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.

Exposure to high thermal conditions for a long time induces apoptosis and decreases total RNA concentration in peripheral blood mononuclear cells among Indian Zebu-Jersey crossbreds.

Background and Aim: Global warming has grave consequences on livestock production systems and profound negative effects on animal production. This study aimed to carry out an in vitro thermal stress stimulation (TSS) of bovine peripheral blood mononuclear cells (PBMCs) using different thermal assault conditions (TACs), including normal to extreme temperatures and varying durations of thermal exposure (DTE) to understand how PBMCs of Indian Zebu-Jersey crossbreds respond to various levels and durations of heat shock. Materials and Methods: Ten milliliters of blood were collected from 70 Indian Zebu-Jersey crossbreds under aseptic conditions and were sampled for isolating PBMCs. Peripheral blood mononuclear cells were divided into seven groups, each comprising 10 PBMC samples isolated from 10 different animals. Aliquots of 500 µL of PBMCs were stressed by exposure to different TACs (37, 40, and 45°C) for DTEs of 3 or 6 h. Subsequently, the cells were harvested. The control unstressed samples (500 µL aliquots of PBMCs) were exposed to no TAC (0°C) and zero DTE (0 h). Total RNA from all the treatment groups of PBMCs were isolated and quantitated. Results: We found a very strong association between TACs and RNA levels. In addition, PBMCs viability was negatively affected by heat shock. This led to an exponential reduction in PBMC count as TACs toughened. Only 3.59 × 105 ± 0.34 cells/mL were viable after exposure to 45°C for a 6 h DTE. This cell viability was lower than that measured in controls subjected to no stress and zero time DTE (2.56 × 107 ± 0.22 cells/mL). We also observed a reduction in the concentration of RNA isolated from thermally stressed PBMCs. Conclusion: In vitro TSS of PBMCs provided biological information on the response of cellular systems to heat shock after exposure to TACs. This will help to mitigate and manage the effects of thermal stress in bovine species. The association between the reduction in PBMC count after in vitro TSS and the expression of heat shock protein 70 gene will be investigated in the future to further understand how Indian Zebu-Jersey crossbreds respond to in vitro thermal conditions. This will be used to determine the in vivo response of Indian Jersey crossbreds to different environmental thermal conditions and will further enable the in vivo understanding of thermotolerance potentials of bovine species for better adaptation, survival, and production performance.

Heterozygous Single-Nucleotide Polymorphism Genotypes at Heat Shock Protein 70 Gene Potentially Influence Thermo-Tolerance Among Four Zebu Breeds of Nigeria.

Genetic variants at heat shock protein 70 gene and their influence on heat stress (HS) tolerance were studied among selected Nigeria zebu, namely, 25 White Fulani (WF), 21 Sokoto Gudali (SG), 21 Red Bororo (RB), and 23 Ambala (AM). Detection of single nucleotide polymorphism (SNP) followed by determination of genotype and genotypic frequency was made among the selected breeds. The heat tolerance coefficient (HTC) was determined from thermo-related parameters including body temperature, rectal temperature, and respiratory rate. Thermo-Tolerance was evaluated through the SNP-thermo-parameter relationship. Statistical analyses were done using the GLM procedure in SAS. A quantitative real-time/high-resolution melting-based assay detected twelve genetic variants. Five of these were common and shared across all breeds of cattle. Of the remaining seven variants, three were specifically identified in AM, two in SG, and two in RB. Also, SNPs were evaluated and four unique SNPs (C151T, C146T, G90A, and C219A) were identified. Heterozygous animals had lower HTC suggesting their potential to withstand HS than homozygous counterparts. The WF and RB animals had significantly lower values for all parameters (BT, RT, RR, and HTC) compared to AM and SG breeds. Thermo-related parameters were significantly different (P < 0.001), and it is recommended that screening of SNPs in zebu is needed to enable selection for improved thermo-tolerance.

Development of a practical framework for sustainable surveillance and control of ticks and tick-borne diseases in Africa.

A workshop on ticks and tick-borne diseases (T&TBDs) was held on June 25 and 26, 2019, at the Tropical Pesticides Research Institute, Division of Livestock and Human Diseases Vector Control, Arusha, Tanzania. The objectives of the workshop were to discuss the current situation and to formulate actionable strategies to improve surveillance and control of T&TBDs in Africa. The workshop was funded by the National Research Foundation and the Cape Peninsula University of Technology and attended by livestock health providers, farmers, and researchers from East, West, and Southern African countries. During the workshop, experts presented recent surveillance data focused on T&TBDs; participants discussed research opportunities and community engagement. The primary outcome of the workshop was the creation of a new research consortium known as The African Consortium for T&TBDs. The consortium is intended to function as a community for researchers, students, farmers, policymakers, extension workers, and community members who are interested in the advancement of T&TBD control. The consortium will engage in research activities that focus on comprehensive surveillance of T&TBDs, developing tick acaricide resistance, alternative tick control programs, and policy development and education. These areas were identified as top priorities to be developed to improve T&TBD control on the continent.

