Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

AbstractRapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce CT values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region

Foot-and-mouth disease virus serotypes detected in Tanzania from 2003 to 2010: Conjectured status and future prospects

This study was conducted to investigate the presence of foot-and-mouth disease virus (FMDV) in different geographic locations of Tanzania. Epithelial tissues and fluids (n = 364) were collected from cattle exhibiting oral and foot vesicular lesions suggestive of FMD and submitted for routine FMD diagnosis. The analysis of these samples collected during the period of 2002 and 2010 was performed by serotype-specific antigen capture ELISA to determine the presence of FMDV. The results of this study indicated that 167 out of 364 (46.1%) of the samples contained FMDV antigen. Of the 167 positive samples, 37 (28.4%) were type O, 7 (4.1%) type A, 45 (21.9%) SAT 1 and 79 (45.6%) SAT 2. Two FMDV serotypes (O and SAT 2) were widely distributed throughout Tanzania whilst SAT 1 and A types were only found in the Eastern zone. Our findings suggest that serotypes A, O, SAT 1 and SAT 2 prevail in Tanzania and are associated with the recent FMD outbreaks. The lack of comprehensive animal movement records and inconsistent vaccination programmes make it difficult to determine the exact source of FMD outbreaks or to trace the transmission of the disease over time. Therefore, further collection and analysis of samples from domestic and wild animals are being undertaken to investigate the genetic and antigenic characteristics of the circulating strains, so that a rational method to control FMD in Tanzania and the neighbouring countries can be recommended

Ecological and epidemiological findings associated with zoonotic rabies outbreaks and control in Moshi, Tanzania, 2017–2018

Approximately 1500 people die annually due to rabies in the United Republic of Tanzania. Moshi, in the Kilimanjaro Region, reported sporadic cases of human rabies between 2017 and 2018. In response and following a One Health approach, we implemented surveillance, monitoring, as well as a mass vaccinations of domestic pets concurrently in >150 villages, achieving a 74.5% vaccination coverage (n = 29, 885 dogs and cats) by September 2018. As of April 2019, no single human or animal case has been recorded. We have observed a disparity between awareness and knowledge levels of community members on rabies epidemiology. Self-adherence to protective rabies vaccination in animals was poor due to the challenges of costs and distances to vaccination centers, among others. Incidence of dog bites was high and only a fraction (65%) of dog bite victims (humans) received post-exposure prophylaxis. A high proportion of unvaccinated dogs and cats and the relative intense interactions with wild dog species at interfaces were the risk factors for seropositivity to rabies virus infection in dogs. A percentage of the previously vaccinated dogs remained unimmunized and some unvaccinated dogs were seropositive. Evidence of community engagement and multi-coordinated implementation of One Health in Moshi serves as an example of best practice in tackling zoonotic diseases using multi-level government e orts. The district-level establishment of the One Health rapid response team (OHRRT), implementation of a carefully structured routine vaccination campaign, improved health education, and the implementation of barriers between domestic animals and wildlife at the interfaces are necessary to reduce the burden of rabies in Moshi and communities with similar profiles.The USAID funded project—OSRO/GLO/507/USA on Global Health Security Agenda for the control of zoonosis in Africa.http://www.mdpi.com/journal/ijerpham2020Veterinary Tropical Disease

Genotyping sorghum germplasm in Tanzania using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. In this study microsatellite markers were used to quantify the genetic diversity within as well as among 200 accessions sampled from sorghum germplasm collection at Tanzania National gene bank germplasm collection of sorghum. Although all methods did not provide similar description of relationships between accessions, there existed some consistency in discriminating accessions which are closely related and the ones which were distantly related. But, considerable variation was found at the 39 microsatellite markers analysed, with an average number of alleles per locus equal to 9.49 within accessions, the lowest was 2.0 from Xtxp114, Xcup61 and the highest number of allele was 25 as for Xgap206 marker. The collection of sorghum appeared moderately structured genetically with about 59% of the average gene diversity occurring among accessions. The SSR markers were moderately polymorphic, with diversity indices ranging from 0.07 to 0.91 with mean of 0.55.The UPGMA dendrogram based on SSR marker data clearly discriminated among clusters, even though some consistency in classification was observed among clusters. However, differentiation among morphologically accessions of sorghum, or among geographic origins, accounted for less than 35% of the total genetic diversity. Data in this study demonstrated that accessions of Tanzania sorghum contain a great deal of genetic diversity as indicated by the observed number of alleles. These results are in global agreement with those obtained previously with allozyme markers. It was also possible to show that microsatellite data are useful iniii identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Therefore, from the result outcome the inventory was compiled that will be used in future to characterize the rest of the sorghum germplasm and make use of the identified potential parental genotypes for mapping populations and marker assisted selection programs.ICRISAT/Bec

Genotyping sorghum germplasm in Tanzania using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. In this study microsatellite markers were used to quantify the genetic diversity within as well as among 200 accessions sampled from sorghum germplasm collection at Tanzania National gene bank germplasm collection of sorghum. Although all methods did not provide similar description of relationships between accessions, there existed some consistency in discriminating accessions which are closely related and the ones which were distantly related. But, considerable variation was found at the 39 microsatellite markers analysed, with an average number of alleles per locus equal to 9.49 within accessions, the lowest was 2.0 from Xtxp114, Xcup61 and the highest number of allele was 25 as for Xgap206 marker. The collection of sorghum appeared moderately structured genetically with about 59% of the average gene diversity occurring among accessions. The SSR markers were moderately polymorphic, with diversity indices ranging from 0.07 to 0.91 with mean of 0.55.The UPGMA dendrogram based on SSR marker data clearly discriminated among clusters, even though some consistency in classification was observed among clusters. However, differentiation among morphologically accessions of sorghum, or among geographic origins, accounted for less than 35% of the total genetic diversity. Data in this study demonstrated that accessions of Tanzania sorghum contain a great deal of genetic diversity as indicated by the observed number of alleles. These results are in global agreement with those obtained previously with allozyme markers. It was also possible to show that microsatellite data are useful iniii identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Therefore, from the result outcome the inventory was compiled that will be used in future to characterize the rest of the sorghum germplasm and make use of the identified potential parental genotypes for mapping populations and marker assisted selection programs.ICRISAT/Bec

