Detection and Molecular Characterization of Foot-and-mouth Disease Virus Isolates

Abstract

Pakistan is an agriculture-based country and in agriculture sector, livestock is contributing lions‘ share. The livestock has great potential of increments in dairy products if the losses due to infectious diseases, lower management, and lack of recognition of this sector as industry may be reduced to minimum level. As foot-and-mouth disease (FMD) is a severe, clinically acute, contagious vesicular disease of cloven-hoofed animals including wild and domesticated ruminants. Foot-and-mouth disease virus (FMDV) types O, A and Asia I are endemic in Pakistan. The losses due to this disease only have reached to billions of rupees every year. In first part of the study, suspected FMD samples from 374 animals (buffaloes and cattle) were collected including mouth swabs, vesicles, and serum and tongue tissue from dead animals from field outbreaks occurred in three provinces of Pakistan (Punjab, Khyber Pakhtunkhwa and Balochistan) during 2005-2009. The samples were subjected to virus isolation, Ag-ELISA, non-structural protein (NSP) ELISA, real-time RT-PCR (rRT-PCR), reverse transcriptase PCR (RT-PCR), amplification of VP1, P1 and whole genome, sequencing of VP1, P1 and whole genome. The sequences were then used in phylogenetic analysis using MEGA 4.0, to assess the movement of virus. Thirty eight viruses were isolated on cell culture. Thirty six viruses were confirmed as FMDV serotype O and two as serotype A. The viral isolates were sequenced and that include FMDV type O (32 VP1, 2 whole genome and 2 P1 sequences) and type A (1 VP1 and 1 P1 sequences). Phylogenetic analysis revealed that all the isolates fall in PanAsia II lineage of a broader group of PanAsia lineage. The type A viral isolates were closely related to Iranian sequences of 2001, 2002 and 2003. The second part of the study was the development and evaluation of an ELISA system for the early detection of specific FMD IgM antibodies in the serum of infected, carrier and vaccinated animals. Experimental and field outbreak serum samples (n=3440) were used for this purpose. The experimental samples were from cattle and sheep experiments carried out at Institute for Animal Health (IAH), United Kingdom in the past and field outbreak serum samples were from UK 2001 and 2007 outbreaks of FMD. Naïve samples (n=236 cattle & 36 sheep) were tested to find out the frequency distribution. It was found that at a cutoff value of 0.3, 100% specificity can be achieved. Furthermore, the test also had the potential to detect the infected, vaccinated and carrier animals. It was concluded from the study that FMD was endemic in Pakistan and upon phylogenetic analysis it was revealed that same viruses with minor nucleotide changes were the cause of repeated outbreaks in the areas under study. To our knowledge, this is the first report on whole length genome sequence including S, L, and complete open reading frame of FMD viral isolate from Pakistan. Overall, the results of the IgM ELISA may correlate with the infection status and possibly the carrier status of cattle. Therefore, the IgM ELISA could be a tool to identify infected cattle and in particular probable carrier cattle in a vaccinated population. However, the IgM ELISA cannot, sensu strict, detect carrier animals, which is only possible by virus isolation. Further studies with vaccinated, non-infected cattle should be carried out to clarify whether the IgM ELISA can reliably detect infected cattle in a vaccinate population.