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The National Academies
500 5th St NW - KWS 502
Washington, DC 20001
Tel: (202) 334-2800
Fax: (202) 334-2139
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
Researchers on the project at the UVAS Diagnostic Lab (photo courtesy of Dr. Mazhar Khan).
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
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.
During the reporting period, 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.
In the upcoming months, the team will continue sequencing of both Mycoplasma gallisepticum (MG) and MS PCR DNA products of local isolates. MG vaccine will be developed and tested in field experiments.
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2011 Show summary || Hide summary || Download full report
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 || Download full report
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 || Download full report