Validation of a diagnostic tool for the diagnosis of lumpy skin disease.

BACKGROUND: Lumpy skin disease (LSD) is caused by LSD virus which is a member of the Capripoxvirus (CaPV) genus. Although PCR provides for a rapid and sensitive diagnosis, it has limited use due to its complexity in terms of cost, time and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, specific and cost-effective method with a diagnostic accuracy similar to PCR. OBJECTIVES/HYPOTHESIS: To compare the detection rate (DR) of two LAMP assays versus PCR for the detection of CaPV. ANIMALS: This study used 105 apparently health animals (AHA) and 59 clinically sick animals (CSA). METHODS AND MATERIALS: PCR and LAMP assays (LAMP1 and LAMP 2) were compared for detection of CaPV from AHA and CSA using blood and tissue samples. The detection was confirmed by sequencing of PCR positive samples. Analytical sensitivity and specificity of LAMP assays also were assessed. RESULTS: The DR in CSA was 13.6% for PCR whereas for LAMP it was 39.0% and 25.4% for LAMP 1 and 2 methods, respectively. In AHA, the LAMP assay DR was 14.3% and 1.9% for LAMP 1 and 2, respectively. Phylogenetic tree analysis confirmed the identity of CaPV. Analytic sensitivity showed a detection limit of 8 copies/µL. The analytic specificity test showed no cross detection with other infectious agents. CONCLUSION AND CLINICAL IMPORTANCE: Good sensitivity and specificity results for LAMP assay support its application in the routine diagnosis of LSD, whereas its ability to detect LSDV in apparently healthy animals shows its usefulness in identifying populations at risk of LSD.

Molecular detection of Rift Valley fever virus in serum samples from selected areas of Tanzania.

Rift Valley fever (RVF) is an acute mosquito-borne viral zoonotic disease affecting domestic animals and humans caused by the Rift Valley fever virus (RVFV). The virus belongs to the genus Phlebovirus of the family Bunyaviridae. The main aim of this study was to detect the presence of antibodies to RVFV as well as the virus in the serum samples that were collected from livestock during the 2006/2007 RVF outbreaks in different locations in Tanzania. Analysis of selected samples was done using a RVF-specific inhibition enzyme-linked immunosorbent assay (I-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Genomic viral RNA was extracted directly from serum samples using a QIAamp Viral RNA Mini Kit (QIAGEN), and a one-step RT-PCR protocol was used to amplify the S segment of RVFV. Positive results were obtained in 39.5% (n = 200) samples using the RVF I-ELISA, and 17.6% (n = 108) of samples were positive by RT-PCR. I-ELISA detected 41 (38.7%), 32 (39.0%), and 6 (50.0%) positive results in cattle, goats, and sheep sera, respectively, whereas the RT-PCR detected 11 (0.2%), 7 (0.2%), and 1 (0.1%) positive results in cattle, goats, and sheep sera, respectively. These findings have demonstrated the presence of RVFV in Tanzania during the 2006/2007 RVF outbreaks. To our knowledge, this is the first report to detect RVFV in serum samples from domestic animals in Tanzania using PCR technique. Therefore, a detailed molecular study to characterize the virus from different geographical locations in order to establish the profile of strains circulating in the country and develop more effective and efficient control strategies should be done.

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 < 0.0001), followed by Kibondo at 2.3% (CI 95% = 0.5% - 6.5%; 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.

Molecular survey for foot-and-mouth disease virus in livestock in Tanzania, 2008-2013.

Phylogeography data are of paramount importance in studying the molecular epidemiology dynamics of foot-and-mouth disease virus (FMDV). In this study, epithelial samples and oesophageal-pharyngeal fluids were collected from 361 convalescent animals (cattle and buffaloes) in the field throughout Tanzania between 2009 and 2013. The single plex real-time RT-PCR (qRT-PCR) assay for rapid and accurate diagnosis of FMDV employing the Callahan 3DF-2, 3DF-R primers and Callahan 3DP-1 probe were used. Preparation of the samples was performed according to the OIE manual, with a Kenya O serotype obtained from the attenuated vaccine serving as a positive control and samples collected from healthy animals serving as true negatives. The results indicated that 53.49% of samples (n = 176) were positive for FMDV genome by qRT-PCR, with Ct values ranging from 14 to 32. In addition, molecular typing of the FMDV genome positive samples using serotype specific primers revealed the existence of several serotypes: serotype South Africa Territory 1 (SAT1) (34.25%, n = 60), serotype A (68.92%, n = 98), serotype O (59.20%, n = 98) and SAT2 (54.54%, n = 96). The virus protein 1 sequences analysis for 35 samples was performed and the collective results indicated: 54.28% serotype O, 25.71% serotype A, 14.28% serotype SAT1 and 2.85% serotype SAT2. Therefore in this study, both the phylogenetic trees and spatial distribution of serotypes elucidated the phylodynamics of multiple FMDV field strains in Tanzania and neighbouring countries.

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.

On the origin and diversity of Newcastle disease virus in Tanzania.

Free-range rural chickens (FRCs) dominate the poultry industry in developing countries and chickens are exposed to multi-host infections, including Newcastle disease virus (NDV). The knowledge about the characteristics of NDV from FRCs is limited. This study investigated the persistence, spread and risks of NDV from FRCs. NDV isolates (n = 21) from unvaccinated FRCs in Tanzania were characterised by conventional intracerebral pathogenicity index (ICPI) and sequence analysis of a partial region of the deduced fusion protein encompassing the cleavage site. Results showed that five isolates were screened as lentogenic, nine as mesogenic and six as velogenic. Phylogenetic analysis of the 21 isolates compared to reference sequences revealed three, four, nine and five isolates in genotypes 1, 2, 3c and 4a, respectively. Genotype 3c also included published sequences of Tanzanian isolates obtained from exotic birds and chicken isolates from Uganda. The analysis showed that NDV were persistently present among chicken populations and possibly spread through live chicken markets or migration of wild birds. Differences in amino acid sequences detected around the cleavage site separated the isolates in six types. However, cleavage site pattern could not fully differentiate mesogenic isolates from velogenic isolates.