Plague in Tanzania: first report of sylvatic plague in Morogoro region, persistence in Mbulu focus, and ongoing quiescence in Lushoto and Iringa foci.

Objectives: Plague has been a threat to human health in Tanzania since 1886. This zoonotic disease has established several endemic foci in the country, posing a risk of outbreaks. This study was conducted to investigate the presence of Yersinia pestis in small mammals in five districts. These districts were selected because of recent (Mbulu), past (40-18 years ago: Lushoto) and historic (>100 years ago: Iringa and Kilolo) human cases of plague. In addition, one region that has not had any reported human cases of plague was included (Morogoro-Mvomero). Methods: Blood from 645 captured small mammals was screened for antibodies against the fraction 1 (F1) antigen of Y. pestis using indirect enzyme-linked immunosorbent assay (ELISA) and competitive-blocking ELISA. Results: Specific antibodies against Y. pestis F1 antigens were detected in six (0.93%) animals belonging to Mastomys natalensis. Of these, four animals were captured in the active focus in Mbulu, and two animals were captured from an area with no history of human plague (Morogoro-Mvomero). Conclusion: These results provide evidence of the circulation of Y. pestis in small mammals in Tanzania. Furthermore, evidence of the circulation of Y. pestis in Morogoro-Mvomero highlights the importance of carrying out plague surveillance in areas with no history of human plague, which can help to predict areas where future outbreaks may occur.

Performance characteristics and costs of serological tests for brucellosis in a pastoralist community of northern Tanzania.

The control of brucellosis across sub-Saharan Africa is hampered by the lack of standardized testing and the use of tests with poor performance. This study evaluated the performance and costs of serological assays for human brucellosis in a pastoralist community in northern Tanzania. Serum collected from 218 febrile hospital patients was used to evaluate the performance of seven index tests, selected based on international recommendation or current use. We evaluated the Rose Bengal test (RBT) using two protocols, four commercial agglutination tests and a competitive enzyme-linked immunosorbent assay (cELISA). The sensitivity, specificity, positive predictive value, negative predictive value, Youden's index, diagnostic accuracy, and per-sample cost of each index test were estimated. The diagnostic accuracy estimates ranged from 95.9 to 97.7% for the RBT, 55.0 to 72.0% for the commercial plate tests, and 89.4% for the cELISA. The per-sample cost range was $0.69-$0.79 for the RBT, $1.03-$1.14 for the commercial plate tests, and $2.51 for the cELISA. The widely used commercial plate tests performed poorly and cost more than the RBT. These findings provide evidence for the public health value of discontinuing the use of commercial agglutination tests for human brucellosis in Tanzania.

Molecular characterization of Brucella species detected in humans and domestic ruminants of pastoral areas in Kagera ecosystem, Tanzania.

Brucellosis is a zoonotic disease of importance to both public health and the livestock industry. The disease is likely to be endemic in Tanzania and little is reported on molecular characterization of Brucella species in pastoral settings. This study aimed at characterizing Brucella species (targeting genus Brucella) infecting humans, cattle and goat in Kagera region (Ngara and Karagwe districts) using real-time PCR, PCR amplification of 16S rRNA genes and Sanger sequencing. Brucella spp. were detected in 47 samples (19 sera and 28 milk) out of 125 samples (77 sera, 35 milk and 13 aborted materials) using real-time PCR. All aborted materials (13 samples) were negative to real-time PCR. Out of the 47 real-time PCR positive samples (28 milk and 19 sera), 20 samples (10 milk and 10 sera) showed an expected 16S rRNA gene PCR product. Sequence analysis and blasting confirmed the presence of Brucella spp. in pastoral areas of Kagera region. The Brucella spp. from Kagera were phylogenetically grouped in two clades and three branches all closer to B. melitensis, B. abortus and B. suis from USA, Sudan and Iran. However, they were distinct from other species isolated also in USA, New Zealand, Germany and Egypt. This was expected based on the distance between the geographical regions from which the data (nucleotides sequences from 16S gene sequencing) for the phylogeny reconstruction were obtained. This is the first study to report Brucella species identified using 16S rRNA gene sequencing in East and Central Africa. A livestock vaccination program re-inforced with a high index of Brucella diagnosis is needed to eradicate brucellosis in animals and minimize suffering from Brucella infections in humans in Tanzania.

Evidence of chikungunya virus infection among febrile patients seeking healthcare in selected districts of Tanzania.

