Sabenzia Nabalayo Wekesa

Laboratory capacity for diagnosis of foot-and-mouth disease in Eastern Africa: implications for the progressive control pathway

BackgroundAccurate diagnosis is pertinent to any disease control programme. If Eastern Africa is to work towards control of foot-and-mouth disease (FMD) using the Progressive Control Pathway for FMD (PCP-FMD) as a tool, then the capacity of national reference laboratories (NRLs) mandated to diagnose FMD should match this task. This study assessed the laboratory capacity of 14 NRLs of the Eastern Africa Region Laboratory Network member countries using a semi-structured questionnaire and retrospective data from the World Reference Laboratory for FMD annual reports and Genbank® through National Centre for Biotechnology Information for the period 2006–2010.ResultsThe questionnaire response rate was 13/14 (93%). Twelve out of the 13 countries/regions had experienced at least one outbreak in the relevant five year period. Only two countries (Ethiopia and Kenya) had laboratories at biosecurity level 3 and only three (Ethiopia, Kenya and Sudan) had identified FMD virus serotypes for all reported outbreaks. Based on their own country/region assessment, 12/13 of these countries /regions were below stage 3 of the PCP-FMD. Quarantine (77%) and vaccination (54%) were the major FMD control strategies employed. The majority (12/13) of the NRLs used serological techniques to diagnose FMD, seven used antigen ELISA and three of these (25%) also used molecular techniques which were the tests most frequently requested from collaborating laboratories by the majority (69%) of the NRLs. Only 4/13 (31%) participated in proficiency testing for FMD. Four (31%) laboratories had no quality management systems (QMS) in place and where QMS existed it was still deficient, thus, none of the laboratories had achieved accreditation for FMD diagnosis.ConclusionsThis study indicates that FMD diagnostic capacity in Eastern Africa is still inadequate and largely depends on antigen and antibody ELISAs techniques undertaken by the NRLs. Hence, for the region to progress on the PCP-FMD, there is need to: implement regional control measures, improve the serological diagnostic test performance and laboratory capacity of the NRLs (including training of personnel as well as upgrading of equipment and methods, especially strengthening the molecular diagnostic capacity), and to establish a regional reference laboratory to enforce QMS and characterization of FMD virus containing samples.

Genetic diversity of serotype A foot-and-mouth disease viruses in Kenya from 1964 to 2013; implications for control strategies in eastern Africa

Serotype A is the most genetically and antigenically diverse of the foot-and-mouth disease virus (FMDV) serotypes. Records of its occurrence in Kenya date back to 1952 and the antigenic diversity of the outbreak viruses in this region is reflected by the current use of two different vaccine strains (K5/1980 and K35/1980) and previous use of two other strains (K18/66 and K179/71). This study aimed at enhancing the understanding of the patterns of genetic variation of serotype A FMDV in Kenya. The complete VP1 coding region sequences of 38 field isolates, identified as serotype A FMDV, collected between 1964 and 2013 were determined. Coalescent-based methods were used to infer times of divergence of the virus strains and the evolutionary rates alongside 27 other serotype A FMDV sequences from Genbank and the World Reference Laboratory (WRL). This study represents the first comprehensive genetic analysis of serotype A FMDVs from Kenya. The study detected four previously defined genotypes/clusters (termed G-I, G-III, G-VII and G-VIII), within the Africa topotype, together with a fifth lineage that has apparently emerged from within G-I; these different lineages have each had a countrywide distribution. Genotypes G-III and G-VIII that were first isolated in 1964 are now apparently extinct; G-VII was last recorded in 2005, while G-I (including the new lineage) is currently in widespread circulation. High genetic diversity, widespread distribution and transboundary spread of serotype A FMDVs across the region of eastern Africa was apparent. Continuous surveillance for the virus, coupled to genetic and antigenic characterization is recommended for improved regional control strategies.

