Organism: Local isolate of A. paragallinarum was received from the Department of Pathology, BAU, Mymensingh. For the growth of A. paragallinarum in artificial media, the colony of Staphylococcus aureus is needed to supply NAD required for A. paragallinarum. A. paragallinarum was cultured in blood agar media in association with S. aureus colonies.
Inoculum preparation: One single colony of A. paragallinarum was picked up from blood agar and placed in 250 ml of nutrient broth and cultured for 48 hours. Inoculation dose of A. paragallinarum (1ml/bird) was prepared for inoculation on 14 days old chicks.
Experimental design: A total of 24 (day 1 old) chicks of cobb 500 broiler chicks were collected and equally divided into two groups (A and B, N = 12). On day 14 of age, the chicks of group A were inoculated with pure nutrient broth whereas the chicks of group B were inoculated with A. paragallinarum at the dose rate of 1ml inoculum/bird (Islam, 2010) through the intranasal route. Clinical signs observation, reisolation of A. paragallinarum and pathological study were performed at interval of day 3, 5 and 7. At each time, 4 birds were sacrificed. Different tissues were collected for histopathology in 10% formalin on day 3, 5 and 7 of post inoculation.
Gross lesion study: All the internal organs including nasal passage were examined and gross lesions were recorded. The inflammatory lesions of different organs (congestion, hemorrhage, swelling, mucus, etc.) were graded as ± = almost absence of lesion, + = mild lesion, ++ = moderate lesion and +++ = severe lesion.
Histopathological study: Decalcification of nasal bone, processing of tissues and routine H&E staining were performed. The histopathological lesions (congestion, hemorrhage, necrosis, extent of lesions, inflammatory cells etc.) in different organs were graded as ± = almost absence of lesion, + = mild lesion, ++ = moderate lesion and +++= severe lesion.
Re-isolation of Avibacterium paragallinarum: Re-isolation of A. paragallinarum was performed from nasal passage at day 7 of PI where prominent rhinitis was found following the procedures followed by Akter (2012).