Introduction: Chikungunya virus (CHIKV) infection is an emerging mosquito-borne disease that has been associated with frequent epidemics in the world. However, there is a dearth of information on its magnitude and associated risk factors in Tanzania. Objective: A study was conducted to determine seroprevalence of CHIKV among febrile patients seeking medical care at health facilities in Karagwe, Sengerema, Kilombero and Kyela districts. Methods: Structured questionnaires were administered and 728 serum samples were collected between May and June, 2015 and tested for the presence of CHIKV-IgM and IgG-specific antibodies using Enzyme-linked immunosorbent assay. Results and discussion: The common clinical characteristics exhibited by outpatients were fever, headache and joint pains (100%, 70%, and 68.3% respectively). Out of 728 outpatients screened for CHIKV, 105 (14%) tested CHIKV IgG positive whilst 11 (1.5%) tested CHIKV IgM positive. Chikungunya seropositivity was significantly higher than previously reported in Tanzania. The most affected age group was 20-29 years. Our results indicate that CHIKV infection is prevalent and contributes to the burden of febrile illnesses in Tanzania. The seroprevalence varies between districts, reflecting variation in mosquito vector transmission dynamics in different parts of the country. Abbreviations: CHIKV: Chikungunya virus; EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; IgG: Immunoglobulin G; IgM: Immunoglobulin M; NIMR: National Institute for Medical Research; RU: Relative Units; SACIDS: Southern African Centre for Infectious Disease Surveillance; USA: United States of America.

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

Rapid, 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.

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.

Preliminary investigation on presence of peste des petits ruminants in Dakawa, Mvomero district, Morogoro region, Tanzania.

Peste des petits ruminants (PPR) is an acute viral disease of small ruminants characterised by the sudden onset of depression, fever, oculonasal discharges, sores in the mouth, foul-smelling diarrhoea and death. For many years, in Africa, the disease was mainly confined to West and Central Africa but it has now spread southwards to previously PPR-free countries including Tanzania, Democratic Republic of Congo and Angola. The disease was first reported in Tanzania in 2008 when it was confined to the Northern Zone districts bordering Kenya. Presence of the disease has also been confirmed in southern Tanzania especially Mtwara region. Recently, a suspected outbreak of PPR in Dakawa area, Mvomero district, Morogoro region was reported. Clinical samples (lungs, intestines, lymph nodes, whole blood and sera) from suspected goats (n = 8) and sheep (n = 1) were submitted to Sokoine University of Agriculture for analysis. Molecular diagnosis by amplification of the nucleoprotein gene and the fusion gene of PPR virus (PPRV) using PPRV specific primers was done. Five goats and the sheep were positive for PPRV after performing RT-PCR. To our knowledge, this is the first report confirming the presence of PPR in the Mvomero district of the Morogoro region, Tanzania. Hence, more efforts should be put in place to prevent the spread of PPR in Tanzania.

Spatial and temporal distribution of foot-and-mouth disease virus in the lake zone of Tanzania.

This study was conducted to determine the spatiotemporal distribution of foot-and-mouth disease (FMD) virus (FMDV) serotypes and evaluate the awareness of livestock keepers about FMD in Tanzania. An observational prospective study involving serological analysis, FMDV antigen detection and questionnaire survey was carried out in the lake zone of Tanzania. Seroprevalence of antibodies to the nonstructural protein 3ABC of FMDV and serotype-specific antigen detection were investigated by using SVANOVIR® FMDV 3ABC-Ab ELISA and indirect-sandwich ELISA (sELISA), respectively, whilst a structured questionnaire was used to evaluate the awareness of livestock keepers about FMD. During the period of 2010-2011, both serum and tissue (foot-and-mouth epithelia) samples were collected from cattle suspected of FMD in 13 districts of the four regions of the lake zone. A total of 107 (80.5%) out of 133 tested serum samples were seropositive to nonstructural protein 3ABC, with at least one sample being positive from all 10 districts screened. Fifteen (53.6%) out of 28 tissue epithelial samples collected from FMD cases in eight districts during the course of this study were positive to serotype O FMDV antigen. Of these eight districts, serotype O FMDV antigens were detected from seven districts and no other serotypes were recovered from animal samples screened. Questionnaire surveys in six districts indicated that livestock keepers in the lake zone were aware of the clinical manifestations (26/29 = 90.0%) and economic impact (23/29 = 79.0%) of FMD in the region. The questionnaire data showed that FMD outbreaks often occurred after rainy seasons (22/29 = 75.9%), with the highest peaks predominantly occurring just after the long rains in May and June, and at the end of the short rains in November and December of each year. The spatial distribution of the FMD cases suggested that serotype O virus exposure was the only widespread cause of the 2010-2011 outbreaks in the lake zone.

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.

Rapid, sensitive and effective diagnostic tools for foot-and-mouth disease virus in Africa.

Speed is paramount in the diagnosis of highly infectious diseases, such as foot-and-mouth disease (FMD), as well as for emerging diseases; however, simplicity is required if a test is to be deployed in the field. Recent developments in molecular biology have enabled the specific detection of FMD virus (FMDV) by reverse-transcription loop-mediated isothermal amplification (RT-LAMP), real-time reverse-transcription polymerase chain reaction (RT-qPCR) and sequencing. RT-LAMP enables amplification of the FMDV RNA-dependent RNA polymerase 3D(pol) gene at 63 °C (in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase) for 1 h, whilst RT-qPCR amplifies the same gene in approximately 2 h 30 min. In this study, we compared the sensitivity and effectiveness of RT-LAMP against RT-qPCR for the detection of the FMDV 3D(pol) gene in 179 oesophageal-pharyngeal scraping samples (collected by probang) obtained from clinically healthy cattle and buffalo in Malawi, Mozambique and Tanzania in 2010. The FMDV detection rate was higher with RT-LAMP (30.2%; n = 54) than with RT-qPCR (17.3%; n = 31). All samples positive by RT-qPCR (Cq ≤ 32.0) were also positive for the RT-LAMP assay; and both assays proved to be highly specific for the FMDV target sequence. In addition, the VP1 sequences of 10 viruses isolated from positive samples corresponded to the respective FMDV serotypes and genotypes. Our findings indicate that the performance of RT-LAMP is superior to RT-qPCR. Accordingly, we consider this test to have great potential with regard to the specific detection and surveillance of infectious diseases of humans and animals in resource-compromised developing countries.

