Antigenic variation is a very widespread process for pathogen evasion of mammalian adaptive immunity, involving the continuous change of exposed antigens. In many single-celled pathogens, antigen expression during antigenic variation is monoallelic: just a single gene from a large family is expressed in one cell at a time. In African trypanosomes, antigenic variation relies on expression of Variant Surface Glycoprotein (VSG), and in Trypanosoma brucei there is detailed understanding of the machinery that dictates that only one of approximately 15 VSG expression sites is actively transcribed at a time. In the closely related African trypanosome, T. congolense, which remains a significant blight on agriculture productivity in sub-Sharan Africa, we have no such understanding of VSG gene expression control or dynamics. Here, we have investigated the mechanics of antigenic variation in T. congolense, first examining the patterns of VSG expression at the transcript and protein level in small parasite populations in vitro. Surprisingly, this analysis revealed much greater VSG diversity than seen in T. brucei, with such expression diversity unaltered in mutants that impair homologous recombination. Using single cell transcriptomics, we explain this diversity, since we find no evidence for monoallelic transcription of T. congolense VSGs, but show instead that each parasite can dynamically express up to ~40 different VSGs in a single cell both in vitro and in vivo. VSG co-expression occurs from VSG genes distributed across the genome, indicating the lack of a dedicated locus for VSG transcription. Thus, comparing two trypanosome species that rely on the same class of surface antigen for immune evasion has revealed highly distinct mechanisms for controlling antigen gene expression, challenging the assumed common operation of antigenic variation across African trypanosome species.

Abstract Trypanosoma brucei exploits an extreme form of antigenic variation to escape the mammalian immune response. This involves the progressive expression of antigenically distinct variant surface glycoproteins (VSGs) on the surface of individual parasites in the population, generating waves of parasitaemia that are successively cleared by host antibodies. Current paradigms were established using in vitro studies and acute rodent infections characterized by high parasitaemia, but natural livestock infections are characterized by low parasitaemia and chronicity. Here, we analysed the infection dynamics of isogenic parasites in mice and cattle in blood during early and chronic infections, quantitating VSG expression diversity within and between hosts, antigen type persistence in vivo and their timing of appearance. This revealed enhanced antigenic diversity in cattle but with a surprisingly reproducible temporal expression hierarchy of related VSGs between independent chronic infections. Analyses demonstrated the unexpected dominance of a single telomeric VSG expression site irrespective of host species and time of infection. Detailed prediction of mosaic VSG assembly reveals exceptional parasite genome diversification within infections involving extensive macro and micro-homology-based recombination to evolve the antigen repertoire. This diversity was restricted but not eliminated in homologous recombination mutants, which could nonetheless sustain chronic infections in mice. These data provide the first comprehensive insight into trypanosome antigenic variation in the clinically-relevant host.

Tsetse-transmitted Trypanosoma parasites infect a wide host range and cause Human African Trypanosomiasis and Animal African Trypanosomosis. The dominant hosts of Trypanosoma brucei sensu lato are non-human mammals, including agriculturally important cattle. In rodent infections, T. brucei transitions from proliferative slender to tsetse-transmissible stumpy forms at high parasitaemia in a density-dependent quorum sensing-type process. However, chronic bovine infections are characterised by markedly lower blood parasitaemia levels; mostly substantially below the density assumed to trigger slender-to-stumpy differentiation. This challenges the current (rodent-based) assumptions and quantitative parameter estimations around stumpy form generation in the bloodstream. By combining scRNA-seq and microscopy we observe mixed populations of parasites with both slender- and stumpy-associated transcriptomes in cattle blood. The appearance of the latter coincides with fewer detectably dividing parasites and parasites with shortened flagellum indicative of differentiation, despite the absence of stumpy morphology or developmental marker protein expression. Comparisons with murine infections and in vitro culture demonstrates conserved transcriptomic signatures for both slender- and stumpy-like forms, as well as host specific differences, including a subpopulation of slender-like parasites upregulating pyruvate metabolism and TCA cycle transcripts in cattle samples. These similarities and differences are key to understanding parasite development and transmission in its natural host.

Despite the key role genetic mutations play in shaping phenotypic differences between species, little is currently known about the evolution of germline mutation spectra across mammals. Domesticated species are likely particularly interesting case studies because of their high mutation rates and complex evolutionary histories, which can span multiple founding events and genetic bottlenecks. Here we have developed a new reusable workflow, nSPECTRa, that can undertake the key steps in characterising mutation spectra, from determining ancestral alleles to characterising multiple forms of variation. We apply nSPECTRa to seven species, including several that have undergone domestication, and highlight how nSPECTRa can provide important insights into mutation rate evolution. While mutation spectra most often show marked differences between species and even breeds, certain mutation types have risen to a high frequency in subpopulations of different species, indicative of convergent evolution in mutation rates. This includes the previously characterized TCC- > TTC change enriched among European humans, which is also enriched among East Asian cattle. We show Indicine cattle are particularly interesting examples of how different mutation spectra segregate within a population and subsequently spread across the globe. Together, this work has important implications for understanding the control and evolution of mammalian mutation rates.

