~ 221 ~ The Pharma Innovation Journal 2021; SP-10(11): 221-223 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.23 TPI 2021; SP-10(11): 221-223 © 2021 TPI www.thepharmajournal.com Received: 22-09-2021 Accepted: 24-10-2021 Poornima Gumasta Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India RC Ghosh Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India Charlee Porte Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India Deepak Prajapati Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India Hemant Sahu Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India Devesh Kumar Giri Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India DK Jolhe Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India Corresponding Author Poornima Gumasta Department of Veterinary Pathology, DSVC, Kamdhenu Veterinary University, Durg, Chhattisgarh, India Isolation of Mycoplasma gallisepticum from the natural cases of chronic respiratory disease Poornima Gumasta, RC Ghosh, Charlee Porte, Deepak Prajapati, Hemant Sahu, Devesh Kumar Giri and DK Jolhe DOI: https://doi.org/10.22271/tpi.2021.v10.i11Sd.8708 Abstract Present work is a postmortem study. Total 428 dead birds, suspected to have died of chronic respiratory disease were considered for sampling. Swabs collected from the respiratory organs of the chickens and subjected for culture of the organism. Collected swabs were inoculated into PPLO broth medium allowed to incubate for 2-3 days. After removal of the contamination, culture from broth medium were inoculated over enriched PPLO agar plate. Typical “fried egg” like colonies appeared after 7 days of incubation. Colonies were confirmed as Mycoplasma gallisepticum (MG) by PCR. MG has been proven to cause serious respiratory illness in the chickens under present investigation. Keywords: Mycoplasma gallisepticum, natural cases, chronic respiratory disease Introduction Chronic respiratory disease (CRD) is a bacterial illness caused by one of the pleuro pneumonia-like organisms (PPLO). However, the organism directly linked to CRD is Mycoplasma gallisepticum (MG), which can also cause secondary problems (Ley, 2008) [1] . Chronic respiratory disease has been recorded all throughout the world, resulting in significant financial losses in huge commercial enterprises. The infection may be undetectable or cause varied degrees of respiratory distress, including rales, trouble breathing, coughing, and sneezing. In uncomplicated instances, morbidity is high and death is low (Bahatti et al., 2013) [2] . Typical visual and histological lesions, serological assays to detect antibody generation, and/or isolation and identification of the organism can all be used to diagnose avian mycoplasma infection in chickens (OIE, 2008; Uddin et al., 2010) [3, 4] . Present study incorporated the isolation of Mycoplasma gallisepticum from the suspected cases of chronic respiratory disease. Materials and Methods Total of 428 dead birds included under present investigation. Birds suspected to have died due to chronic respiratory disease were considered for sample collection. All the dead birds were subjected for detailed post mortem examination and gross lesions were recorded carefully. Swabs collected from the birds showed lesions suggestive of CRD i.e., swollen head, sinusitis, air sacculitis, pneumonic lungs and tracheitis (Figure 1). Samples collected from the nostrils, choanal cleft, mouth, trachea, lungs and air sacs from the dead birds with respiratory lesions. All work involving handling of Mycoplasma organism was performed by taking appropriate bio-safety precautions and strict aseptic conditions. Culture of Mycoplasma spp. organism Collected swabs were pooled area wise and firstly inoculated in the PPLO (Pleuro-pneumonia like organism) broth medium for the isolation of the avian mycoplasmas. Broth was prepared as per the manufacturer guidelines. Briefly, 2.1gm of PPLO broth base (Himedia, M267) was added in 70 ml of distilled water in a conical flask. Media was autoclaved at 15 lbs pressure at 121 °C for 15min. After autoclaving the media was allowed to cool down up to 45 °C. Afterwards, one vial (30 ml) of mycoplasma enrichment supplement (Himedia, FD075) was added in the autoclaved broth media and mixed properly. Prepared enriched broth media than dispensed into the sterile centrifuge tubes aseptically and stored at 4 °C for further use.