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.

Foot and mouth disease in Zambia: spatial and temporal distributions of outbreaks, assessment of clusters and implications for control.

Zambia has been experiencing low livestock productivity as well as trade restrictions owing to the occurrence of foot and mouth disease (FMD), but little is known about the epidemiology of the disease in these endemic settings. The fundamental questions relate to the spatio-temporal distribution of FMD cases and what determines their occurrence. A retrospective review of FMD cases in Zambia from 1981 to 2012 was conducted using geographical information systems and the SaTScan software package. Information was collected from peer-reviewed journal articles, conference proceedings, laboratory reports, unpublished scientific reports and grey literature. A space-time permutation probability model using a varying time window of one year was used to scan for areas with high infection rates. The spatial scan statistic detected a significant purely spatial cluster around the Mbala-Isoka area between 2009 and 2012, with secondary clusters in Sesheke-Kazungula in 2007 and 2008, the Kafue flats in 2004 and 2005 and Livingstone in 2012. This study provides evidence of the existence of statistically significant FMD clusters and an increase in occurrence in Zambia between 2004 and 2012. The identified clusters agree with areas known to be at high risk of FMD. The FMD virus transmission dynamics and the heterogeneous variability in risk within these locations may need further investigation.

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.

Existence of variant strains Fowlpox virus integrated with Reticuloendotheliosis virus in its genome in field isolates in Tanzania.

Fowlpox virus (FPV) is one example of poultry viruses which undergoes recombination with Reticuloendotheliosis virus (REV). Trepidation had been raised, and it was well established on augmented pathogenicity of the FPV upon integration of the full intact REV. In this study, we therefore intended at assessing the integration of REV into FPV genome of the field isolates obtained in samples collected from different regions of Tanzania. DNA extraction of 85 samples (scabs) was performed, and FPV-specific PCR was done by the amplification of the highly conserved P4b gene. Evaluation of FPV-REV recombination was done to FPV-specific PCR positively identified samples by amplifying the env gene and REV long terminal repeats (5' LTR). A 578-bp PCR product was amplified from 43 samples. We are reporting for the first time in Tanzania the existence of variant stains of FPV integrated with REV in its genome as 65 % of FPV identified isolates were having full intact REV integration, 21 % had partial FPV-REV env gene integration and 5 % had partial 5' LTR integration. Despite of the fact that FPV-REV integrated stains prevailed, FPV-REV-free isolates (9 %) also existed. In view of the fact that full intact REV integration is connected with increased pathogenicity of FPV, its existence in the FPV genome of most field isolates could have played a role in increased endemic, sporadic and recurring outbreaks in selected areas in Tanzania.

Challenges and prospects for the control of foot-and-mouth disease: an African perspective.

The epidemiology of foot-and-mouth disease (FMD) in Africa is unique in the sense that six of the seven serotypes of FMD viruses (Southern African Territories [SAT] 1, SAT2, SAT3, A, O, and C), with the exception of Asia-1, have occurred in the last decade. Due to underreporting of FMD, the current strains circulating throughout sub-Saharan Africa are in many cases unknown. For SAT1, SAT2, and serotype A viruses, the genetic diversity is reflected in antigenic variation, and indications are that vaccine strains may be needed for each topotype. This has serious implications for control using vaccines and for choice of strains to include in regional antigen banks. The epidemiology is further complicated by the fact that SAT1, SAT2, and SAT3 viruses are maintained and spread by wildlife, persistently infecting African buffalo in particular. Although the precise mechanism of transmission of FMD from buffalo to cattle is not well understood, it is facilitated by direct contact between these two species. Once cattle are infected they may maintain SAT infections without the further involvement of buffalo. No single strategy for control of FMD in Africa is applicable. Decision on the most effective regional control strategy should focus on an ecosystem approach, identification of primary endemic areas, animal husbandry practices, climate, and animal movement. Within each ecosystem, human behavior could be integrated in disease control planning. Different regions in sub-Saharan Africa are at different developmental stages and are thus facing unique challenges and priorities in terms of veterinary disease control. Many science-based options targeting improved vaccinology, diagnostics, and other control measures have been described. This review therefore aims to emphasize, on one hand, the progress that has been achieved in the development of new technologies, including research towards improved tailored vaccines, appropriate vaccine strain selection, vaccine potency, and diagnostics, and how it relates to the conditions in Africa. On the other hand, we focus on the unique epidemiological, ecological, livestock farming and marketing, socioeconomic, and governance issues that constrain effective FMD control. Any such new technologies should have the availability of safe livestock products for trade as the ultimate goal.