Seropidemiology and Molecular Characterization of Avian Mycoplasmosis in Major Districts of Khyber Pakhtunkhwa

Abstract

Avian mycoplasmosis is a main threat to poultry industry in Pakistan and all over the World. The most important species responsible for avian mycoplasmosis are Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). MG and MS are the most virulent and economically significant pathogens of poultry. A lot of studies had been designed to identify and isolate these pathogens. Serological as well as molecular diagnostic assays have been developed for the prevalence and characterization of these wall less avian bacteria to describe the epidemiological factors involved in spreading of mycoplasma infections within and across the farms environment. The current study was designed to evaluate the serological as well as molecular prevalence of MG and MS in broiler birds in five districts of Khyber Pakhtunkhwa. The serum samples were examined by (SPA) test using stained MG & MS antigens separately. Out of total (n=648) broiler serum samples MG and MS prevalence was recorded as 35% and 16.7 % respectively. The highest prevalence of MG was recorded in Dera Ismail Khan 43.3% (65/150) followed by Tank 40% (60/150), Peshawar 36.7 % (33/90), Abbottabad 31.5% (45/143) and Mansehra 20.86% (24/115) while in case of MS the highest prevalence was reported in Peshawar 23.3% (21/90) followed by Dera Ismail Khan 20% (30/150), Mansehra 18.26% (21/115), Tank 12.7% (19/150) and Abbottabad 11.9% (17/143). In open shed the prevalence of MG & MS was comparatively high (40.5% & 17.9%) than in closed shed (26.7 &14.7%) respectively. Moreover, prevalence of MG and MS was higher during winter season (38.2% &18.5%) than summer (31.8% &14.8%). Combined MG/MS prevalence percentage was recorded as Peshawar (17.8%), Dera Ismail Khan (14%), Tank (12%), Mansehra (10.4%) and Abbottabad (7.7%) and it was found slightly higher (12.7%) in winter than (11.3%) in summer. Similarly in open shed reared birds MG/MS prevalence was high (14.1%) than in closed shed (8.9%). The SPA positive 40 broiler farms were selected for specimen sampling. Tracheal, choanal, cloacal, synovial fluid and nasal swabs were collected for cultural isolation on PPLO media. After morphological studies the biochemical tests were applied for differentiation of MG and MS. Lastly confirmatory study was conducted through PCR assay. PCR technique was used for molecular characterization of MG and MS using species specific primers for the xv amplification of 16SrRNA gene of MG and vlhA gene of MS. As a result of PCR amplification an amplicon of 185bp was generated for MG and for MS an amplicon of 392bp was produced. In PCR study three MG isolates (MG-1 MG-2 and MG-3) were recovered from field samples. Similarly two MS isolates (MS-1 & MS-2) were isolated from specimen samples. The heterologous and homologous cross reactivity of these isolates was studied in rabbits using Haemagglutination inhibition (HI) test. The minimum antibody titer against MS-1 antigen in Group I MS rabbits was 1:32 and 1:64 was maximum titer while in case of Group II MS rabbit antibody titer ranged between 1:32 and 1:64. The antigenic cross reactivity existing among the indigenous isolates of MS was also evaluated. The homologous antibody titers against MS-1 and MS-2 was same as 42.2. While the heterologous GMT value for MS-1 was low (27.9) and in case of MS-II it was 38.6. No titer was noticed in control group III. The field isolates of MS also showed strong cross immunity. This feature of cross immunity in field isolates may be useful in immunization against MS.