Molecular Detection of Hemoplasma in animals in Tamil Nadu, India and Hemoplasma genome analysis.

Hemoplasma are small pleomorphic wall-less Gram-positive bacteria that infect erythrocytes of various mammalian hosts. They generally cause asymptomatic or chronic anaemia but occasionally causes overt life-threatening hemolytic anaemia. In the present study, 316 cattle, 115 sheep, 61 goats and 6 buffalo blood samples were collected from various villages or organized farms located in nine districts of Tamil Nadu to detect the hemoplasma by PCR. Overall prevalence of 43.04%, 65.22%, and 44.26% hemoplasma DNA was observed in cattle, sheep and goats, respectively. In total, 21 hemoplasma positive samples were sequenced for 16S rRNA gene which revealed 8 Mycoplasma wenyonii, 11 'Candidatus Mycoplasma haemobos' and one Mycoplasma ovis infection. Sheep blood samples from Chennai district were infected with 'Ca. M. haemobos' whereas sheep sample from Thiruvannamalai district was infected with M. wenyonii. At least 50% genes in the hemoplasma genomes were paralogous genes whose functions were not known. Only 'Ca. M. haemolamae' genome contained one primitive CRISPR system without any cas genes. Antimicrobial resistance genes (ARG) could not be identified in any of the hemoplasma genomes but homologous ARG were identified in all the genomes. Adhesion related gene EF-Tu was detected in all 14 hemoplasma genomes but enolase gene was detected only in 'Ca. M. haemohominis' SWG34-3 genome. This is the first report on the prevalence of hemoplasma infection in cattle, sheep and goat in India.

Staphylococcus coagulans possesses many virulence factors of Staph. aureus and Staph. pseudintermedius.

AIMS: To understand the Staphylococcus coagulans prevalence in causing skin infections in dogs and detection of various virulence genes in Staph. coagulans isolates. METHODS AND RESULTS: Staph. coagulans was isolated from pus swabs collected from dogs with skin infection and identified by detecting thermonuclease, coagulase, and urease genes. The presence of methicillin-resistant gene (mecA) was performed by PCR. Antimicrobial susceptibility test was carried out by disc diffusion method. In total, 38 Staph. coagulans clinical isolates and 42 Staph. coagulans genomes available in NCBI database were screened for 19 virulence genes by PCR and in silico prediction, respectively. A prevalence of 13.8% (38/275) of Staph. coagulans dog skin infection was observed and 15.8% (6/38) of Staph. coagulans isolates carried mecA gene. Many Staph. coagulans isolates were susceptible to all tested antimicrobials. Twenty nine per cent isolates were resistant to ciprofloxacin. Genes encoding leukotoxins, DNase, exfoliative toxin, superantigen-like exotoxin, immunoglobulin-binding proteins, fibrinogen-binding proteins, autolysin, and rod shape-determining protein were detected in almost all the Staph. coagulans clinical isolates and genomes from NCBI database, whereas anti-adhesin plasma-sensitive protein genes were present in relatively lesser number of Staph. coagulans clinical isolates and genomes from NCBI database. CONCLUSIONS: Staph. coagulans possesses many virulence factors that are present in other coagulase-positive staphylococci, such as Staph. aureus and Staph. pseudintermedius. The presence of two bi-component leukotoxin genes in tandem with other virulence factor genes in a single pathogenic island in the Staph. coagulans genomes explained their eminence in the virulence of Staph. coagulans causing infections. Staph. coagulans was classified as a separate species in the year 2020 and primarily causes skin infections in dogs. Identification of this species is not included in any of the automated bacterial identification systems. Hence, many veterinary laboratories do not have a strategy to identify this bacterium. This study will help in the identification of Staph. coagulans in veterinary laboratories by PCR apart from detecting various virulence factors present in this pathogen. The existence of many virulence factors and prevalence in different animals in varied geographical locations suggest that Staph. coagulans is an important coagulase-positive staphylococcal pathogen in animals.

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38 nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

Defined Antigen Skin Test for Bovine Tuberculosis Retains Specificity on Revaccination With Bacillus Calmette-Guérin.

The Bacillus Calmette-Guérin (BCG) vaccination provides partial protection against, and reduces severity of pathological lesions associated with bovine tuberculosis (bTB) in cattle. Accumulating evidence also suggests that revaccination with BCG may be needed to enhance the duration of immune protection. Since BCG vaccine cross-reacts with traditional tuberculin-based diagnostic tests, a peptide-based defined antigen skin test (DST) comprising of ESAT-6, CFP-10, and Rv3615c to detect the infected among the BCG-vaccinated animals (DIVA) was recently described. The DST reliably identifies bTB-infected animals in experimental challenge models and in natural infection settings, and differentiated these from animals immunized with a single dose of BCG in both skin tests and interferon-gamma release assay (IGRA). The current investigation sought to assess the diagnostic specificity of DST in calves (Bos taurus ssp. taurus × B. t. ssp. indicus; n = 15) revaccinated with BCG 6 months after primary immunization. The results show that none of the 15 BCG-revaccinated calves exhibited a delayed hypersensitivity response when skin tested with DST 61 days post-revaccination, suggesting 100% diagnostic specificity (one-tailed lower 95% CI: 82). In contrast, 8 of 15 (diagnostic specificity = 47%; 95% CI: 21, 73) BCG-revaccinated calves were positive per the single cervical tuberculin (SCT) test using bovine tuberculin. Together, these results show that the DST retains its specificity even after revaccination with BCG and confirms the potential for implementation of BCG-based interventions in settings where test-and-slaughter are not economically or culturally feasible.