The effect of Beauveria bassiana inoculation on plant growth, volatile constituents, and tick (Rhipicephalus appendiculatus) repellency of acetone extracts of Tulbaghia violacea.

AIM: The purpose of the present study was to evaluate the effects of Beauveria bassiana (Hypocreales) inoculum on plant growth, volatile constituents, and tick repellency of the extracts of Tulbaghia violacea (Amaryllidaceae). MATERIALS AND METHODS: Eight-week-old potted seedlings of T. violacea were each inoculated with conidia of B. bassiana (strain SM3) suspended at a concentration of 1×106 conidia mL-1. Tissue colonization by fungal conidia was assessed after 3 weeks. Plant growth, volatile constituents, and tick repellency were assessed after 12 weeks post-treatment. RESULTS: B. bassiana conidia successfully colonized leaf and root tissues of T. violacea. The growth of fungal hyphae out of the leaf and root sections occurred in 75% and 91.6% of plants, respectively. Inoculation of the plants with B. bassiana significantly (p<0.05) influenced root length and plant height but did not have substantial effects on weights and leaf number of T. violacea. While the fungus did not have significant effects on overall number of the volatile chemical constituents, significant variations in the quantity (area ratio) were observed in at least four compounds that were detected. In the tick repellency bioassay, high concentration (20 w/v%) of acetone extract from fungus-exposed plants produced the least repellent effect on Rhipicephalus appendiculatus larvae (Ixodidae), while at lower concentrations (5 w/v% and 10 w/v%) of acetone extracts of T. violacea, tick repellent activity of the extract of the fungus treatment was significantly improved and was comparable to commercial N,N-Diethyl-m-toluamide and the other treatments. CONCLUSION: Experimental fungal inoculation positively influenced plant growth in height and root length and tick (R. appendiculatus) repellency of acetone extracts of T. violacea at a concentration of 10 w/v% compared to the control treatment.

Single nucleotide polymorphisms at heat shock protein 90 gene and their association with thermo-tolerance potential in selected indigenous Nigerian cattle.

Heat shock protein (HSP) 90 gene provides protection and adaptation to thermal assault and certain polymorphisms have been associated to heat tolerance in humans and animals. Single nucleotide polymorphisms (SNPs) of HSP 90 gene were used to evaluate the scientific basis of heat tolerance in four zebu breeds of Nigeria. The DNA was extracted from skin tissue of 90 adult bulls representing White Fulani (WF), Sokoto Gudali (SG), Red Bororo (RB), and Ambala (AM). The SNPs were determined in DNAs using PCR, sequencing, and visualization and bio-editing by chromatogram in SeqMan Ngen tool. Subsequently, respective genotypes were constructed and genotypic and allelic frequencies were computed. Also, body parameters related to heat stress (HS) including body temperature (BT), rectal temperature (RT), and respiratory rates (RR) were taken for each animal before biological sampling and heat tolerance coefficient (HTC) was calculated. We detected four SNPs distinct/specific for each breed as follows: change from thymine (T) to guanine (G) at position 116 (T116G) in RB, G to cytosine (C) at 220 (G220C) in SG, G to adenine (A) at two positions, 346 (G346A) and 390 (G390A) in AM and WF, respectively. Heterozygous SNPs showed significantly lower values (P < 0.0001) for BT, RT, RR, and HTC than homozygous genotypes at all positions. We hypothesize that animals with heterozygous SNPs in exon 3 of HSP 90 may be tolerant to HS. These SNPs can be used as bio-markers for screening large populations of cattle for tolerance to hot tropical conditions in Nigeria and other sub-humid places.

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.

Seasonal variations in the availability of fodder resources and practices of dairy cattle feeding among the smallholder farmers in Western Usambara Highlands, Tanzania.