Analysis of Recent Serotype O Foot‐and‐Mouth Disease Viruses from Livestock in Kenya: Evidence of Four Independently Evolving Lineages

Foot-and-mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these viruses were determined to ascertain whether these were independent outbreaks or one single strain spreading throughout the country. The sequences of the complete VP1-coding region were analysed from viruses sampled within different areas of Kenya during 2010 and 2011. The results indicated that the 2010 to 2011 outbreaks in Kenya were caused by four independent strains. By comparison with earlier type O isolates from Eastern Africa, it was apparent that the outbreaks were caused by viruses from three different lineages of topotype EA-2 and a fourth virus strain belonging to topotype EA-4. The topotypes EA-1 and EA-3 were not detected from these outbreaks. Implications of these results for FMD control in Eastern Africa are discussed.

Characterization of Foot-And-Mouth Disease Viruses (FMDVs) from Ugandan Cattle Outbreaks during 2012-2013: Evidence for Circulation of Multiple Serotypes

To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012–2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.

Foot-and-Mouth Disease Virus Serotype SAT 3 in Long-Horned Ankole Calf, Uganda

After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closest relatives isolated previously from buffalo in Uganda.

Characterisation of recent foot-and-mouth disease viruses from African buffalo (Syncerus caffer) and cattle in Kenya is consistent with independent virus populations

BackgroundUnderstanding the epidemiology of foot-and-mouth disease (FMD), including roles played by different hosts, is essential for improving disease control. The African buffalo (Syncerus caffer) is a reservoir for the SAT serotypes of FMD virus (FMDV). Large buffalo populations commonly intermingle with livestock in Kenya, yet earlier studies have focused on FMD in the domestic livestock, hence the contribution of buffalo to disease in livestock is largely unknown. This study analysed 47 epithelia collected from FMD outbreaks in Kenyan cattle between 2008 and 2012, and 102 probang and serum samples collected from buffalo in three different Kenyan ecosystems; Maasai-Mara (MME) (n = 40), Tsavo (TSE) (n = 33), and Meru (ME) (n = 29).ResultsAntibodies against FMDV non-structural proteins were found in 65 of 102 (64%) sera from buffalo with 44/102 and 53/102 also having neutralising antibodies directed against FMDV SAT 1 and SAT 2, respectively. FMDV RNA was detected in 42% of the buffalo probang samples by RT-qPCR (Cycle Threshold (Ct) ≤32). Two buffalo probang samples were positive by VI and were identified as FMDV SAT 1 and SAT 2 by Ag-ELISA, while the latter assay detected serotypes O (1), A (20), SAT 1 (7) and SAT 2 (19) in the 47 cattle epithelia. VP1 coding sequences were generated for two buffalo and 21 cattle samples. Phylogenetic analyses revealed SAT 1 and SAT 2 virus lineages within buffalo that were distinct from those detected in cattle.ConclusionsWe found that FMDV serotypes O, A, SAT 1 and SAT 2 were circulating among cattle in Kenya and cause disease, but only SAT 1 and SAT 2 viruses were successfully isolated from clinically normal buffalo. The buffalo isolates were genetically distinct from isolates obtained from cattle. Control efforts should focus primarily on reducing FMDV circulation among livestock and limiting interaction with buffalo. Comprehensive studies incorporating additional buffalo viruses are recommended.

Time Clustered Sampling Can Inflate the Inferred Substitution Rate in Foot-And-Mouth Disease Virus Analyses

With the emergence of analytical software for the inference of viral evolution, a number of studies have focused on estimating important parameters such as the substitution rate and the time to the most recent common ancestor (t MRCA) for rapidly evolving viruses. Coupled with an increasing abundance of sequence data sampled under widely different schemes, an effort to keep results consistent and comparable is needed. This study emphasizes commonly disregarded problems in the inference of evolutionary rates in viral sequence data when sampling is unevenly distributed on a temporal scale through a study of the foot-and-mouth (FMD) disease virus serotypes SAT 1 and SAT 2. Our study shows that clustered temporal sampling in phylogenetic analyses of FMD viruses will strongly bias the inferences of substitution rates and t MRCA because the inferred rates in such data sets reflect a rate closer to the mutation rate rather than the substitution rate. Estimating evolutionary parameters from viral sequences should be performed with due consideration of the differences in short-term and longer-term evolutionary processes occurring within sets of temporally sampled viruses, and studies should carefully consider how samples are combined.