Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/963
Title: BIOLOGICAL, IMMUNOLOGICAL AND MOLECULAR STUDIES ON CAPRINE MYCOPLASMA IN BALOCHISTAN
Authors: AWAN, MOHAMMAD ARIF
Keywords: Natural Sciences
Chemistry & allied sciences
Biochemistry
Issue Date: 2010
Publisher: University of Balochistan, Quetta Pakistan
Abstract: Mycoplasma species belong to the class Mollicutes, and are well known for inflicting respiratory diseases in animals including goats. Respiratory mycoplasmosis particularly caprine pleuropneumonia or contagious caprine pleuropneumonia (CCPP) is well known to inflict significant economic losses to goat production in many parts of the world including Balochistan, Pakistan. Mycoplasma diseases especially respiratory problems are characterized with fever, nasal discharge, cough, dyspnoea, pleuropneumonia with higher rates of morbidity and mortality. Caprine pleuropneumonia and associated respiratory problems caused by members of the Mycoplasma mycoides cluster members particularly with Mycoplasma mycoides subspecies mycoides large colony (MmmLC), Mycoplasma mycoides subspecies capri (Mmc), and Mycoplasma capricolum subspecies capripneumoniae (Mccp) have been known since long in Balochistan, Pakistan. The studies on the prevalence, isolation, identification, sero-molecular diagnosis, and immuno-prophylactic control for the frequently reported mycoplasma diseases are not well carried out and addressed. The clinico-bacteriological investigation for the isolation and characterization of Mycoplasma species from the nasal swabs of the randomly selected goats from fifteen districts of Balochistan has been found with varying proportions of clinical symptoms among goats with highest (27.2%) for nasal discharge and least (7.2%) for diarrhea. Overall 3.7% prevalence of Mycoplasma species by nasal swab cultures was observed in goats. Highest isolation (78.1%) was observed for single Mycoplasma species than the concurrent isolation (21.9%) of MmmLC with other Mycoplasma species. The highest rate of isolation was observed for Mmc (16%), followed by Mcc (15.9%), Mycoplasma arginini (13.7%) and concurrent infection of MmmLC with M.arginini organisms (12.2 %). Surprisingly Mycoplasma putrefacience (Mp) as 9.2 percent was isolated from the nasal swabs of goats. Statistically highest prevalence of mycoplasma species in goats by the nasal swab culture was observed in Loralai district followed by Pishin, and Quetta as compared to Nasirabad district. The gross-pathologic and bacteriological study for the lungs collected from goats slaughtered at locally organized butcher shops in districts of Balochistan has shown 11% prevalence of pneumonic lesion among goats. Statistically significant difference in prevalence of pneumonic lesions among goats between the districts was observed. Of the pneumonic lesions, highest frequency was noticed for broncho-pneumonia (31.9%) followed by fibrinous-pneumonia (23.5%), while the lowest frequency was noted for congested lungs (7.0%). Overall mycoplasmas were isolated from 4.7% of the lung samples from goats. The highest rate of isolation was 43.6% for MmmLC, 18.5% for Mmc, and 10.9% for Mcc, while lowest prevalence of 1.9% was observed for M. arginini. Statistically significant difference for the isolation of mycoplasma species between the districts was found. The clinico-pathologic and bacteriological study for the pleuropneumonia suspected goats (n=210) from fifteen districts of Balochistan has shown highest frequency of clinical symptoms for nasal discharge (82.4%) followed by cough (47.6%), anorexia (26.7%), pyrexia (20.5%), dyspnoea (19%) and swollen fore-limb joints (15.2%). The pneumonic lesions were present as 87.1% and statistically non-significant difference was observed in the occurrence of pneumonic lesions between the districts. Highest frequency of gross lesions was observed for pneumonia (87.1%), followed by lung adhesions (27.1%), and hydrothorax (8.6%). Pneumonic lesions further showed highest frequency for lobar pneumonia (29.5%), followed by fibrinous pneumonia (27.9%), and broncho-pneumonia (20.2%). Mycoplasma were positive in 31% of the samples (n=1890) from different body sites of the pleuropneumonia suspected goats. Overall, 34.23% mycoplasma isolates were recovered and characterized. The highest isolation