Animal Trypanosomosis (AT) is a significant disease affecting cattle across sub-Saharan Africa, Latin America, and Asia, posing a major threat to economic productivity and animal welfare. The absence of reliable diagnostic tests has led to an over-reliance on widespread pre-emptive drug treatments, which not only compromise animal health but also heighten the risk of drug resistance. The chronic nature of AT, characterized by cyclical low or undetectable parasite levels, and the necessity for field-applicable tests that can distinguish between active infection and prior exposure, present considerable challenges in developing effective diagnostics. In previous work, we identified a parasite-specific small RNA, 7SL-sRNA, which is detectable in the serum of infected cattle, even during the chronic stages of infection. However, existing methods for detecting sRNA require specialized equipment, making them unsuitable for field use. In this study, we have developed both a fluorescence-based and a lateral flow diagnostic test utilizing Cas10 technology for the detection of 7SL-sRNA from Trypanosoma congolense and Trypanosoma brucei. The fluorescence assay detects 10 - 100 fM T. congolense 7SL-sRNA and 1 pM T. brucei 7SL-sRNA, and the lateral flow assay showed a limit of detection of 1 - 10 pM for both species. Either assay can effectively identify active infections in cattle, including during chronic phases (with positive signals observed up to the experimental end point, 63 days post infection). This also highlights the effective use of Cas10 for small RNA detection, paving the way for a cost-effective, user-friendly, and field-deployable diagnostic test for AT, while establishing Cas10 technology for the detection of small RNAs in general.

African animal trypanosomosis (AAT) in cattle is primarily managed through trypanocide administration and insecticide application. Trypanocides can be used for both treatment and prophylaxis, but failure is often reported; this may occur due to resistance, substandard drugs, or inappropriate administration. This study in Tanzania aims to quantify reasons for trypanocide failure. An observational year-long longitudinal study was conducted in high-risk AAT areas in Serengeti District between June 2021-October 2022. Purposive sampling targeted herds with high utilization of the prophylactic trypanocide isometamidium chloride (ISM). When a farmer administered a trypanocide (ISM, diminazine aceturate, homidium), the project veterinarian assessed administration and treatment outcomes were determined based on PCR results from blood samples. A multivariable mixed model was utilized to evaluate risk factors for prophylaxis failure. Quality analysis was performed on trypanocide samples using High Performance Liquid Chromatography. A total of 630 cattle from 21 farms were monitored for a year-long period. A total of 295 trypanocide administrations were reported, predominantly being ISM (56%) used for prophylaxis (87%). One-third of trypanocide administrations were not given adequately, and many trypanocides were given to animals that tested negative for trypanosome infections by PCR. Failures occurred in 7% (95% CI 3.0-14%) of curative treatments, and 44% (95% CI 35-42%) of prophylactic administrations. The brand of ISM was significantly associated with odds of prophylaxis failure (p = 0.011). On quality analysis, two ISM samples had no detectable ISM isomers, but the remainder of ISM and DA samples (n = 46) fell within the range of acceptable levels. Drug counterfeiting, inadequate use of trypanocides, and resistance are all contributing to trypanocide failure, limiting effective AAT control and with implications for human disease risk. In order to curb trypanocide failure a multi-modal approach to managing the use of trypanocides is required to address all contributing factors.

One of the principal limitations on livestock productivity in sub-Saharan Africa is the constraining effect of infectious diseases, including tick-borne blood pathogens. Currently, diagnostic markers for these pathogens are species or genus specific, making it challenging to implement high-throughput screening methods. The aim of this study was to develop and validate a novel high-throughput diagnostic tool capable of detecting a range of important haemopathogens in livestock. To achieve this, we developed a high-throughput diagnostic tool that can detect all species of Anaplasma, Ehrlichia, Theileria and Babesia present in a sample. The approach involves targeting the 16S/18S rDNA region by PCR and subjecting amplicons to deep sequencing, which allows for the identification of species present in a sample, and the exploration of haemopathogen communities. To validate the accuracy of this Next Generation Sequencing method, we compared the amplicon sequencing results with species-specific PCR and reverse line blot (RLB) test data of both control and field samples. The Haemabiome tool demonstrated the successful resolution of positive and negative samples, and highlighted the power of this diagnostic tool in identifying multiplicity of infections. The Haemabiome tool can therefore generate valuable insights regarding the understanding of the true diversity of species composition and the distribution of pathogen communities in field samples.

