Genetic diversity and genomic analysis of G3P[6] and equine-like G3P[8] in children under-five from Southern Highlands and Eastern Tanzania.

Rotavirus group A genomic characterization is critical for understanding the mechanisms of rotavirus diversity, such as reassortment events and possible interspecies transmission. However, little is known about the genetic diversity and genomic relationship of the rotavirus group A strains circulating in Tanzania. The genetic and genomic relationship of RVA genotypes was investigated in children under the age of five. A total of 169 Fecal samples were collected from under-five with diarrhea in Mbeya, Iringa and Morogoro regions of Tanzania. The RVA were screened in children under five with diarrhea using reverse transcription PCR for VP7 and VP4, and the G and P genotypes were determined using Sanger dideoxynucleotide cycle sequencing. Whole-genome sequencing was performed on selected genotypes. The overall RVA rate was 4.7% (8/169). The G genotypes were G3 (7/8) and G6 (1/8) among the 8 RVA positives, while the P genotypes were P[6] (4/8) and P[8] (2), and the other two were untypeable. G3P[6] and G3P[8] were the identified genotype combinations. The genomic analysis reveals that the circulating G3P[8] and G3P[6] isolates from children under the age of five with diarrhea had a DS-1-like genome configuration (I2-R2-C2-M2-axe-N2-T2-E2-H2). The phylogenic analysis revealed that all 11 segments of G3P[6] were closely related to human G3P[6] identified in neighboring countries such as Uganda, Kenya, and other African countries, implying that G3P[6] strains descended from a common ancestor. Whereas, G3P[8] were closely related to previously identified equine-like G3P[P8] from Kenya, Japan, Thailand, Brazil, and Taiwan, implying that this strain was introduced rather than reassortment events. We discovered amino acid differences at antigenic epitopes and N-linked glycosylation sites between the wild type G3 and P[8] compared to vaccine strains, implying that further research into the impact of these differences on vaccine effectiveness is warranted. The phylogenic analysis of VP7 also identified a bovine-like G6. For the first time in Tanzania, we report the emergence of novel equine-like G3 and bovine-like G6 RVA strains, highlighting the importance of rotavirus genotype monitoring and genomic analysis of representative genotypes.

Prevalence and genomic characterization of rotavirus group A genotypes in piglets from southern highlands and eastern Tanzania.

Animals have been identified as the potential reservoirs of rotavirus group A (RVA) for human infection. However, very little is known regarding the genotype and genomic profiles of circulating RVA in Tanzanian piglets. The rotavirus genetic diversity and genome analysis was assessed among piglets from Southern highlands and Eastern Tanzania. A total of 241 faecal samples were collected from piglets in the regions of Mbeya, Iringa, and Morogoro. RVA was detected and genotyped using reverse transcription polymerase chain reaction (RT-PCR). Sanger dideoxynucleotide cycle sequencing of the viral protein (VP) 4 and VP7 genes was afterwards performed to confirm the RT-PCR results. Selected genotypes were subjected to whole genome sequencing. The overall prevalence of RVA was 35.26% (85/241) in piglets (30.58% in Mbeya, 43.75% in Iringa and 31.16% in Morogoro). Upon genotyping, the G genotypes were G4 (26), G9 (10), G3 (6), G5 (3) and the remaining 40 were untypeable, while the P genotype, were P[6] (35), P[13] (3) and the remaining 47 were untypeable. The G4P[6] were the predominant genotype followed by G3P[6], G3P[13], G4P[13] and G5P[13] were most common genotypes combinations. On phylogenetic analysis, G4 was grouped to lineage V, sublineages VIIa and VIIc, G9 to lineage I, G5 to lineage II, G3 to lineage IV, P[6] to lineage V and sublineage Ic and the P[13] to lineage IV. We revealed amino acid differences between the circulating G4 and the G4 in the ProSystems RCE vaccine used in pigs. The whole genome reveals genomic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1, G5-P[x]-I5-R1-C1-M1-A8-N1-Tx-E1-H1, G3/G4-P[13]/P[6]-Ix-R1-C1-M1-A8-N1-T1-E1-H1, G3-P[6]-Ix-R1-C1-M1-A8-N1-Tx-E1-H1 and G9-P[x]-Ix-R1-C1-M1-Ax-N1-Tx-E1-H1. The VP7 gene of G9, the VP4 gene of P[6] and NSP4 (E1) gene of some genotypes clustered together and closely related to humans origin or porcine-human reassortant strains with nucleotide similarities ranging from 97.90% to 99.74% from neighboring countries, implying possibility intragenogroup reassortment and interspecies transmission. The higher strain diversity observed within the gene segments highlight the importance of genomic analysis and continuous monitoring of RVA genotypes. Further research is needed to determine the risk factors associated with RVA infection in Tanzanian pigs in order to properly design a control program.

Rotavirus Burden, Genetic Diversity and Impact of Vaccine in Children under Five in Tanzania.

