxacin (102.97 ± 0.19, 21.00 ± 1.25, 90.00 ± 2.58, respectively) treated groups. By hour 144, all the groups showed restoration of clinical parameters of normal health and diminishing signs of CCPP, viz. fever, dyspnea, coughing, nasal discharge, weakness, and pleurodynia. Significant (P ≤ 0.05) decrease in levels of TNF-α and non-significant (P > 0.05) decrease in levels of TOS and an increase in levels of TAS were noted from hour 0 to 144 in all the test groups. Within the groups, no significant (P > 0.05) change was noted in TNF-α, TOS, and TAS levels; however, TNF-α levels were comparatively lower in group III. Hematological parameters did not differ significantly (P > 0.05). From these findings, it can be inferred that tylosin-based combination therapy is relatively better for early, rapid, and safe recovery besides minimizing inflammatory and oxidative cascade in CCPP affected Pashmina goats compared to oxytetracycline- and enrofloxacin-based therapies.