What is needed to achieve effective and sustainable control of African animal trypanosomosis?

A welcome resurgence in African animal trypanosomosis (AAT) research has resulted in advances in capabilities, foundational datasets, and understanding. Additionally, there is the prospect of the first new trypanocide in >60 years. However, it is vital to ensure that advances translate to improved and sustainable control in the field. A recent meeting, the Symposium on African Livestock Trypanosomes - Tanzania, convened stakeholders from across the spectrum of AAT research and control to ask how this can be achieved. Current constraints on progress were defined, as were critical gaps and opportunities that need addressing. There is a requirement and opportunity for the AAT research community to communicate, collaborate, and coordinate to maintain momentum and achieve the ultimate goal of sustainable AAT control.

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection.

BACKGROUND: Tsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naïve tsetse with T. brucei DNA via faeces when co-housed. METHODOLOGY/PRINCIPLE FINDINGS: Insectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). CONCLUSIONS/SIGNIFICANCE: Our results show that infected tsetse can contaminate naïve flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.

Continent-wide genomic analysis of the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided little support for the currently described four subspecies. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management.

Aedes albopictus invasion across Africa: the time is now for cross-country collaboration and control.

The distribution of Aedes albopictus across west Africa is well documented. However, little has been done to synthesise data and establish the current distribution of this invasive vector in central and east Africa. In this Viewpoint, we show that A albopictus is establishing across Africa, how this is potentially related to urbanisation, and how establishment poses risks of near-term increases in arbovirus transmission. We then use existing species distribution maps for A albopictus and Aedes aegypti to produce consensus estimates of suitability and make these estimates accessible. Although urban development and increased trade have economic and other societal gains, the resulting potential changes in Aedes-borne virus epidemiology require a discussion of how cross-country collaboration and mitigation could be facilitated. Failure to respond to species invasion could result in increased transmission of Aedes-associated pathogens, including dengue, chikungunya, and Rift Valley fever viruses.

An outbreak of Rift Valley fever among peri-urban dairy cattle in northern Tanzania.

BACKGROUND: Human and animal cases of Rift Valley fever (RVF) are typically only reported during large outbreaks. The occurrence of RVF cases that go undetected by national surveillance systems in the period between these outbreaks is considered likely. The last reported cases of RVF in Tanzania occurred during a large outbreak in 2007-2008. METHODS: Samples collected between 2017 and 2019 from livestock suffering abortion across northern Tanzania were retrospectively tested for evidence of RVF virus infection using serology and reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: A total of 14 RVF-associated cattle abortions were identified among dairy cattle in a peri-urban area surrounding the town of Moshi. RVF cases occurred from May to August 2018 and were considered to represent an undetected, small-scale RVF outbreak. Milk samples from 3 of 14 cases (21%) were found to be RT-qPCR positive. Genotyping revealed circulation of RVF viruses from two distinct lineages. CONCLUSIONS: RVF outbreaks can occur more often in endemic settings than would be expected on the basis of detection by national surveillance. The occurrence of RVF cases among peri-urban dairy cattle and evidence for viral shedding in milk, also highlights potentially emerging risks for RVF associated with increasing urban and peri-urban livestock populations.

Pharma to farmer: field challenges of optimizing trypanocide use in African animal trypanosomiasis.

Trypanocides are a key control component of African animal trypanosomiasis (AAT) in tsetse-infested areas of sub-Saharan Africa. While farmers are dependent upon trypanocides, recent research highlights their inappropriate and ineffective use, problems with drug quality, and treatment failure. There are currently gaps in knowledge and investment in inexpensive AAT diagnostics, understanding of drug resistance, and the effective use of trypanocides in the field. Without this important knowledge it is difficult to develop best practice and policy for existing drugs or to inform development and use of new drugs. There needs to be better understanding of the drivers and behavioural practices around trypanocide use so that they can be incorporated into sustainable solutions needed for the development of effective control of AAT.

Antigenic Diversity in Theileria parva Populations From Sympatric Cattle and African Buffalo Analyzed Using Long Read Sequencing.

