Isolation, Characterization and Pathogenesis of Capripox Virus

Abstract

This study was conducted in selected areas of Punjab, namely northern, central and southern regions, to investigate the Goatpox virus under a series of studies: i) prevalence of pox lesions in goats in Punjab (and sheep for comparison purposes); ii) in a subset of goats, conducted confirmation of the lesions as Goatpox virus using virus isolation, cell culture, and PCR, and optimization of PCR; and iii) using the field-isolated Goatpox virus, follow the pathogenesis, histopathology and immunohistochemistry of the Goatpox virus in experimentally infected goats. Study i): For this purpose, the samples were collected from four types of sources, slaughter houses, tanneries, cattle markets and hide markets, located in three regions, northern, arid and southern regions of Punjab, Pakistan. Total samples collected for goatpx from the various regions were 15452, 7231 and 8791, respectively. The overall prevalence of pox disease observed in goats at cattle markets of northern irrigated, arid and southern irrigated regions was 5%, 5.79% and 5.34%, respectively. Similarly for sheeppox 2831, 3112 and 3341 samples were collected from northern, arid and southern regions. Sheep, pox disease observed in cattle markets was 3.133%, 4.11% and 2.67% from northern irrigated, arid and southern irrigated regions, respectively. At slaughter houses, the highest prevalence of pox lesions in goats was 9.93% in arid regions followed by 8.69% and 7% in southern and northern irrigated regions, respectively. The prevalence of pox disease in sheep at slaughter house was highest (8.54%) in the northern irrigated region, and 7.69% and 6.62% in arid and southern irrigated regions, respectively. The prevalence of pox lesions recorded in sheep in the hide markets was highest in arid regions (7.29%), followed by southern irrigated region (6.22%) and northern irrigated regions (3.84%). In sheep, the overall prevalence of pox disease in hide markets was 0.51%, 4.44% and 1.66% in northern irrigated, arid and southern irrigated regions, respectively. The prevalence of pox lesions in goats in tanneries was 3.96%, 4.06% and 4.09% in northern irrigated, arid and southern irrigated regions, respectively. In sheep, the prevalence of pox disease in tanneries was 9.58%, 2.41% and 10% in northern irrigated, arid and southern irrigated regions, respectively. The prevalence of pox disease in goats was significantly different (P< 0.05) in various ecological zones included in the study, while the prevalence of pox lesions in sheep was insignificantly different (P>0.05) in various ecological zones included in the study. It was concluded that the prevalence of pox disease was more at slaughter houses as compared to other collection sources, probably due to attraction of sick/ culled animals for slaughtering purpose. Furthermore it was concluded that i the disease was more prevalent in arid region, followed by southern and northern regions, which were probably due to the climatic stress and poor socio economic status of the owners leading to depress immunity. Disease affects the quality of skin and causes economic losses to leather industries as the prevalence was higher in the low grade skin examined in the hide markets Study ii): From the goats with pox lesions in Study i, 100 clinical specimens of skin lesions were randomly collected from goatpox virus suspected field cases and were investigated by virus isolation in vero cell culture and PCR tests for confirmation of the isolated virus. These samples were consisting of 55 scabs and 45 skin tissue samples. The cell culture results were positive in 60% of scab samples from cattle markets, 20% from hide markets and tanneries and 40% from slaughter houses. The cell culture results were positive in 100% of skin tissue samples from cattle markets, 30% from hide markets and tanneries and 60% from slaughter houses. Total cell culture results for scabs and skin tissue samples from all sources were 41.82% and 51.11%, respectively. Total PCR results for scabs and skin tissues from all areas were 76.36% and 75.56%, respectively. The results of both cell culture and PCR were compared and were found that cell culture gave 46% while PCR gave 76% positive results in cattle markets, hide markets and tanneries and slaughter houses. The primer OLIGO KS-1. 5: KS 1. 6 offered good detection of 149 bp product for Capripoxvirus infection. The specific 149 bp PCR products were visualized on agarose gel electrophoresis. It was concluded that PCR is a good technique as compared to other techniques. Study iii): After confirmation through PCR, the pathogenesis of Goatpox virus was recorded by inoculating the virus in two groups of 4 goats each through two different routes, intratracheal (Group C) and intradermal routes (Group B). Maximum temperature was noted on day 13, then it gradually decreased until day 29 when it attained the normal temperature. After recording the complete daily clinical picture, one goat from all groups was slaughtered at weekly interval for recording gross and histopathological changes. The samples of different organs from all goats of infected and control groups were subjected to detailed histopathological studies and immunohistochemistry. Immunohistochemical (IHC) examination was performed on tissues including skin, lungs, lymph node, liver, spleen, kidney and intestine from all goats of infected and control groups but only skin, lymph nodes and lungs of infected goat gave positive reactions. The positive reaction in skin and lymph nodes was found in all goats except 1 of both infected groups. While the lung positive reaction was mostly found in the goats infected intra-tracehaly(but also in those goats infected intra-dermally but were slaughtered at 3rd and 4th week of post-inoculation). From the present study, it was concluded that the incubation period of disease was 5 to 7 days after inoculation of virus via intratracheal and intradermal routes. The histopathological changes were more severe in the lungs in the late period of the disease. During the experimental trials, it was found out that the pox disease adopted the same pattern of pathogenesis in experimental animals as in natural outbreaks, and the tracheal route of inoculation produced more severe disease. Immunohistochemistry was successfully used for detection of goat pox viral ii antigen. Reaction sites in the germinal centers of lymph nodes, alveolar macrophages of lungs and dermal leukocytes were found, indicative of disease confirmation. In conclusion, it was indicated from the present study that pox disease is a widespread disease goats (and sheep) in Pakistan, often caused by Goatpox virus, as confirmed by PCR and cell culture, and the disease was efficiently reproduced in experimental animals that developed the acute form of the disease, occasionally with lethal outcomes. The immunohistochemistry technique was a rapid and sensitive tool for diagnosis of Goatpox virus and the immunohistochemical technique was tested for the first time in Pakistan for detection of Goatpox virus antigen within tissues. Key Words: Capripox virus, PCR, Immunohistochesitry, Histopathology, Cell Culture.