Quality Operations Laboratory
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Foot and Mouth Disease (FMD) is a viral problem of cloven footed animals such as buffaloes, cows, sheep, goat etc. It is characterized by rise of temperature, off feed, lameness, ulcerative lesions on dorsum of tongue, dental pad, nares, on skin of the feet and teats. It causes mitigation of milk production, working efficiency, weight gain, mortality in young stock, abortions etc. Signs and symptoms of FMD infected animals from 59 outbreaks were recorded. Severity of the disease is more in cattle as compared to buffalo.

Tongue epithelial tissue from FMD cases were collected in glycerol buffer from the animals of each of the outbreaks. Causative agent from 51 samples grew successfully on bovine thyroid primary (BTY) cell culture and 25 out of 51 virus isolates adapted and grew on Baby Hamster Kidney 21 (BHK-21) cell line. Tissue culture infective dose 50 (TCID50) titer of the virus isolates was not more than 103 units and increases with successive passage in BHK-21 cell line. Each of the virus isolates was characterized through indirect sandwich ELISA, complement fixation test (CFT), and virus neutralization test (VN), using imported serotype specific anti-sera.

The FMD virus isolates were typed as ?O? (88%), ?A? (4%) and ?Asia-1? (8%). However, each of the tissue sample as well as BHK-21 adapted virus isolate was also confirmed as FMD virus through RT-PCR method. Moreover, antigen capture ELISA confirmed serotype ?C? of FMD virus as cause of 2 outbreaks of the disease in cattle in Pakistan. Trivalent FMD virus vaccine using local isolates was prepared and evaluated in laboratory experimental calves as well as in field animals. Vaccinated animals showed protective titer of the anti-FMD virus CFT antibodies up to 8 months and resistance to natural exposure of field virus up to one year. Non-vaccinated animals on the same manger contract the disease 7 months post vaccination. Effect of immunogen amount, type of in-activant and adjuvant, and vaccine storage on efficacy of the vaccine in rabbits was also evaluated.

It is concluded that it is possible to prepare oil based trivalent FMD vaccine using local virus isolates. The vaccine was found quite effective and protected dairy animals against field exposure of challenge infection over a period of one year.

Acknowledgement: Principal Investigator (P.I.) and other staff of the project have honor to express their deep sense of obligations to Directorate of Agriculture Linkage Program, Pakistan Agriculture Research Council, Islamabad, Pakistan for providing funds (Rs 5.9 millions) for research proposal ? Immunoprophylaxis of Foot and Mouth Disease in Bovine? and Government of Punjab for financial support (Rs 99.998 million) for development project ?Establishment of Quality Operations Laboratory, in UVAS, Lahore? in the best interest of Dairy Industry, Pakistan.





Enzyme Linked Immuno-Sorbent Assay (ELISA) is one of the most sensitive, rapid and reliable techniques for diagnosis of infectious diseases. For execution of ELISA, antibody-peroxidase conjugate is the fundamental reagent. Maximum activity of Peroxidase was determined in turnip as compared to horse radish and carrot. Peroxidase was purified from turnips that included homogenization, inactivation of catalase, ammonium sulphate precipitation and size exclusion chromatography on Sephadex G-25-80. The purified peroxidase had Rz value of 1.7, total protein 0.9 mg/ml and total enzyme activity 36152 units/liter. The maximum enzyme extraction and working pH was recorded as 5.0. The buffalo serum lg-G was fractionated using 40 percent final concentration of ammonium sulphate followed by anion exchange chromatography.

The salt fractionated serum globulin (10 ml) was depleted of its lg-G in less than 25 minutes on DEAE cellulose packed column followed by suitable elution. The lg-G solution (1.0 gm/dl) was mixed in four times volume of oil base (Liquid paraffin and emulsifiers). Rabbits were primed and boosted (0.25ml/: subcut) with buffalo lg-G antigen with 21 days interval. The immune serum was harvested on 21 day post-boosting. The serum contained 2048 agar gel precipitation (AGP) units and 10,000 ELISA units. Rabbit anti buffalo lg-G was purified with salt precipitation followed by anion exchange chromatography. The peroxidase was linked with the rabbit anti-buffalo lg-G using the sodium metaperiodate. The conjugate was titrated against buffalo lg-G and working dilution for execution of ELISA was 1: 1200.


An indirect ELISA was standardized for titration of anti-FMD-serotype-specific antibodies. In this test polystyrene plates were coated with known FMD serotype ?O? virus using carbonate/bicarbonate buffer. The blank spaces were blocked with horse serum. The immunoplate was coated with anti-FMD ?O? virus specific anti-serum from vaccinated calves. After washing, the plate was coated with rabbit anti-bovinge-lg-specific-antibodies-horse radish peroxidase conjugate. After washing, the plate was coated with HRP specific substrate. Development of color was recorded in form of OD value using ELISA reader. During the standardization of ELISA, U-shape titration plates among all types of plates, 1:10 diluted virus among different dilution of FMD ?O? type virus, 1:10 diluted serum from buffalo calves vaccinated with FMD ?O? type specific vaccine, 1:4000 dilution of conjugate and incubation of 4o C for coating the virus showed good results. In each experiment, plateau region, test back ground and plate back ground was recorded.

Results of the study will help development of an economical, sensitive, reliable indirect ELISA conjugate that will subsequently be used in ELISA for monitor prevalence of FMD virus in Pakistan and efficacy of FMD vaccines.

Acknowledgement: Principal Investigator (P.I.) and other staff of the project have honor to express their deep sense of obligations to Directorate of National Commission on Biotechnology, Islamabad, Pakistan, for providing funds (Rs 2.02 millions) to execute the research in the best interest of Dairy Industry, Pakistan.


Quality Operations Laboratory