In Tanzania, rotavirus infections are responsible for 72% of diarrhea deaths in children under five. The Rotarix vaccine was introduced in early 2013 to mitigate rotavirus infections. Understanding the disease burden and virus genotype trends over time is important for assessing the impact of rotavirus vaccine in Tanzania. When assessing the data for this review, we found that deaths of children under five declined after vaccine introduction, from 8171/11,391 (72% of diarrhea deaths) in 2008 to 2552/7087 (36% of diarrhea deaths) in 2013. Prior to vaccination, the prevalence of rotavirus infections in children under five was 18.1-43.4%, 9.8-51%, and 29-41% in Dar es Salaam, Mwanza and Tanga, respectively, and after the introduction of vaccines, these percentages declined to 17.4-23.5%, 16-19%, and 10-29%, respectively. Rotaviruses in Tanzania are highly diverse, and include genotypes of animal origin in children under five. Of the genotypes, 10%, 28%, and 7% of the strains are untypable in Dar es Salaam, Tanga, and Zanzibar, respectively. Mixed rotavirus genotype infection accounts for 31%, 29%, and 12% of genotypes in Mwanza, Tanga and Zanzibar, respectively. The vaccine effectiveness ranges between 53% and 75% in Mwanza, Manyara and Zanzibar. Rotavirus vaccination has successfully reduced the rotavirus burden in Tanzania; however, further studies are needed to better understand the relationship between the wildtype strain and the vaccine strain as well as the zoonotic potential of rotavirus in the post-vaccine era.

Toxigenic Vibrio cholerae O1 in vegetables and fish raised in wastewater irrigated fields and stabilization ponds during a non-cholera outbreak period in Morogoro, Tanzania: an environmental health study.

BACKGROUND: Cholera, one of the world's deadliest infectious diseases, remains rampant and frequent in Tanzania and thus hinders existing control measures. The present study was undertaken to evaluate the occurrence of toxigenic Vibrio cholerae O1 in wastewater, fish and vegetables during a non-outbreak period in Morogoro, Tanzania. METHODS: From October 2014 to February 2015, 60 wastewater samples, 60 fish samples from sewage stabilization ponds and 60 wastewater irrigated vegetable samples were collected. Samples were cultured for identification of V. cholerae using conventional bacteriological methods. Isolates were confirmed as V. cholerae by detection of the outer membrane protein gene (ompW) using polymerase chain reaction (PCR). Isolates were further tested for antibiotic susceptibility and presence of virulence genes including, cholera enterotoxin gene (ctx), the toxin co-regulated pilus gene (tcpA) and the haemolysin gene (hlyA). RESULTS: The prevalence of V. cholerae in wastewater, vegetables and fish was 36.7, 21.7 and 23.3 %, respectively. Two isolates from fish gills were V. cholerae O1 and tested positive for ctx and tcpA. One of these contained in addition the hlyA gene while five isolates from fish intestines tested positive for tcpA. All V. cholerae isolates were resistant to ampicillin, amoxicillin and some to tetracycline, but sensitive to gentamicin, chloramphenicol, and ciprofloxacin. CONCLUSIONS: Our results show that toxigenic and drug-resistant V. cholerae O1 species are present and persist in aquatic environments during a non-cholera outbreak period. This is of public health importance and shows that such environments may be important as reservoirs and in the transmission of V. cholerae O1.

Quantitative analysis of risk factors associated with brucellosis in livestock in the Katavi-Rukwa ecosystem, Tanzania.

Brucellosis is a neglected contagious bacterial disease of public health and economic importance. Nevertheless, its spread is not well known to many livestock farmers. Unmatched case control study was carried out to identify risk factors associated with brucellosis in cattle and goats at the herd level in Mpanda, Mlele and Nsimbo districts of Katavi region, in Tanzania between September 2012 and July 2013. A total of 138 adult respondents were selected randomly for the interview using a structured questionnaire. The criterion for inclusion was to have at least one Brucella-positive animal in the herd while the control was chosen from among the herds which these animals tested negative. The presence of seropositive herds were statistically linked (P < 0.1) by univariate analysis with such variables as lack of formal education among the herders; slaughtering of cattle or goats in the household; the history of occurrence of abortion in the herd; and the history of occurrence of retained foetal membranes in the herd following parturition. However, in the multivariate analysis, lack of formal education among the herders and the previous history of occurrence of retained foetal membrane in the herd following parturition were significantly associated with Brucella seropositive (P < 0.05). This study suggests the need for raising community awareness on the risk factors associated with the transmission of brucellosis between animals and humans and the implementation of disease prevention and control programmes.

Epidemiology of Brucella infection in the human, livestock and wildlife interface in the Katavi-Rukwa ecosystem, Tanzania.