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite Theileria parva, transmitted by the three-host tick Rhipicephalus appendiculatus. The African buffalo (Syncerus caffer) is the natural host for T. parva but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between T. parva populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of T. parva populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three T. parva antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8+ and CD4+ T cells in immunized cattle, were used to characterize genetic diversity of T. parva in cattle (n = 126) and buffalo samples (n = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between T. parva populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo T. parva population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of T. parva is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo T. parva population dynamics is needed, as only a comprehensive appreciation of the population genetics of T. parva populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination.

A cross-sectional survey to establish Theileria parva prevalence and vector control at the wildlife-livestock interface, Northern Tanzania.

East Coast fever (ECF) in cattle is caused by the protozoan parasite Theileria parva, transmitted by Rhipicephalus appendiculatus ticks. In cattle ECF is often fatal, causing annual losses >$500 million across its range. The African buffalo (Syncerus caffer) is the natural host for T. parva but the transmission dynamics between wild hosts and livestock are poorly understood. This study aimed to determine the prevalence of T. parva in cattle, in a 30 km zone adjacent to the Serengeti National Park, Tanzania where livestock and buffalo co-exist, and to ascertain how livestock keepers controlled ECF and other vector-borne diseases of cattle. A randomised cross-sectional cattle survey and questionnaire of vector control practices were conducted. Blood samples were collected from 770 cattle from 48 herds and analysed by PCR to establish T. parva prevalence. Half body tick counts were recorded on every animal. Farmers were interviewed (n = 120; including the blood sampled herds) using a standardised questionnaire to obtain data on vector control practices. Local workshops were held to discuss findings and validate results. Overall prevalence of T. parva in cattle was 5.07% (CI: 3.70-7.00%), with significantly higher prevalence in older animals. Although all farmers reported seeing ticks on their cattle, tick counts were very low with 78% cattle having none. Questionnaire analysis indicated significant acaricide use with 79% and 41% of farmers reporting spraying or dipping with cypermethrin-based insecticides, respectively. Some farmers reported very frequent spraying, as often as every four days. However, doses per animal were often insufficient. These data indicate high levels of acaricide use, which may be responsible for the low observed tick burdens and low ECF prevalence. This vector control is farmer-led and aimed at both tick- and tsetse-borne diseases of livestock. The levels of acaricide use raise concerns regarding sustainability; resistance development is a risk, particularly in ticks. Integrating vaccination as part of this community-based disease control may alleviate acaricide dependence, but increased understanding of the Theileria strains circulating in wildlife-livestock interface areas is required to establish the potential benefits of vaccination.

Assessing the effect of insecticide-treated cattle on tsetse abundance and trypanosome transmission at the wildlife-livestock interface in Serengeti, Tanzania.

In the absence of national control programmes against Rhodesian human African trypanosomiasis, farmer-led treatment of cattle with pyrethroid-based insecticides may be an effective strategy for foci at the edges of wildlife areas, but there is limited evidence to support this. We combined data on insecticide use by farmers, tsetse abundance and trypanosome prevalence, with mathematical models, to quantify the likely impact of insecticide-treated cattle. Sixteen percent of farmers reported treating cattle with a pyrethroid, and chemical analysis indicated 18% of individual cattle had been treated, in the previous week. Treatment of cattle was estimated to increase daily mortality of tsetse by 5-14%. Trypanosome prevalence in tsetse, predominantly from wildlife areas, was 1.25% for T. brucei s.l. and 0.03% for T. b. rhodesiense. For 750 cattle sampled from 48 herds, 2.3% were PCR positive for T. brucei s.l. and none for T. b. rhodesiense. Using mathematical models, we estimated there was 8-29% increase in mortality of tsetse in farming areas and this increase can explain the relatively low prevalence of T. brucei s.l. in cattle. Farmer-led treatment of cattle with pyrethroids is likely, in part, to be limiting the spill-over of human-infective trypanosomes from wildlife areas.

Symptomatic and asymptomatic cases of African swine fever in Tanzania.

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub-clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease-endemic regions.

Ecological and Epidemiological Findings Associated with Zoonotic Rabies Outbreaks and Control in Moshi, Tanzania, 2017-2018.

