PATHOGENESIS, MOLECULAR CHARACTERIZATION, CHEMOTHERAPY AND VACCINE DEVELOPMENT FOR MYCOPLASMOSIS IN SMALL RUMINANTS

Abstract

The project was designed to investigate the pathogenesis, molecular characterization, chemotherapy and vaccine development for Mycoplasmosis in small ruminants. The project was completed in four different studies as described below: In study-1, the isolation and molecular characterization of Mycoplasma spp. was carried out in small ruminants suffering from respiratory syndrome in natural infection suspected for Contagious Caprine Pleuropneumonia (CCPP). A total of 1980 samples were collected from different sources comprising of nasal discharge (n=1500), tracheal swab (n=300), lungs tissue (n=147) and pleural fluids (n=33) from animals exhibiting respiratory signs suspected for CCPP. A detail history about specie, age, sex of animals was also documented on preformed questionnaire. Out of total, 737 (37.22%) were positive for Mycoplasma growth, showing mass turbidity, whirling movement in culture broth and typical fried egg colonies on agar media. The results revealed that significantly (P˂0.001) higher isolation rate (% age) of Mycoplasma isolates was noted in northern zone (43%) followed by southern (34.6%). Similarly, significantly (P˂0.01) higher ocuurenace was observed in goats (58.8%) as compared to sheep (41.2%). On PCR analysis, a total of 553 (27.92%) isolates were confirmed as Mycoplasma with species distribution of 13.53%, 5.5% and 7.97% Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capripneumoniae and Mycoplasma putrefaciens, respectively. It was revealed that highest isolates were recovered from pleural fluids (63.6%) followed by lungs tissue (58.5%), nasal discharge (25.5%) and least from tracheal swab (21%). The phylogenetic study of all the three species were also documented having distinct nucleotide sequence as compared with the available isolates at National Center for Biotechnology Information (NCBI). This revealed the local isolates of Mycoplasma capricolum subsp. capripneumoniae (Mccp) different from the strains of USA and France but having close homology with the strain of neighboring countries i.e. China and India. In study-2, a total of 1800 diseased and 180 necropsied animals were surveyed for recording of the clinico-pathological picture of contagious caprine pleuropneumonia (CCPP) in naturally infected sheep and goats. Out of total examined animals, pneumonia was recorded in 61.55% followed by pyrexia (58.2%), cough (56.83%) watery nasal discharge (52.22%) and lacrimation (40.77%). The most frequent lesions were recorded in the lungs 53.88%, followed by trachea 37.7% and pleural effusion 18.33%. On histopathological examination majority of lung sections showed emphysema, atelectasis, thickning of alveolar wall and extensive leukocytic infiltration. Some section also showed chronic inflammatory changes consisted of aggregation of xii macrophages, fibroblast and plasma cells. The multi-systemic involvement is the common feature of the present findings. The internal organs including liver, spleen, kidneys and intestine revealed congestion, hemorrhages and leukocytic infiltration. Few brain sections showed mild congestion and few inflammatory cells; however most of the brains were showing normal histological details. The gross and microscopic lesion scoring revealed that maximum lesions were observed in respiratory tissue. The overall lesion scoring indicated more severe nature of disease in goat as compared to sheep. In study-3, chemotherapeutic trials were conducted to investigate the effects of commonly used antimicrobial agents and three indigenous medicinal plants against the local isolates of Mycoplasma recovered from small ruminants. Five different commercially available antimicrobial agents like tylosin, oxytetracycline, enrofloxacin, gentamycin and ceftofer sodium and three medicinal plants including Calotropis procera, Azadirachta indica and Artemisia herba-alba were tested invitro as broth micro dilution and disc diffusion assay. The results of disc diffusion assay revealed that maximum zone of inhibition 19.0±0.71 mm was produced by enrofloxacin, followed by gentamycin 11.0±0.45 mm and tylosin 6.8±0.37 mm against Mmc. The isolates showed resistance against oxytetracycline and ceftofer sodium. The results of broth micro dilution revealed that enrofloxacin exhibited strong antibacterial activity with minimum inhibitory concentrations (MICs) value of 0.001, 0.001 and 0.01 mg/mL against Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mycoplasma putrefaciens (Mp), respectively. Gentamycin was moderately effective against all isolates of Mycoplasma. Tylosin, oxytetracycline and ceftofer sodium exhibited high MICs value against the tested isolates. Among the tested methanolic plant extracts A. herba-alba showed maximum zone of inhibition 16.3±0.33, 14.0±0.44 and 15.4±0.12 mm at 30 mg against Mmc, Mccp and Mp, respectively. It was concluded that enrofloxacin is the most potent agent for the treatment of caprine mycoplasmosis. Among the tested medicinal plants A. herba-alba was showing high anti-mycoplasmal activity against the local tested isolates of Mycoplasma. The study-4 was aimed to prepare a saponized vaccine from the local isolates of Mycoplasma mycoides subsp. capri (Mmc). The PCR confirmed local isolates of Mmc having 0.2 mg/mL protein content was inactivated with saponin at the dose rate of 3.0 mg/mL. The indigenous saponized vaccine and commercially available lyophilized Mmc vaccine were inoculated in experimental animals consisted of sheep and goats for evaluation and comparison of its immunogenic potential. In sheep the maximum antibodies titer was achieved with geometric mean titer (GMT) values of 147.1 and 128 for saponized and lyophilized vaccine on day 35 post vaccination. The antibodies titer with highest GMT values of 224 and 192 was recorded on day 28 post vaccination in a challenged group vaccinated with saponized and lyophilized vaccine, respectively. No abnormal signs were observed in all experimental animals throughout the experimental trial. This study confirmed that the vaccine prepared from local field strain of Mmc confer better protection as compared with the commercially available vaccine. Key words: Mycoplasma, goat, nasal discharge, lungs, PCR, lesions, antimicrobial agents, disc diffusion, medicinal plants, saponin, vaccine.