Studies on Pathogenesis and Molecular Characterization of Contagious Caprine Pleuropneumonia in Small Ruminants

Abstract

This study was conducted in selected areas of Khyber Pukhtoonkhwa Pakistan, namely northern, central and southern regions, with the objective to determine clinico-pathological manifestation of contagious Caprine pleuropneumonia in field outbreak and documented its pathogenesis in experimental animals. The first study included isolation and identification of Mycoplasma species from field outbreaks by usage of a selective differentiating hay flick medium, growth inhibition test and a Polymerase Chain Reaction (PCR) test. Out of 120 inoculated samples, 30% and 22.5% were positive on culture from lungs and pleura. Isolates were identified as Mycoplasma mycoides subspecies capri by a growth inhibition test and PCR. Similarly, tissue samples that were negative on culture were also subjected to PCR analysis. Out of 120 samples 62.5% and 54.16% from lungs and pleura, respectively, were positive. On statistical analysis, a significant difference (P<0.05) was found between results of PCR and culture. This difference reflects that the PCR technique is more sensitive than the culture method. Based upon these findings, disease was prevalent in almost all selected regions of province. The predominant clinical findings include pyrexia, nasal discharges catarrhal initially turned into mucopurulent in the advance stage, excessive lacremation, unilateral and bilateral conjunctivitis with corneal opacity, painful cough, dysponia, weakness, reluctant movement, extended neck, abduction of the elbow and diarrhea. The majority of animals presented pathological lesions in the form of consolidation and marbled appearance of lungs with fibrinopurulent membrane on pleural surface. Straw colored pleural fluid was present in pleural cavity with pleural adhesion, hydro pericardial fluid in pericardial sac, necrotic foci on surface of the liver and pus in the pelvis of kidneys. Histopathological lesions revealed emphysema, atelactasis with interstitial and bronchopneumonia and thickening of interlobular septa with extensive infiltration of polymorph nucleated cells. In second experiment, isolated Mycoplasma mycoides subspecies capri was inoculated into twelve goats to study detailed pathogenesis and usage of immunohistochemical techniques for detection and confirmation of Mycoplasma antigen within paraffin-embedded tissue sections. Almost all animals exhibited signs of disease. Signs of disease appeared in acute septicemic form with fever and nasal discharges on sixth day after inoculation and became more pronounced and severe at by the third and fourth weeks and then progressed to moderate and chronic forms. The pattern of disease development was similar as in a field outbreak, but was more severe in nature. On scoring clinical signs of disease, it presented a specific pattern of infection mild at the beginning and became more severe at the third and fourth weeks and then progressed to moderate and chronic forms. Similarly, gross and microscopic lesions were also recorded in selected organs. In experimental infection, the disease adopted the same pattern of clinical course as in natural outbreak. Four animals were found dead and three developed nervous signs during the course of study. Gross and histopathological lesions were recorded in almost all organs. To demonstrate Mycoplasma antigens in tissues, a special immunohistochemical technique called the labeled streptavidin biotin (LSAB) method was used with hyper-immune serum raised in rabbits against the reference species. Antigen of Mycoplasma mycoides subspecies capri was detected in tissue sections of lungs and lymph nodes. Out of 12 samples, 7 were positive for the immunohistochemical reaction. Mycoplasma antigen was detected in cytoplasm of alveolar macrophages and in the walls of alveoli. This positive result indicated the importance of these cells in host defense mechanism against Mycoplasma. The result also confirmed that the antigen was the same as that inoculated in experimentally infected animals. Samples of all infected goats were found positive by PCR for confirmation of antigens. By comparing results of IHC and PCR, significant difference (P<0.05) was found. This result revealed that PCR is a more sensitive and effective tool for confirmation of the antigen. In conclusion, it was indicated from the present study that CCPP is a wide spread disease in Pakistan, caused by Mycoplasma mycoides subspecies capri and the disease was efficiently iireproduced in experimental animals that adopted acute septicemic form with lethal outcomes. The PCR technique was a more rapid and sensitive tool for diagnosis of CCPP and the immunohistochemical technique was optimized for the first time in Pakistan for detection of antigen within tissues. Key words: Contagious caprine pleuropneumonia, growth inhibition test, PCR, Mycoplasma mycoides subspecies capri, immunohistochemistry, hyperimmune serum