Approximately 1500 people die annually due to rabies in the United Republic of Tanzania. Moshi, in the Kilimanjaro Region, reported sporadic cases of human rabies between 2017 and 2018. In response and following a One Health approach, we implemented surveillance, monitoring, as well as a mass vaccinations of domestic pets concurrently in >150 villages, achieving a 74.5% vaccination coverage (n = 29, 885 dogs and cats) by September 2018. As of April 2019, no single human or animal case has been recorded. We have observed a disparity between awareness and knowledge levels of community members on rabies epidemiology. Self-adherence to protective rabies vaccination in animals was poor due to the challenges of costs and distances to vaccination centers, among others. Incidence of dog bites was high and only a fraction (65%) of dog bite victims (humans) received post-exposure prophylaxis. A high proportion of unvaccinated dogs and cats and the relative intense interactions with wild dog species at interfaces were the risk factors for seropositivity to rabies virus infection in dogs. A percentage of the previously vaccinated dogs remained unimmunized and some unvaccinated dogs were seropositive. Evidence of community engagement and multi-coordinated implementation of One Health in Moshi serves as an example of best practice in tackling zoonotic diseases using multi-level government efforts. The district-level establishment of the One Health rapid response team (OHRRT), implementation of a carefully structured routine vaccination campaign, improved health education, and the implementation of barriers between domestic animals and wildlife at the interfaces are necessary to reduce the burden of rabies in Moshi and communities with similar profiles.

Geostatistical models using remotely-sensed data predict savanna tsetse decline across the interface between protected and unprotected areas in Serengeti, Tanzania.

Monitoring abundance is essential for vector management, but it is often only possible in a fraction of managed areas. For vector control programmes, sampling to estimate abundance is usually carried out at a local-scale (10s km2), while interventions often extend across 100s km2. Geostatistical models have been used to interpolate between points where data are available, but this still requires costly sampling across the entire area of interest. Instead, we used geostatistical models to predict local-scale spatial variation in the abundance of tsetse-vectors of human and animal African trypanosomes-beyond the spatial extent of data to which models were fitted, in Serengeti, Tanzania.We sampled Glossina swynnertoni and Glossina pallidipes >10 km inside the Serengeti National Park (SNP) and along four transects extending into areas where humans and livestock live. We fitted geostatistical models to data >10 km inside the SNP to produce maps of abundance for the entire region, including unprotected areas.Inside the SNP, the mean number of G. pallidipes caught per trap per day in dense woodland was 166 (± 24 SE), compared to 3 (±1) in grassland. Glossina swynnertoni was more homogenous with respective means of 15 (±3) and 15 (±8). In general, models predicted a decline in abundance from protected to unprotected areas, related to anthropogenic changes to vegetation, which was confirmed during field survey. Synthesis and applications. Our approach allows vector control managers to identify sites predicted to have relatively high tsetse abundance, and therefore to design and implement improved surveillance strategies. In East and Southern Africa, trypanosomiasis is associated with wilderness areas. Our study identified pockets of vegetation which could sustain tsetse populations in farming areas outside the Serengeti National Park. Our method will assist countries in identifying, monitoring and, if necessary, controlling tsetse in trypanosomiasis foci. This has specific application to tsetse, but the approach could also be developed for vectors of other pathogens.

A tsetse Glossina pallidipes harbors the pathogenic trypanosomes circulating in Liwale district, Tanzania.

African Animal Trypanosomiasis (AAT) is among several constraints hindering development of the livestock sector in Tanzania. A survey was conducted in Liwale district located in southern Tanzania in 2013 to determine the population density of Glossina species, distribution pattern and Trypanosome species infection rate in tsetse flies. A total of 200 flies were collected from the study area and three Glossina species were identified. The proportional abundance of all trapped flies was 90% (180) for Glossina pallidipes, 6% (12) for G. brevipalpis and 4% (8) for G. m. morsitans with apparent densities (fly/trap/day - FTD) of 0.44. Higher density of Glossina pallidipes was observed in villages closer to than those far from the Selous game reserve. Trypanosomes were detected and identified by microscopy and ITS1 polymerase chain reaction (PCR) assay on DNA purified from 200 flies. Glossina pallidipes was the only fly found infected by three Trypanosoma species, namely T. vivax (60%), T. simiae (10%) and T. brucei (30%) with an overall infection rate of 10% (20/200). A higher proportion of trypanosome infections were observed in female tsetse flies than in males. Results of this study show that G pallidipes is the major Glossina species harboring pathogenic trypanosomes in Liwale district and that the Selous game reserve is a potential reservoir of trypanosomes in terms of parasite abundance and species diversity.

