Molecular epidemiological study of foot-and-mouth disease virus in Tanzania

A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF SOKOINE UNIVERSITY OF AGRICULTURE. MOROGORO, TANZANIA.

Foot-and-mouth disease (FMD) is endemic in Tanzania since it was first reported in 1954. Foot-and-mouth disease is caused by FMD virus (FMDV) that presents in seven serotypes. The FMD outbreaks that occur almost each year are caused by different serotypes in the country. With its large area of 945 000 km2 and its climate which varies from tropical to temperate, large number of livestock including 25.8 million cattle (Ministry of Agriculture, Livestock and Fisheries, 2016) and high density of wild animals, it is very difficult to control the spread of this economically important viral disease. This is compounded by the lack of the comprehensive studies that could indicate the spatial distribution of genetically divergent FMDV field strains. This study was conducted to investigate the distribution of FMDV strains circulating in the country, with the main focus on the molecular epidemiology of FMDV in endemic settings of Tanzania using the molecular approach and field epidemiological data. The overriding research question for this study was “what is the contribution of genetic variability of FMDV to the occurrence of FMD in Tanzania?” The general objective of this study was to determine the epidemiological information and molecular and genetic diversity of circulating FMDV field strains in Tanzania. A cross sectional study design was used for this research in a total of 361 (n=361) epithelial tissue samples from tongue and interdigital space of feet were purposively collected from FMD suspected cases/outbreaks in different geographic areas of Tanzania. Laboratory analysis of samples collected between 2008 and 2013 was performed at the Centre for Infectious Diseases and Biotechnology (CIDB) and Faculty of Veterinary Medicine, Sokoine University of Agriculture using the optimized protocols adopted by the FAO World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD). A real-time RT-PCR was used to screen epithelial samples to ascertain the presence of the FMDV genome in the sample. Conventional RT-PCR employing serotype-specific oligo-nucleotides (primers) was used to amplify genome fragments of FMDV in the vesicular epithelium. Two assays real-time RT-PCR and antigen detection ELISA were evaluated for their specificity and sensitivity as tests of choice for detection of FMDV from field samples. The genetic characterization of 56 field samples was performed by VP1 sequencing and phylogenetic analysis using Neighbour joining method. A sensitivity result for rRT-PCR assay was 86.6% (n= 26) and specificity was 70.0% (n= 21) while for Antigen-ELISA had a sensitivity of 70.0% (n= 21) with a specificity of 70.0% (n= 21). These results were still moderate as most of the time Antigen-ELISA can give positive results with only about 70-80% of epithelial suspensions that contain virus due to its low sensitivity. The phylogenetic analysis of type O revealed that all samples (n= 39) clustered into the EA-2 topotype while, serotype A (n=12), viruses were classified into one major genetic clade (G-I) within topotype AFRICA and SAT1 viruses (n= 5) were placed within the topotype 1 (NWZ). For SAT1 the nucleotide differences in VP1 coding region was between 15-20.0%; the nucleotide number of composition was 663 and the average nucleotide match was from 645-660 with the other Tanzanian strains. The molecular epidemiological results from this study indicated the increase of genetic variability of FMD viruses that are independently maintained within Tanzania and the east African region. These findings suggest that outbreaks that took place during the last six years were scattered throughout the country/region and there is suggestive evidence of the history of introductions, exchange and spread of FMDV within Tanzania and its neighbouring border countries. Phylogenetic analysis of complete VP1sequences revealed evidence for the presence of distinct strains within the country and historical genetic relationship of some strains within the Great Lakes countries of Africa. This study provides a ‘valuable pilot’ report on genetic characterization, epidemiological situation and phylogeography of FMDV circulating in Tanzania from 2008-2013. Also, due to gradual increase of the amount of diversity within Tanzanian strains (e.g. serotype O) it is advisable that the future study should be based on molecular evolution of FMDV with the main focus of understanding the rates of nucleotide substitution. Further studies are required to investigate the possible sources of FMDV, transmission ability of field strains and maintenance of viruses in Tanzania. Therefore, further studies should be done to ascertain the possible sources of FMDV and how it is maintained in these endemic settings.