Involvement of opportunistic pathogens in the respiratory distress cases of poultry was detected from the poultry flocks scattered in some districts of Punjab, Pakistan. Serum plate agglutination (SPA) test for serological screening of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were performed separately and it revealed 80% flocks were sero-positive among respiratory distress cases. Specimen samples including trachea (n=157), lungs (n=40), spleen (n=93), oral (n=201) and nasal swabs (n=147) were collected from both sero-positive and sero-negative flocks. On the basis of multiplex PCR, the percentage of opportunistic pathogens in sero-positive flocks was found maximum with Newcastle disease virus (28.6%), followed by E. coli (24.4%), Avibacterium paragallinarum (5.7%), Infectious bronchitis virus (4.7%) and Avian Influenza (1.6%), while in case of sero-negative flocks highest distribution was found in NDV (12.6%), followed by E. coli (8.4%), Av. Paragallinarum (3.8%), IBV (3.1%) and AIV (1.1%). The Frequency of respiratory tract pathogens including NDV and E. coli in combination with MG and MS was recorded significantly high.

Key words: Avian Mycoplasma, Multiplex polymerase chain reaction, Opportunistic pathogens, Serum Plate Agglutination