Abstract
Foot and Mouth Disease (FMD) is a highly infectious, fatal, and economically important disease of cattle, sheep, goat, swine, and wild ruminants. FMD is clinically characterized by fever and vesicle on the mouth, feet and udder, and high fatality in young animals. The disease is cosmopolitan and the economic loss is immense, while OIE recognized it as 'list A infectious disease'. This study aimed at isolation and molecular characterization of FMD virus serotypes circulating in Bangladesh; with a view to develop two new vaccines namely BAU FMD Vac-1 and -2 with these field isolates. Antibody titers of these experimental vaccines were evaluated in sero-negative calves which were compared with immune antibody titers of other five commercially available vaccines in Bangladesh. Efficacy test of BAU FMD Vac- 1 and 2 vaccines were carried out in guinea pigs, reared in Laboratory Animal Centre for Microbiological Research (LACMR) of the Department of Microbiology and Hygiene, BAU; and found 100% potent against FMD virus serotypes O, A and asia-1. A total of 151(84 tongue epithelium and 67 inter-digital tissues) clinical samples were collected during the year of 2012-2014 from different districts of Bangladesh and subjected to tissue culture screening in BHK-21 cell for virus isolation. Out of 151 samples tested, 85(56.29%) was adapted in BHK-21 cell, and showed characteristic cytopathic effect (CPE) specific for FMD virus that included rounding, swelling, clumping of the cells, break down of intercellular bridge and cell death. After 2-3 passages in confluent BHK-21 cell culture out of 84 tongue epithelium tested, 67(79.76%) was successfully adapted and exhibited 100% CPE within 24-36 hours of inoculation. On the contrary out of 67 inter-digital space tissues tested, only 18(26.86%) was successfully adapted after 3-4 serial passages and produce 100% CPE. Viral RNA was extracted from 85 FMD virus infected BH-21 cell culture fluid using RNA extraction kit (Promega®, USA). All 85 RNA samples were used for amplification of VP1 gene with specific primers by RT-PCR for FMD virus serotyping and 71 (83.53%) was found positive for different serotypes of FMD virus. Among these 71 positive samples, 31 (43.67%) were positive for serotype A, 26 (36.62%) for serotype O, 10 (14.08%) for serotype Asia 1, and 4 (5.63%) for mixed. Nucleotide sequencing of VP1 gene was performed two from each serotypes (O, A & Asia-1) and comparison conducted using the BLAST search and 92-99%, 92-100% and 96-98% homology were found with some FMD virus serotypes O, A and Asia-1 isolates of Bangladesh, India, Pakistan, Nepal and Bhutan respectively. Neighbor joining trees were constructed by using software package "Lasergene" (Modules - EditSeq and MegAlign; DNASTAR Inc., USA) and MEGA6, with the nucleotide sequences of FMD virus were obtained from Genbank. Isolates of this study were belonged to PanAsia-02 sub-lineage of ME-SA topotype (serotype O), genotype VII(18) of ASIA topotype (serotype A) and Lineage C (serotype Asia-1) respectively. Immune responses of these vaccines in sero-negative calves were assessed by ELISA and SNT, and along with five commercial FMD vaccines in Bangladesh. All vaccines were administered in single and booster dose order; out of which BAU FMD Vac-1 produced better immune response than other vaccines including BAU FMD Vac-2. Highest antibody titers were found in all vaccines at 60 dpv, and after 60 dpv the antibody titers gradually decline. Protective immunity persist upto 5 months (single dose) and 6 months (booster dose) in case of BAU FMD Vac-1. On the other hand in case of with BAU FMD Vac -2, RakshaR and Aftovaxpur® protective immunity persisted only 4 months (single dose) and 5 months (booster dose), but in case of LRI (Mohakhali), LRI (Comilla) and Bangla FMD vac.® protective immunity persisted only 2 months (single dose) and 3 months (booster dose) respectively. Statistically highly significant antibody titers were found in calves vaccinated with BAU FMD Vac -1, (P<0.01) at 150 dpv (single dose) and 180 dpv (booster dose) when compare with BAU FMD Vac -2, RakshaR and AftovaxpurR. On the other hand antibody titers in calves vaccinated with BAU FMD Vac-1 were found statistically highly significant (P<0.01) at 21, 60, 90, 120, 150 and 180 dpv in comparison to LRI (Mohakhali), LRI (Comilla) and Bangla FMD vac.®, both in single dose and booster respectively. Statistically highly significant (P<0.01) antibody titers were also found in calves vaccinated with booster dose in comparison to single dose, in all experimental groups. On basis of antibody titers of vaccines used in this study including two experimental vaccines BAU FMD Vac-1 and -2 were ranked by (DMRT) and rank were-i). BAU FMD Vac-1, ii). Aftovaxpur®, iii). BAU FMD Vac-2 and Raksha®, and iv). LRI (Comilla), LRI (Mohakhali), Bangla FMD vac® respectively. Present study demonstrated that antibody titers induced by BAU FMD Vac-1 in calves were highly effective both in single and booster dose and could successfully be used to maintain herd immunity against FMD virus serotypes O, A and Asia.