was observed for MmmLC (43.74%), followed by Mcc (13.45%), Mmc (11.59%), and Mycoplasma arginini (7.11%). Of the pleuropneumonia suspected goats, the highest isolation of mycoplasmas was observed from the lung tissues (27.1%), followed by nose (25.3%), while, the lowest isolation was observed from intestine (1.7%). Significant difference in the isolation of mycoplasmas from different body sites of the goats was observed. The odds ratio value analysis showed higher isolation of mycoplasmas from the lung tissues followed by nose, trachea, liver, spleen, and fore- limbs joint fluid than the intestine samples. Polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and denaturing gradient gel electrophoresis (DGGE) were used for the identification of Mycoplasma species isolated from goats in Balochistan. Of the mycoplasma isolates (n=1120) previously characterized by the classical biochemical and growth inhibition tests, highest number (78%) was identified by the Mycoplasma mycoides cluster PCR, followed by Mycoplasma mycoides sub-cluster PCR (63.3%), and Mp PCR (6.3%), while none of the isolate was identified as Mccp by the Mccp-specific PCR. Among the mycoplasma isolates highest percentage was observed for MmmLC (44.8%), followed by Mmc (18.5%), and Mcc (14.7%), while the lowest was found for Acholeplasma (2.5%), followed by Mp (6.3%), and Mycoplasma arginini (13.1%). The Mycoplasma mycoides sub-cluster PCR results for MmmLC/Mmc were also validated by specific RFLP. Of the 20 mycoplasma isolates, 8 (44.4%) were specifically identified as MmmLC, 10 (55.5%) for MmmLC with unknown type of microorganism profile, while two of the mycoplasma isolates could not be analyzed by DGGE at Veterinary Laboratories Agencies (VLA), Weybridge, United Kingdom. Whole genomic DNA was extracted directly from 5760 nasal swabs (randomly selected field goats), 4522 lung tissues (goats slaughtered in butcher shops) and 210 lung tissues (pleuropneumonia suspected goats) from the goats from fifteen districts of Balochistan to highlight the prevalence of Mycoplasma species in goats using PCR reactions. The PCR results for the nasal swabs samples showed highest prevalence for Mycoplasma mycoides cluster (6.6%), followed by Mycoplasma mycoides sub-cluster (5.6%), and Mcc (1%), whereas the lowest prevalence was noted for Mp (0.7%). The PCR results for the lung tissue samples (goats slaughtered in butcher shops) has shown highest prevalence for Mycoplasma mycoides cluster (11.7%), followed by Mycoplasma mycoides sub-cluster (10.2%), and Mcc (1.6%), while the lowest prevalence was observed for Mp (0.8%). The lung samples (pleuropneumonia suspected goats) showed highest prevalence for Mycoplasma mycoides cluster (76.7%), followed by Mycoplasma mycoides sub-cluster (56.2%), and Mcc (19%), while the lowest prevalence for Mp (5.2%) was observed. Statistically highly significant difference was observed for the prevalence of Mycoplasma species in all the samples by PCRs. The PCR results for the Mycoplasma mycoides cluster and Mccp organisms were validated by specific RFLP for MmmLC/Mmc and Mccp organisms. Only 1.4% prevalence of Mccp was observed in lung tissue samples from pleuropneumonia suspected goats by the Mccp- specific PCR. Latex agglutination test (LAT) and indirect haemaggluitination (IHA) assay were standardized and used to highlight the sero-prevalence of MmmLC infection in goats in Quetta, Pishin and Loralai districts of Balochistan. Of the 50 serum samples from the healthy goats (negative for Mycoplasma mycoides cluster and Mp by PCRs and mycoplasma culture), 90% and 74% specificity was observed by LAT and IHA (titer <1:64 considered negative titer) respectively with statistically significant difference. Further, of the 59 serum samples from the pleuropneumonia suspected goats (positive for MmmLC by the PCRs and mycoplasma culture), 92% and 70% sensitivity was calculated by LAT and IHA (the cut-off value for the antibody titer ≥1:64 taken as positive) respectively with statistically significant difference. IHA and LAT has shown 18.5%, 14.3%, 13.5% and 21.9%, 18% 16.1% sero-prevalence to MmmLC in goats from Quetta, Pishin and Loralai districts in Balochistan respectively. Statistically non-significant difference by IHA, and LAT was observed in the sero-prevalence among the three