Epidemiological study of Rift Valley fever virus in Kigoma, Tanzania

Rift Valley fever virus (RVFV) is an acute, zoonotic viral disease caused by a  Phlebovirus, which belongs to the Bunyaviridae family. Among livestock, outbreaks of the disease are economically devastating. They are often characterised by large, sweeping abortion storms and have significant mortality in adult livestock. The aim of the current study was to investigate RVFV infection in the Kigoma region, which is nestled under the hills of the western arm of the Great Rift Valley on the edge of Lake Tanganyika, Tanzania. A region-wide serosurvey was conducted on non-vaccinated small ruminants (sheep and goats, n = 411). Sera samples were tested for the presence of anti-RVFV antibodies and viral antigen, using commercial enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction, respectively. The overall past infections were detected in 22 of the 411 animals, 5.4% (Confidence Interval (CI) 95% = 3.5% – 8.1%). The Kigoma rural area recorded the higher seroprevalence of 12.0% (CI 95% = 7.3% – 18.3%; p 0.05) and the Kasulu district at 0.8% (CI 95% = 0.0% – 4.2%; p > 0.05). The prevalence was 12.5% and 4.7% for sheep and goats, respectively. Reverse transcriptase polymerase chain reaction results indicated that only eight samples were found to be positive (n = 63). This study has confirmed, for the first time, the presence of the RVFV in the Kigoma region four years after the 2007 epizootic in Tanzania. The study further suggests that the virus activity exists during the inter-epizootic period, even in regions with no history of RVFV

Serosurveillance of foot-and-mouth disease virus in selected livestock-wildlife interface areas of Tanzania

Foot-and-mouth disease (FMD) is caused by a virus of the genus Aphthorvirus of the family Picornaviridae. There is great scientific need for determining the transmission dynamics of FMD virus (FMDV) by drawing more attention to the livestock-wildlife interface areas. A variety of literature suggests that buffalo could serve as reservoir of FMDV in wildlife and cattle. However, many FMDV research studies conducted on experimentally infected cattle as carriers and groups of animal highly susceptible to FMDV (i.e. bovine calves) have shown lower chances of transmission of the virus between carriers and the susceptible groups. These findings underscore the importance of continued research on the role played by carrier animals on FMDV transmission dynamics under natural conditions. The aim of this research study was to determine FMDV infection status among buffalo and cattle herds in selected livestock-wildlife interface areas. The sampled areas included Mikumi, Mkomazi and Ruaha national parks, where a total of 330 buffalo and bovine sera samples were collected. Laboratory analysis of the samples was done through the NSP ELISA technique using the PrioCHECK® FMDV NS Kit for detection of antibodies directed against 3ABC non-structural proteins and confirming natural infections. Results showed that 76.3% of tested sera samples were positive for FMDV. However, serotyping of NSP ELISA seroreactors with LPBE is yet to be done. This information is important for further epidemiological studies towards developing effective FMD control strategies

Investigation of foot-and-mouth disease outbreaks in the Mbala and Kazungula districts of Zambia

Foot-and-mouth disease (FMD) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. It is known to be endemic in Zambia, with periodic outbreaks occurring in different geographical areas of the country. This study was conducted to investigate the presence of FMD virus (FMDV) in reported FMD-suspected cases in cattle from the Kazungula and Mbala districts of Zambia. Sixty epithelial tissues or oesophageal-pharyngeal (OP) scrapings (probang samples) were collected from Mbala (n = 51) and Kazungula (n = 9) and examined for FMDV. The FMDV viral RNA and serotypes were examined by realtime reverse transcription polymerase chain reaction (qRT-PCR) and antigen Enzyme- linked immunosorbent assay (ELISA), respectively. Twenty-two samples (36.7%) were positive for the FMDV genome by qRT-PCR with Cycle threshold (Ct) values ranging from 13 to 31. The FMDV-positive samples from epithelial tissues showed relatively higher Ct values compared to those obtained from OP scrapings, irrespective of geographical location. Forty percent (40%; n = 4) of epithelial tissues from Mbala were serotyped into SAT 2 serotype by antigen ELISA. Kazungula samples were serotyped into SAT 1. These findings indicated that Mbala and Kazungula districts had FMD outbreaks in 2012 that were ascribed to at least FMDV serotype SAT 2 and SAT 1 field strains. Furthermore, regular interaction between buffalos from the Mosi-o Tunya Park and domestic animals from surrounding areas could contribute to the occurrence of regular FMD outbreaks in Kazungula, whilst the uncontrolled animal movements across borders between Mbala and Nsumbawanga could be responsible for disease outbreaks in Mbala. In-depth molecular biological studies, including sequencing and phylogeny of the viruses, should be conducted to elucidate the complex epidemiology of FMD in Zambia, thereby providing valuable information needed for the rational control strategy of FMD in Zambia and neighbouring countries