Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva.

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

Monitoring vaccinated cattle for induction and longevity of persistent tick-transmissible infection: Implications for wider deployment of live vaccination against East Coast fever in Tanzania.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

Molecular species identification of bushmeat recovered from the Serengeti ecosystem in Tanzania.

Bushmeat harvesting and consumption represents a potential risk for the spillover of endemic zoonotic pathogens, yet remains a common practice in many parts of the world. Given that the harvesting and selling of bushmeat is illegal in Tanzania and other parts of Africa, the supply chain is informal and may include hunters, whole-sellers, retailers, and individual resellers who typically sell bushmeat in small pieces. These pieces are often further processed, obscuring species-identifying morphological characteristics, contributing to incomplete or mistaken knowledge of species of origin and potentially confounding assessments of pathogen spillover risk and bushmeat offtake. The current investigation sought to identify the species of origin and assess the concordance between seller-reported and laboratory-confirmed species of origin of bushmeat harvested from in and around the Serengeti National Park in Tanzania. After obtaining necessary permits, the species of origin of a total of 151 bushmeat samples purchased from known intermediaries from 2016 to 2018 were characterized by PCR and sequence analysis of the cytochrome B (CytB) gene. Based on these sequence analyses, 30%, 95% Confidence Interval (CI: 24.4-38.6) of bushmeat samples were misidentified by sellers. Misreporting amongst the top five source species (wildebeest, buffalo, impala, zebra, and giraffe) ranged from 20% (CI: 11.4-33.2) for samples reported as wildebeest to 47% (CI: 22.2-72.7) for samples reported as zebra although there was no systematic bias in reporting. Our findings suggest that while misreporting errors are unlikely to confound wildlife offtake estimates for bushmeat consumption within the Serengeti ecosystem, the role of misreporting bias on the risk of spillover events of endemic zoonotic infections from bushmeat requires further investigation.

Detection of carrier state and genetic diversity of Theileria parva in ECF-vaccinated and naturally exposed cattle in Tanzania.

Infection and Treatment Method (ITM) has been practiced in Tanzania for over 20 years as a prevention measure against East Coast Fever disease. It is known that ITM, like natural ECF infection, leads to a carrier state, whereby vaccinated cattle become asymptomatic carriers of the parasite. It is expected that ECF vaccination using ITM also leads to generation of combinations of vaccine specific Theileria parva and local strains that circulate in the field what contributes to an unknown level of parasite diversity. Moreover, the long term impact of ITM on carrier state and parasite diversity in cattle are largely unknown. To address this question blood was collected from ECF-vaccinated (n = 239) and unvaccinated (n = 97) cattle from Loiborsoit, Emboreet, Esilalei, Manyara ranch and Mswakini villages in the Maasai steppe of northern Tanzania, as well as Mruazi and Leila farms in Tanga in eastern Tanzania. Screening for T. parva using nested PCR revealed an overall prevalence of T. parva to be 34.5%, with a significant higher prevalence among ECF-vaccinated cattle. Using three VNTR markers (ms2, ms5 and MS7) higher parasite genetic diversity in terms of higher number of alleles and expected heterozygosity was shown in vaccinated than unvaccinated cattle. These parameters were highest in cattle from Manyara ranch. Nevertheless, the principle component analysis (PCoA) showed no distinct clustering patterns as most T. parva alleles clustered together throughout the four quadrants implying parasite homogeneity among the sampled populations. However, some of the parasite alleles closely clustered with Muguga vaccine alleles in two of the quadrants, consistent with closer genetic relatedness between the vaccine strains and the T. parva populations from the Maasai steppe. Likewise analysis of molecular variance (AMOVA) revealed most of the genetic variation (93%) being contained within populations with only 7% being among populations. This study therefore confirms the role of ECF vaccination in enhancing carrier state and T. parva diversity in vaccinated cattle populations. Higher T. parva diversity may play an important role in carrier cattle by way of restricting breakthrough infections from field parasite strains.