The aim of this study was to assess the seasonal effects on quantity and quality of fodder resources and associated utilization practices among smallholder dairy farmers in Western Usambara Highlands (WUHs) in Tanzania. The WUHs are among the major milk producing areas under smallholder dairy farming systems (SDFS) in Tanzania. Dry season fodder scarcity is a widespread problem affecting the East African SDFS and has been shown to contribute to over 40% reduction in milk yield. There is limited information with regard to seasonal fodder fluctuation and its effects on productivity of dairy cows in different landscape levels of Tanzania. Field and household surveys were conducted in 150 dairy cattle farming households from five villages in three wards located in WUHs. Survey data were analyzed using IBM SPSS version 21. In addition, remote sensing techniques were employed on gap-filled and smoothed Landsat data to generate land cover maps and bimonthly normalized difference vegetation index-time series for the 2009-2016. SDFS landscape was highly heterogeneous typified by crops, bushes, and forests. On average, the household landholding was 1.3 ha, while herd size was three cattle. About 87% of household land was devoted to crop growing with limited pasture along the farm margins and contour strips. Fodder scarcity was the major challenge during the dry season (July to October) as indicated by 87% of the respondents. On-farm fodder resources contributed most of the cattle diet (73%) while rangeland, forest, and purchased feed provided small amount. Natural pasture and napier grass (Pennisetum purpureum) were the most important feeds in wet season while maize stover was most significant during the dry season. Maize stover was profusely stored for dry season feeding and neither silage nor hay making was practiced. The nutritional values of the fibrous feeds declined during the dry season, whereby the metabolizable energy and crude protein contents were 6.0 MJ/kg and 10.1% dry matter, respectively, during wet season compared to 4.8 MJ/kg and 7.8% dry matter, respectively, during the dry season. Consequently, milk yield drops from 5.6 l per cow per day in the wet season to 3.0 l in the dry season. It is concluded that dry season fodder scarcity is a major problem in the WUHs and it hinders sustainable dairy production. It is therefore suggested that increase in fodder production as well as adoption of fodder conservation and feeding technologies are inevitable if sustainable dairy production is to be met in the Western Usambara Highlands and elsewhere with similar environments.

WITHDRAWN:Phylogenetic relationship and variation among four strains of Small East African goats of Tanzania based on mitochondrial DNA.

Ahead of Print article withdrawn by publisher.

Determination of Genetic Structure and Signatures of Selection in Three Strains of Tanzania Shorthorn Zebu, Boran and Friesian Cattle by Genome-Wide SNP Analyses.

BACKGROUND: More than 90 percent of cattle in Tanzania belong to the indigenous Tanzania Short Horn Zebu (TSZ) population which has been classified into 12 strains based on historical evidence, morphological characteristics, and geographic distribution. However, specific genetic information of each TSZ population has been lacking and has caused difficulties in designing programs such as selection, crossbreeding, breed improvement or conservation. This study was designed to evaluate the genetic structure, assess genetic relationships, and to identify signatures of selection among cattle of Tanzania with the main goal of understanding genetic relationship, variation and uniqueness among them. METHODOLOGY/PRINCIPAL FINDINGS: The Illumina Bos indicus SNP 80K BeadChip was used to genotype genome wide SNPs in 168 DNA samples obtained from three strains of TSZ cattle namely Maasai, Tarime and Sukuma as well as two comparative breeds; Boran and Friesian. Population structure and signatures of selection were examined using principal component analysis (PCA), admixture analysis, pairwise distances (FST), integrated haplotype score (iHS), identical by state (IBS) and runs of homozygosity (ROH). There was a low level of inbreeding (F~0.01) in the TSZ population compared to the Boran and Friesian breeds. The analyses of FST, IBS and admixture identified no considerable differentiation between TSZ trains. Importantly, common ancestry in Boran and TSZ were revealed based on admixture and IBD, implying gene flow between two populations. In addition, Friesian ancestry was found in Boran. A few common significant iHS were detected, which may reflect influence of recent selection in each breed or strain. CONCLUSIONS: Population admixture and selection signatures could be applied to develop conservation plan of TSZ cattle as well as future breeding programs in East African cattle.

Tick burden and prevalence of Theileria parva infection in Tarime zebu cattle in the lake zone of Tanzania.

This study was carried out to assess the distribution, abundance of different tick genera and prevalence of Theileria parva infection in Tarime zebu cattle kept in selected wards of Serengeti and Tarime districts in Mara region. Adult ticks were identified and counted from half body parts of 360 animals which were extensively managed in communal land with natural pastures. Concurrently, blood samples were collected and thereafter DNA extracted and a nested polymerase chain reaction (nPCR) was done using primers specific for p104 gene to detect the presence of T. parva DNA. Ticks were identified into four groups: Amblyomma genus, Boophilus sub-genus of Rhipicephalus genus, other species of Rhipicephalus, and Hyalomma genus. Rhipicephalus genus accounted for 71.8 % of the total ticks, whereas Amblyomma, Boophilus sub-genus of Rhipicephalus genus and Hyalomma constituted 14.1, 14.0 and 0.1 %, respectively. There were more animals (p < 0.05) infested with ticks in Tarime district (96.1 %) than in Serengeti (61.7 %). The average counts of ticks were higher in adult animals (p < 0.05) than in young animals. The overall prevalence of T. parva was 27.7 % and was higher (p < 0.05) in Serengeti (38.3 %) than in Tarime district (16.7 %). However, all animals tested positive for T. parva did not show any clinical signs of East Coast fever (ECF), suggesting the existence of subclinical infection in Tarime zebu. These results suggest that Tarime cattle can tolerate ECF infection and are likely to serve as potential carriers of T. parva to other less-tolerant cattle breeds in mixed herds. Since Tarime cattle are preferred by most farmers with mixed herds, routine screening for T. parva is highly recommended to minimize introduction of infected cattle into an immunologically naive population.

