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Phase 4 (2009 Deadline)
Characterization of Mycoplasma gallisepticum Isolates from Pakistan and Their Use in Production of Diagnostic Antigen and Vaccine
Mazhar Khan, University of Connecticut
Masood Rabbani, University of Veterinary and Animal Sciences (UVAS)
Pakistani Funding (Department of State): $225,440
US Funding (Department of State): $63,450
Project Dates on US Side: November 15, 2010 - November 14, 2013 (Extended through September 30, 2014)
Project Overview
Mycoplasma gallisepticum (MG) causes chronic respiratory disease in domestic poultry, and without proper diagnostic and preventive measures, a case of MG infection could put a farmer out of business. Up to now, only limited information has been available on the identification and molecular characterization of Pakistani MG isolates. False positive results often arise when commercially available imported diagnostic kits are used, indicating that different strains of MG pathogens are prevalent among Pakistani poultry. In addition, production of biologics using indigenous isolates of MG has not yet been initiated, and the high cost and poor availability of MG biologics in the country has limited their use by poor farmers. These circumstances have helped to perpetuate mycoplasmosis in Pakistani poultry flocks. The objective of this project is to make use of conventional and modern techniques for prompt diagnosis of MG. After successful characterization of the disease agent, the research team will work to produce diagnostic reagents and vaccine using indigenous Pakistani MG isolates. These biologics will then be standardized, and qualified local vaccine meeting international standards will be released to selected governmental and private poultry farms for field trials. If successful, this project should help to address a serious livestock disease and ultimately assist Pakistani poultry farmers.
Quarterly Update
One of the main objectives of this project was development of local cost effective MG antigen for screening of poultry flocks. Antigen has been successfully prepared by local scientists with guidance and coordination of PI’s from both Pak & US side. During the reporting period reproducibility of results was evaluated using local antigen that was found equally good compared to commercially available MG antigen. Other objective was development and evaluation of inactivated MG vaccine. Vaccine has been prepared and evaluated on experimental birds. Feld trial discussions are in process with stake holders to finalize the poultry flock for trail. It is pertinent to mention here that vaccine produced in this project is cost effective and efficacious (observed in experimental trail). About 5 MG isolates have been confirmed and for their sequencing, a foreign lab has been contacted and soon samples will be sent for sequencing. Many postgraduates are receiving training on the isolation and identification of MG in MG lab established at UDL under this project. One post graduate student from Agriculture University Peshawar (khyber Pakhtunkhwa) currently working in this project and two post graduate students from Microbiology department of University of Veterinary and Animal Sciences (UVAS) are working on “Effects of Newcastle disease virus glycoprotein on antibodies of broiler to MG vaccine “ and “antibody response of broilers to oil based combined MG and AI virus vaccine“ at UDL. One abstract titled as “Factors affecting sensitivity of rapid serum agglutination antigen of Mycoplasma gallisepticum” was presented and published in National Conference on Current Approaches in Microbiology, organized by Hazara University, Mansehra on June 26-28, 2013.
Progress Report
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2011 Show summary || Hide summary
Dr. Rabbani completed his four-month visit to the United States (May 6 – September 7, 2011), including stops at Dr. Khan’s lab, as well as Pennsylvania State University and the University of California, Davis. His training program at the University of Connecticut was focused on propagation of avian mycoplasma, development of improved serological tests for avian mycoplasma, and multiplex PCR tests for simultaneous detection and differentiation of MG from other avian mycoplasmas. As of October 2011, Dr. Rabbani reported that 153 blood samples and 150 tissue samples had been collected from MG-suspected birds at various Pakistani farms and processed at UVAS according to the standard protocol for isolation studies. Only four of the samples were found positive based on colony characteristics, and further analysis is in process. Dr. Rabbani suspects that either the widespread use of antibiotics in the flocks sampled or else the difficulty of culturing the mycoplasma organism could have kept the number of positive samples down. To address this problem, future sampling efforts will focus on poultry operations where antibiotics are less commonly used, and different isolation media will also be utilized.
2012 Show summary || Hide summary
During the first quarter of 2012, Dr. Rabbani reports that 123 tracheal organ and swab samples were tested from various mycoplasma-suspected poultry flocks in Pakistan, of which only 4 samples revealed MG-suspected colonies. None of the positive colonies was confirmed as MG through PCR testing, although serological examination by indirect ELISA indicated a 7 percent positive rate in the flocks. Three of the research associates in Dr. Rabbani’s group are using their work on the project toward the requirements for the M.Phil., and another research fellow is working toward his PhD under the supervision of Dr. Rabbani and Dr. Khan. Among the M.Phil scholars, one (Mr. Rana Khurram Khalid) has successfully prepared MG antigen using local isolate and has evaluated various factors affecting the sensitivity of local antigen. The second scholar (Mr. Asim Raza) is working on quality control factors affecting the potency of MG bacterin, and the third (Mr. Javed Muhammad) is studying the effect of physiochemical factors on survival of MG. The PhD scholar (Mr. Mushtaq Ahmad) is conducting his final research trials and should complete his dissertation work in the next few months. With regard to improvements in the lab facilities at UVAS, a mini thermocycler/PCR machine and computer were purchased in 2011, and the procurement process is under way for the remaining equipment (stereoscope microscope, ELISA reader, class III safety cabinet, packaging sealing machine, and fermenter).
In 2012, Dr. Khan expects to host up to four members of the UVAS group at the University of Connecticut for training. A training workshop is also being planned at UVAS, with the date tentatively set for July 2012 in connection with a proposed visit by Dr. Khan. The research agenda at UVAS includes further collection and processing of MG-suspected samples for isolation of more mycoplasma bacterial isolates, as well as initial production and evaluation of a bacterin and an antigen.
During the second quarter, a total of 59 tissues/swabs (29 trachea and 30 tracheal swabs) and 234 blood samples were processed for isolation, identification and sero-diagnosis of Mycoplasma gallisepticum (MG) on the Pakistani side. Two M. Phil students and one Ph.D. scholar completed their research and submitted their theses. In the coming months, a few pieces of equipment will be purchased for the Pakistani lab. Four team members from UVAS are expected to visit the U.S. team at University of Connecticut for training on avian serological methods and other molecular rapid diagnostic tools, pending receipt of their visas. Also, plans have been made for the trainees to visit commercial poultry farms and Connecticut Veterinary Medical Diagnostic Laboratory. Meantime, the UVAS researchers are planning to organize a second workshop on MG in September 2012 with participants from the various regional diagnostic laboratories in Pakistan. The US PI Dr. Khan is also planning to attend the workshop.
2013 Show summary || Hide summary
During the first half of 2013, the Pakistani team collected 157 samples from Peshawar, Rawalpindi, Jaranwala, and Lahore. All the samples were processed for Mycoplasma identification through culturing and molecular characterization. Two new equipments were purchased – the Biofermenter equipment and the standardized PCR kits. Three technical meetings have been conducted in spring. Two research articles based on the research done in this project by Mr. Mushtaq Ahmad were accepted to the Journal of Animal and Plant Sciences. Mr. Ahmad’s PhD thesis was also submitted to the University authority for foreign evaluation. Another student Miss Asfa Rasool has also been supporting this project. Two lab technicians (Mr. Muhammad Arshad and Mr. Imran Shafique) received necessary documents for visa application. Along with Pakistani PI Dr. Masood Rabbani, they will be visiting the US lab at University of Connecticut for two-week training on disease diagnostics techniques in June, 2013.
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