districts. Statistically significant difference in the overall sero-prevalence to MmmLC by IHA (15.5%) and LAT (18.7%) was observed in three districts in Balochistan. Mycoplasma species such as MmmLC, Mmc, Mcc, Mp, Mycoplasma arginini (Marg) isolated from the pleuropneumonia suspected goats from districts of Balochistan and Mmc (PG- 3, vaccinal strain) were used by intra-nasal spray and intra-tracheal routes to reproduce experimental mycoplasma disease in goats. Only goats in groups A and B infected with MmmLC by nasal spray and intra-tracheal routes has shown moderate to severe respiratory disease characterized with pyrexia (up to 41.5oC), nasal discharge, cough, swollen and painful fore-limb joints and lameness with 100% mortality before the end of experiment. Gross-pathology revealed lung congestion, fibrinous pleuropneumonia, lung adhesions, and hydrothorax in the necropsied goats. A low grade respiratory disease characterized with slight nasal discharge and slight lameness was seen in few goats infected with Mmc, Mcc, and Mp field isolates with no mortality. The Mmc (PG-3, vaccinal strain) was also found ineffective in causing a clinical respiratory disease in the experimental goats. Of the total samples (n=420) from different body sites of the experimentally infected goats (n=42) with Mycoplasma species from 14 groups (A-N), 16.2 % mycoplasma isolates were re-isolated. Of these 79.4% (MmmLC), 4.4% (for each of Mmc and Mp), 5.9% for each of Mcc and M.arginini) were identified by the classical biochemical, growth inhibition tests and Mycoplasma mycoides cluster, Mycoplasma mycoides sub-cluster, and Mp specific PCR tests. Sero-conversion against MmmLC was seen only in goats from group A by LAT and IHA. Different concentrations of formalin and saponin inactivated MmmLC vaccines containing 2, 1, 0.5, 0.25, 0.125, 0.06 and 0.03 mg/ml protein and adjuvated with saponin (1mg/ml) or aluminum hydroxide gel (5mg/ml) were prepared, used and evaluated for efficacy in 17 groups of goats (5 goats/group). Saponin and formalin were found suitably mycoplasmacidal in 0.5 mg/ml and 0.125% concentrations at 37oC within 12 hours post-exposure respectively. Saponin is found with comparatively higher efficacy than aluminum hydroxide gel adjuvated MmmLC vaccines. Comparatively higher mean antibody titers up to (log2) of 9.5 for saponated vaccines as compared to (log2) of 6.2 for aluminum hydroxide gel based vaccine and (log2) of 4.2 for Mmc vaccine (CASVAB) were observed on 42 days post-vaccinations. The challenge and protection experiment also showed complete protection in mortality, clinical mycoplasma disease, and gross-pathological lesion only in goats for group nos. 1, 2, 3, and 4 inoculated with saponated MmmLC vaccine with 2, 1, 0.5 and 0.25 mg/ml protein for 3 weeks post-challenge period. There was a prompt and sharp increase in (log2) mean antibody titers for the MmmLC saponated vaccines as compared to the aluminum hydroxide gel based vaccines up to 3 weeks post-challenge. The antibody titers in saponated MmmLC vaccinated goats were seen with little decline but nonetheless were suitably maintained as compared to aluminum hydroxide gel based vaccines. Overall the studies carried out in this thesis have shown alarming status of MmmLC as compared to other isolated Mycoplasma species among goats. The vaccine prepared so far against MmmLC is found safe, immunogenic and protective in experimental goats but further needs trials in the field goats. Although Mccp the sole causative agent of CCPP was not isolated from the goats in Balochistan but its molecular detection by Mycoplasma mycoides cluster PCR and confirmation by Mccp-specific PCR-RFLP from the pleuropneumonia suspected goats from areas closer to Afghanistan is highly alarming and needs attempts to isolate the fastidious bacterium for further molecular epidemiological studies and preparation of an effective vaccine against CCPP, an OIE listed highly contagious and infectious disease of goats. It is anticipated that the research out comes presented in this thesis would greatly benefit the researchers, veterinarians and all those directly or indirectly involved in controlling the mycoplasma inflicted diseases.
URI:  http://prr.hec.gov.pk/jspui/handle/123456789//963
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