Location and Period of Study The study was carried out in different areas of Savar Upazila under Dhaka district from January to December 2018. The molecular and serological works were done in Foot and Mouth Disease (FMD) Research laboratory, Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka..
Sample Collection A total of 92 blood serum samples were randomly collected from vaccinated cattle according to their age, sex and breed and 10 clinical samples (tongue epithelium) from infected cattle. Tissue samples were immediately transported with medium containing equal volumes of glycerol and phosphate-buffered saline (pH 7.2-7.6) and 2% antibiotic-antimycotic to the laboratory on ice. Blood samples were collected from the jugular vein of cattle through the venipuncture method by using sterile 10 ml needle and syringe.
Serum preparation and ELISA Test: Collected blood was kept at least 3-4 hours at room temperature in a slightly inclined position to facilitate clotting and separation of serum. The collected sera were stored at -20°C until use. The ELISA test was carried out from serum by using Indirect ELISA kit manufactured by ID.Vet® Innovative Diagnostics, France according to the manufacture’s protocol.
Inoculum Preparation and RNA Extraction Piece of the epithelial tissue was removed from the glycerol buffer, blotted dry on absorbent paper to reduce the glycerol content. Approximately 1-2 gm tissue was weighted by an electric balance and homogenized by grinding with sterilized mortar and pestle. Then 20% suspension was prepared by adding phosphate buffer saline. The suspension of each of the samples was then centrifuged at 3,000 rpm for 10 minutes maintaining the temperature at 4°C. The supernatant of each of the samples was taken for further processing according to the OIE manual. For the sterility test, a little number of inoculums were inoculated into bacteriological media to identify the presence of any type of bacteria. RNA extraction was carried out from FMD inoculums by using the QIAamp® Viral RNA kit (Qiagen, Germany) according to the manufacture’s protocol.
Conventional Reverse Transcription Polymerase Chain Reaction (RT-PCR) The target in the genome was amplified by one step RT-PCR using the FMD universal and serotype specific primer (Reid et al., 2000).
The amplification was performed on a thermal cycler with a one-step RT-PCR kit (Qiagen, Germany) with one cycle of reverse transcription conditions of 50°C for 30 min and 95°C for 10 min and followed by 30 cycles of 94 °C for 1 min, 55°C for 1 sec (type A), 55°C for 30 sec (type O) and 72°C for 1min and finally one cycle of final extension of 72°C for 10 min. After PCR, the amplified products were visualized by agarose gel electrophoresis using 2% agarose gel containing 0.6 mg/ml ethidium bromide at 100V in 1X tris borate EDTA (TBE) buffer. At the end of electrophoresis, the gel was documented on a UV transilluminator (AlphaImager®Mini System, USA).
Grouping of animal for Antibiotic therapy: A total of 30 FMD affected cattle were used for the study. The animals were divided into three treatment groups each containing 10 animals.
Statistical Analysis The data obtained from this study were subjected to descriptive statistics (Tables and charts), Chi-square test of independence and odds ratio (OR) to determine the association of the variables (age, sex, species and breed) with the presence of FMD virus antibodies. The value of p<0.05 was considered significant in this study. Descriptive statistics was carried out using Microsoft excel 2007. Chi square, odds ratio and confidence intervals were calculated using IBM SPSS 15 software.