Abstract
Foot and Mouth Disease (FMD) is a highly contagious viral disease of cattle, sheep, goats, pigs, buffalo and other 70 wild animal species. FMD is characterized clinically by fever, lameness and vesicular lesions on the mouth, tongue, feet, snout and teats of infected animals. About US$62 million/year losses is incurred due to FMD in Bangladesh. The causal agent, is a single-stranded positive sense RNA virus having seven genetically distinct serotypes (O, A, C, SAT1, SAT2, SAT3 and Asial) and about 65 subtypes. Despite vaccination, frequent outbreaks have been occurring in Bangladesh every year. This study was aimed at clinicopathological investigation of FMD and genetic characterization of the circulating viruses. For this purpose, 202 clinically infected cattle were investigated, 7 dead cattle were necropsized for pathological study and 97 tissue samples from infected and dead cattle were collected from different areas of seven divisions of Bangladesh for genetic analysis during 2013 outbreak. Out of 202 clinically infected cattle investigated; salivation, vesicular lesion, lameness and maggot infestation were found in 66%, 80%, 36% and 14% cattle, respectively. At necropsy, characteristic "tiger heart" disease was found in five young and two adult cattle. Hyaline degeneration with lymphocytic infiltration, edema and necrosis were seen in the heart muscle of dead cattle. Interstitial pneumonia with thicker interlobular septa was seen under microscope. The cardiac and pneumonic lesions as seen could have contributed death of infected cattle. The Lpro geen specific reverse transcription polymerase chain reaction (RT-PCR) was designed and successfully detected FMD viruses (FMDV). The viral serotypes were detected by using multiplex RT-PCR. The complete Lpro and VP1 genes of FMD viral serotype O (N=3), Asial (N=2) and A (N=2) were sequenced and sequence data were analyzed by DNASTAR and MEGA6 molecular biology software. Out of 97 samples tested in uniplex and multiplex RT-PCR, 92 and 90 samples generated expected amplicons, respectively. FMD viral Serotype O, Asial, A, O plus Asial and O plus A were detected in 56 (62.22%), 16 (17.78%), 13 (14.44%), 03 (3.33%) and 02 (2.22%) cases, respectively. The FMD viral serotype O (N=9), A (N=1) and Asial (N=2) were successfully isolated in BHK-21 cell culture and make a safe repository. The deduced amino acid (aa) comparison with other downloaded isolates showed variability in 60.7% aa positions in the N terminus end between the first two initiation codons of the Lpro sequences whereas variability in 43% aa positions was observed while compared among the six study isolates. About 51% aa substitutions was found in between the first two initiation codons. All critical amino acids and catalytic triad were found conserved in all isolates except BD_SI_5_2013 isolate, where critical residue L10 was replaced by V10. Specific clustering of viruses was not seen while Lpro gene sequences were used in phylogenetic analysis. About 12 substitutions were seen in VP1 gene of serotype O viruses of which 58% was located in the main antigenic site. A total of 23 mutations were noted in VP1 gene of Asial isolate, 8 of which were clustered in the antigenic site I. An insertion of A was noticed in the 44th aa position of B-C loop. VP1 analysis also revealed the presence of lineage specific signature residues (10 out of 11) unique to isolates of lineage C in relation to lineage D. There were 12 mutations in FMDV A isolate, 4 of which were clustered in B-C loop and one in antigenic sitel. These huge mutations in each serotype as seen in this study could have contributed antigenic heterogeneity and vaccinal failure. Phylogenetic analyses of VP1 gene revealed the presence of single topotype/lineage of FMD viral serotype "O" (Ind-2001 and PanAsia lineage of ME- SA topotype), "A" (Genotype VII of Asia topotype) and Asial (Genetic lineage C) in Bangladesh. Emergence of PanAsia strain of FMDV O is a new threat to control FMD in Bangladesh. The Lpro gene specific RT-PCR protocol designed in this study is unique to detect FMD viruses in general. The sequence information could be of value to select future vaccine candidate and control FMD in Bangladesh.