Genetic diversity of Glossina fuscipes fuscipes along the shores of Lake Victoria in Tanzania and Kenya: implications for management.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are sole vectors for trypanosomiasis, which affect human health and livestock productivity in Africa. Little is known about the genetic diversity of Glossina fuscipes fuscipes, which is an important species in Tanzania and Kenya. The main objective of the study was to provide baseline data to determine the genetic variability and divergence of G. f. fuscipes in the Lake Victoria basin of Tanzania and Kenya in order to guide future vector control efforts in the region. FINDINGS: Two hundred and seventy five G. f. fuscipes from 8 sites along the shores of Lake Victoria were screened for genetic polymorphisms at 19 microsatellite loci. Samples were collected from two sites in Kenya and six sites in Tanzania. Four of the Tanzanian sites were located in the Rorya district, on the eastern shores of Lake Victoria, while the other two sites were from Ukerewe and Bukoba districts from the southern and western Lake Victoria shores, respectively. Four genetically distinct allopatric clusters were revealed by microsatellite analysis, which sorted the sampling sites according to geography, with sites separated by as little as ~65 km belonging to distinct genetic clusters, while samples located within ~35 km from each other group in the same cluster. CONCLUSION: Our results suggest that there is ongoing genetic admixture within sampling sites located ~35 km from each other, while sites located ~65 km apart are genetically isolated from each other. Similar patterns emerged from a parallel study on G. f. fuscipes analyzed from the Lake Victoria Uganda shores. From a control perspective these results suggest that for sites within the same genetic cluster, control efforts should be carried out in a coordinated fashion in order to avoid re-invasions. Future work should focus on better quantifying the extent and spatial patterns of the observed genetic discontinuities of the G. f. fuscipes populations along the Tanzanian shores. This will aid in their control by providing guidelines on the geographical extent of the area to be treated at the same time.

Standardizing visual control devices for tsetse flies: East African species Glossina swynnertoni.

BACKGROUND: Here we set out to standardize long-lasting, visually-attractive devices for Glossina swynnertoni, a vector of both human and animal trypanosomiasis in open savannah in Tanzania and Kenya, and in neighbouring conservation areas used by pastoralists. The goal was to determine the most practical device/material that would induce the strongest landing response in G. swynnertoni for use in area-wide population suppression of this fly with insecticide-impregnated devices. METHODS AND FINDINGS: Trials were conducted in wet and dry seasons in the Serengeti and Maasai Mara to measure the performance of traps and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a simple enumerator at these remote locations to compare trapping efficiencies of devices. Independent of season or presence of chemical baits, targets in phthalogen blue or turquoise blue cloth with adhesive film were the best devices for capturing G. swynnertoni in all situations, catching up to 19 times more flies than pyramidal traps. Baiting with chemicals did not affect the relative performance of devices. Fly landings were two times higher on 1 m(2) blue-black targets as on pyramidal traps when equivalent areas of both were covered with adhesive film. Landings on 1 m(2) blue-black targets were compared to those on smaller phthalogen blue 0.5 m(2) all-blue or blue-black-blue cloth targets, and to landings on all-blue plastic 0.32-0.47 m(2) leg panels painted in phthalogen blue. These smaller targets and leg panels captured equivalent numbers of G. swynnertoni per unit area as bigger targets. CONCLUSIONS: Leg panels and 0.5 m(2) cloth targets show promise as cost effective devices for management of G. swynnertoni as they can be used for both control (insecticide-impregnated cloth) and for sampling (rigid plastic with insect glue or adhesive film) of populations.