Diagnosis of bovine foot and mouth disease virus by real-time polymerase chain reaction and nucleotide sequencing from outbreak herd samples in Ilesha Baruba, Kwara state, Nigeria

Abstract

Aim: Molecular diagnosis of bovine foot and mouth disease virus (FMDV) from outbreak herd in Bukaru-Rontuwa, Sinawu/Tumbunya ward of Ilesha Baruba, in Kwara state-Nigeria was conducted to establish the associated serotypes and disease control plan. Materials and Methods: Purposive study was conducted in cattle outbreak herds during the dry season of January-March, 2011. Random sampling of blood and observed epithelial tissues was collected, stored in accordance with standard methods and subjected to RNA extraction and real-time reverse transcription polymerase chain reaction (rRT-PCR). Positive samples for FMDV were further subjected to reverse transcription polymerase chain reaction (RT-PCR), nucleotide sequencing using sequence primers of serotypes O, A, SAT 1-3 and gel electrophoresis. Obtained data were interpreted based on NCBI BLASTN program. Results: Foot and mouth disease (FMD)-RNA extract was not found in all the blood tested with beta-actin range of Ct = 30-34. rRT-PCR assay showed two positive samples with Ct values of 18.79 and 15.28. Gel electrophoresis identified sequenced PCR amplicons as serotype A and SAT 2 respectively. Direct product sequencing confirmed SAT 2 serotype was closely related to SAT 2 isolate LIB/7/2003. Cloned RT-PCR product in pGEM-T easy vector confirmed serotype A as closely related to sequence of A/NIG/21/2009, though multiple NIG/2009 sequences were also identified as closely related. Both isolates showed marked genetic homogeneity with >93% genetic identity in the VP1 region which confirmed heterogeneity and antigenic variation nature of FMDV. Conclusion: Quasi species and subtypes of FMD serotypes A and SAT 2 similar to A/NIG/21/2009 and SAT 2/LIB/7/2003 respectively caused the reported FMD outbreaks in Fulani livestock herds investigated. A combined real-time and optimized RT-PCR protocols that would facilitate effective and timely FMD outbreak control plan based on identified serotypes is thus suggested.

Keywords

• foot and mouth disease virus
• Ilesha Baruba
• Kwara State
• molecular
• outbreaks
• phylogenetic