BACKGROUND: Brucellosis is a zoonosis of public health importance worldwide. In Tanzania, the disease is underreported due to insufficient awareness, inadequate diagnostic protocols, including lack of appropriate reagents for diagnosis. Livestock and wildlife are considered potential sources of infection to humans; however, the role played by these carriers in the epidemiology of the disease in the ecosystems in Tanzania is not fully understood. The objective of this study was to establish the prevalence of anti-Brucella antibodies in humans, wildlife and livestock; and molecular prevalence of Brucella spp in cattle and goats in the Katavi- Rukwa ecosystem. RESULTS: Anti-Brucella antibodies were detected in humans at 0.6 % (95 % CI: 0.1, 2.1 %); cattle at 6.8 % (95 % CI: 5.4, 8.5 %), goats at 1.6 % (95 % CI: 0.4, 4.1 %) and buffaloes at 7.9 % (95 % CI: 1.7, 21.4 %). One of the two sampled lions tested positive. Cattle had a significantly higher prevalence of anti-Brucella antibodies as compared to goats (P < 0.05). A significantly higher seroprevalence was found in female than in male cattle and in adult than in young cattle (P < 0.05). There was an agreement of 95 and 89 % in cattle and goats, respectively, for the Rose Bengal plate Test (RBPT) and Competitive Enzyme Linked Immunosorbent Assay (c-ELISA) in detecting Brucella infection. Eight (3.5 %) out of 231 milk samples tested were positive for Brucella spp on Polymerase Chain Reaction (PCR), and Brucella abortus biovar 1 was detected in cattle milk. However, no Brucella spp were detected in goat milk. CONCLUSION: This study has shown the presence of anti- Brucella antibodies in humans, livestock, and wildlife in the Katavi- Rukwa ecosystem. Transmission of the infection between wildlife, livestock and humans is likely to continue due to increasing human activities in the human wildlife interface. This information is an important contribution to public health policy development in the human wildlife interface of the Katavi- Rukwa ecosystem.

Molecular characterization of Mycobacterium bovis isolates from pastoral livestock at Mikumi-Selous ecosystem in the eastern Tanzania.

Domestic ruminants and Wild ungulates can act as reservoir hosts for Mycobacterium bovis, causative agent for bovine tuberculosis (BTB) in animals and man. Bovine tuberculosis (BTB) is endemic in pastoral livestock in Tanzania. There is lack of information on genotypic distribution of M. bovis in animals at wildlife-livestock interface areas and in wildlife conserved areas. The objectives of this study were to characterize and determine the spatial distribution of M. bovis isolates. Tissue samples from cattle and wildlife were cultured and analyzed using deletion typing and spoligotyping techniques. Eight (8.9%) M. bovis strains were isolated from cattle and confirmed by RD4 and RD9 polymerize chain reaction (PCR) assays. Spoligotype SB1467 was the dominant pattern 62.5% (n = 8) in infected animals, two isolates belonged to a newly reported type SB2190, and one isolate belonged to SB0133. The spoligotype patterns of SB1467 and SB0133 were closely related (96.9%) while SB2190 was less related (59.4%) to SB0133, the relatedness amongst spoligotypes were associated with spacer position 15. No M. bovis was isolated from hunted wild animals. The current study has identified a novel spoligotype SB2190. The current data suggest that wild animals in Mikumi-Selous ecosystem are at risk of acquiring M. bovis infection due to occasional interaction by sharing of pasture and water sources between wildlife and livestock. Integrated efforts by all stakeholders are crucial for controlling spread of tuberculosis at livestock/wildlife/human interface areas.

Prevalence and significant geospatial clusters of bovine tuberculosis infection at livestock-wildlife interface ecosystem in Eastern Tanzania.

Bovine tuberculosis (BTB) is an important neglected zoonosis that affects livestock, wildlife and human. A study to determine prevalence and geospatial clusters for BTB was conducted from June 2010 to March 2012 at livestock-wildlife interface areas (LWIA). A total of 1,288 cattle located in vicinity of Mikumi-Selous ecosystem Tanzania were tested. Single Intradermal Comparative Tuberculin Test and spatial scan statistic analysis were applied to establish the status of the disease and identify significant spatial BTB clusters. Overall individual prevalence was 3.7 % (n=1,288) (95 % CI=2.8-4.9) and 7.8 % (95 % CI=6.4-9.4) with cut-off of >4 and >2 mm, respectively. Villages with at least one reactor were 55.8 % (n=43). Reactivity was significantly higher in Mvomero and Kilosa districts compared with Kilombero and Ulanga districts (χ (2) =15.9; P<0.001). Significant spatial BTB clusters were revealed at 11 villages. BTB clustering was significant in Kilosa and Mvomero districts compared with Kilombero and Ulanga districts. There was overlap and aggregation of BTB clusters covering south and south-east of Kilosa district bordering Mikumi National Park (MNP) and Mvomero. Generally, clustering occurred around major rivers. The current study provides useful information on the dynamics and epidemiological status of BTB around the wildlife-livestock-human interface, it reveals that the wildlife are at risk of BTB from infected livestock. The study revealed hotspots for BTB that can be applied to guide implementation of participatory intervention at LWIA and control strategies in marginalised pastoralist communities. This study calls for similar studies in other Tanzania's LWIA for efficient intervention of BTB countrywide.