In the first phase of the project, the sampling of diseased animals presumably affected by Black quarter was carried out from six districts of Punjab belonging to three different zones. Around two hundred and fifty samples from each zone were collected and were subjected to bacterial culturing and isolation procedures followed by biochemical identification mechanism. The prevalence of Black quarter in Cattle and buffaloes were thus calculated for each district and zone. Highest prevalence of BQ in Zone II was observed (27.2%) for cattle while in case of Buffaloes highest prevalence (3.2%) was noted in Zone I. similarly higher Prevalence of BQ was noted in 1st quarter of year for Zone I followed by zone II and III while 2nd quarter of season was showing higher prevalence of BQ in zone II and III.
During 2nd phase of experiment tissue samples were inoculated in RCM and blood agar for the re-isolation of C. chauvoei, identified on the basis of colony characteristics and later on subjected to biochemical tests for the confirmation of the isolated organism. Then it was further confirmed through Polymerase chain Reaction for the identification of the causative agent i.e. C. Chauvoei on the basis of 16S rRNA gene sequence. Another set of primers corresponding to alpha toxin gene sequence of C. chauvoeui was also used which strengthened the belief that this strain of C. chauvoei possessed alpha toxin producing ability.
During third phase of project blood samples collected were subjected to hematological estimation for buffaloes and cattle having confirmed as BQ This study revealed significant effect on RBC's count and white blood cells count (P<0.05), while Differential leukocyte count were also showing significant different as compared to Non-infected (P< 0.05). Serum samples were tested for the change in levels of different enzymes. It was found that blood-glucose level and ALT levels were not significantly higher (P>0.05) when compared with control values, Values of AST, CPK and LDH were found significantly higher (P< 0.05) in all infected animals.
Histopathology of affected muscle tissues of both cattle and buffaloes was done to study microscopic changes in the muscle fibers and surrounding tissues. Lesions were somehow disappointing as compared to the magnitude of gross lesions. There were segmental degeneration, Zenker necrosis, discrete edema, occasional neutrophils and emphysema in affected muscle.
Finally, alpha toxin (hemolysin) in culture supernatant of RCM broth was titrated against 2% washed RBC's of cattle, buffalo, sheep, goat, chicken, rabbit and mice to study the hemolytic activity of the toxin. It was found that highest percentage of hemolysis was observed in mice followed by cattle, sheep, buffalo, chicken and rabbit respectively at 25°C. Higher the dilution of toxin, lower the extent of hemolysis. At 37°C variable results were obtained. It showed the biological activity of alpha toxin is also temperature dependant.