Evaluation of PRNP expression based on genotypes and alleles of two indel loci in the medulla oblongata of Japanese Black and Japanese Brown cattle.

BACKGROUND: Prion protein (PrP) level plays the central role in bovine spongiform encephalopathy (BSE) susceptibility. Increasing the level of PrP decreases incubation period for this disease. Therefore, studying the expression of the cellular PrP or at least the messenger RNA might be used in selection for preventing the propagation of BSE and other prion diseases. Two insertion/deletion (indel) variations have been tentatively associated with susceptibility/resistance of cattle to classical BSE. METHODOLOGY/PRINCIPAL FINDINGS: We studied the expression of each genotype at the two indel sites in Japanese Black (JB) and Japanese Brown (JBr) cattle breeds by a standard curve method of real-time PCR. Five diplotypes subdivided into two categories were selected from each breed. The two cattle breeds were considered differently. Expression of PRNP was significantly (p<0.0001) greater in the homozygous deletion genotype at the 23-bp locus in JB breed. Compared to the homozygous genotypes, the expression of PRNP was significantly greater in the heterozygous genotype at the 12-bp locus in JB (p<0.0001) and in JBr (p = 0.0394) breeds. In addition, there was a statistical significance in the PRNP levels between the insertion and the deletion alleles of the 23-bp locus in JB (p = 0.0003) as well as in JBr (p = 0.0032). There was no significance in relation to sex, age, geographical location or due to their interactions (p>0.05). CONCLUSION: Our results suggest that the del/del genotype or at least its del allele may modulate the expression of PRNP at the 23-bp locus in the medulla oblongata of these cattle breeds.

Allele distributions and frequencies of the six prion protein gene (PRNP) polymorphisms in Asian native cattle, Japanese breeds, and mythun (Bos frontalis).

Six polymorphic sites of the bovine prion protein gene (PRNP) were genotyped in 569 animals of Asian native cattle, Japanese breeds, purebred mythun (Bos frontalis), and mythun x cattle composite animals. At the 23-bp indel site, a deletion (23-) allele was a major allele in all populations except mythun. At the 12-bp indel site, an insertion (12+) allele was a major allele in all populations. The 14-bp indel site was polymorphic in all Asian native cattle. In the octapeptide repeat region, a six-repeat allele was a major allele in all populations, and 5/5 and 4/6 genotypes were detected in Japanese Black and Mongolian cattle and in mythun, respectively. Two nonsynonymous single nucleotide polymorphisms (SNPs) (K3T and S154N) were detected in Asian native cattle and mythun. Haplotype analysis using the genotypes of the six sites estimated 33 different haplotypes. The haplotype 23- 12- K 6 S 14+ was found in all populations.

Gene and haplotype polymorphisms of the Prion gene (PRNP) in Japanese Brown, Japanese native and Holstein cattle.

Polymorphisms in the prion protein gene (PRNP) are known to be associated with transmissible spongiform encephalopathies in human, sheep and goats. There is tentative association between PRNP promoter polymorphism and bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, we genotyped for six bovine PRNP polymorphic sites including a 23-bp indel in the promoter, a 12-bp indel in the intron 1, two nonsynonymous single nucleotide polymorphisms (SNPs), octapeptide repeats in the coding region and a 14-bp indel in the 3'-untranslated region in 178 animals representing Japanese Brown, Kuchinoshima feral, Mishima, Japanese Shorthorn and Holstein. In 64 Japanese Brown cattle, three indel sites were polymorphic. All of the six sites were monomorphic in Kuchinoshima. The 23-bp and 12-bp indel sites were polymorphic in Mishima cattle. The 23-bp and 14-bp indel sites were polymorphic in Japanese Shorthorn cattle. Both SNP sites were monomorphic in all cattle examined in this study. At the 23-bp indel site, the genotype frequencies of Japanese Brown and Holstein breeds were similar to that of BSE affected cattle. We estimated 12 different haplotypes from these genotypic data. A '23-12-K6S14+' haplotype was the major haplotype in all populations, whose frequencies ranged from 0.50 to 1.00.