Herbicides, such as MCPA and clopyralid, may be transported to surface waters via runoff, which can have unintended environmental consequences. A split herbicide application strategy, wherein applications are spread across a season, may improve herbicide effectiveness, although impacts of this strategy on runoff mitigation have not been investigated. Therefore, this study aimed to (1) quantify the impact of split-dose applications of MCPA and clopyralid on herbicide losses in surface runoff and (2) assess the impact of split-dose applications of MCPA on the quantity and classification of MCPA-degrading soil bacteria. Intact grassed soil sods were placed in 1 m-long x 0.25 m-wide x 0.1 m-deep laboratory flumes, onto which either MCPA or clopyralid were applied in one full-dose (13.5 kg MCPA ha(-1); 2 kg clopyralid ha(-1)) or two split-doses (each 6.75 kg MCPA ha(-1); 1 kg clopyralid ha(-1)) 42 days apart. On days 2, 7 and 21 following herbicide applications, flumes were subjected to controlled rainfall simulations at an intensity of 11 mm h(-1), and the herbicides in the runoff were quantified. MCPA and clopyralid concentrations in the runoff were highest immediately after the initial application. Both herbicides were below the limit of detection (0.1 mu g l(-1) for MCPA and 0.45 mu g l(-1) for clopyralid) by 44 days. No herbicides were detected in the runoff following the second split-dose application. For MCPA, this was attributed to an adaptation in the microbial community with the emergence of bacteria possessing the tfdA class III gene in the soil. These results support split-dose herbicide application as a strategy for agricultural management.

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.

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.

Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein–Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle.

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.

The control of tick-borne haemoparasites in cattle largely relies on the use of acaricide drugs against the tick vectors, with some vaccination also being used against selected pathogens. These interventions can be difficult in Africa, where accessibility and cost of vaccines can be issues, and the increasing resistance of tick vectors to the widely used acaricides is a complication to disease control. A potential complementary control strategy could be the exploitation of any natural host genetic resistance to the pathogens. However, there are currently very few estimates of the extent of host resistance to tick-borne haemoparasites, and a significant contributing factor to this knowledge gap is likely to be the difficulty of collecting appropriate samples and data in the smallholder systems that predominate livestock production in low- and middle-income countries, particularly at scale. In this study, we have estimated the heritability for the presence/absence of several important haemoparasite species (including Anaplasma marginale, Babesia bigemina, Babesia bovis, and Ehrlichia ruminantium), as well as for relevant traits such as body weight and body condition score (BCS), in 1,694 cattle from four African countries (Burkina Faso, Ghana, Nigeria, and Tanzania). Heritability estimates within countries were mostly not significant, ranging from 0.05 to 0.84 across traits and countries, with standard errors between 0.07 and 0.91. However, the weighted mean of heritability estimates was moderate and significant for body weight and BCS (0.40 and 0.49, respectively), with significant heritabilities also observed for the presence of A. marginale (0.16) and E. ruminantium (0.19). In a meta-analysis of genome-wide association studies (GWAS) for these traits, two peaks were identified as reaching the suggestive significance threshold (p < 1.91 × 10−7 and p < 1.89 × 10−7, respectively): one on chromosome 24 for BCS and one on chromosome 8 for the E. ruminantium infection status. These findings indicate that there is likely to be a genetic basis that contributes to pathogen presence/absence for tick-borne haemoparasite species, which could potentially be exploited to improve cattle resistance in Africa to the economically important diseases caused by these pathogens.

Background Understanding the variation between well and poorly adapted cattle breeds to local environments and pathogens is essential for breeding cattle with improved climate and disease-resistant phenotypes. Although considerable progress has been made towards identifying genetic differences between breeds, variation at the epigenetic and chromatin levels remains poorly characterized. Here, we generate, sequence and analyse over 150 libraries at base-pair resolution to explore the dynamics of DNA methylation and chromatin accessibility of the bovine immune system across three distinct cattle lineages. Results We find extensive epigenetic divergence between the taurine and indicine cattle breeds across immune cell types, which is linked to the levels of local DNA sequence divergence between the two cattle sub-species. The unique cell type profiles enable the deconvolution of complex cellular mixtures using digital cytometry approaches. Finally, we show distinct sub-categories of CpG islands based on their chromatin and methylation profiles that discriminate between classes of distal and gene proximal islands linked to discrete transcriptional states. Conclusions Our study provides a comprehensive resource of DNA methylation, chromatin accessibility and RNA expression profiles of three diverse cattle populations. The findings have important implications, from understanding how genetic editing across breeds, and consequently regulatory backgrounds, may have distinct impacts to designing effective cattle epigenome-wide association studies in non-European breeds.

Golden seaweed tides are a global environmental and social problem, that have been occurring along Ireland's eastern coastline since the 1990s. This study focused on analysing the spatiotemporal dynamics of golden seaweed (Ectocarpus siliculosus) coverage at Dollymount Strand in Dublin Bay (Ireland), between 2016 and 2022, and its relationship with meteorological conditions. Hyperspectral measurements in the field and Sentinel-2 imagery were utilized to monitor macroalgal blooms with a spatiotemporal resolution of 10 m and minimum costs. The normalized difference vegetation index (NDVI) values filtered between 0.1 and 1 were used to identify the coverage of golden seaweeds. The results showed that the golden seaweed coverage extended over 99% of the study area (54 ha) in June 2020 with an average NDVI of 0.25. A seasonal pattern of golden seaweed abundance was observed and modelled from May to October using a Generalized Additive Model. Approximately 28% of the coverage was correlated with daily average global radiance, and 38% of the mean NDVI was associated with daily average maximum air temperatures and wind direction by means of the Generalized Linear Models. A greater biomass of Ectocarpus spp. was accumulated on the beach when the wind direction was from the northeast and south-east. The results also suggested that freshwater nutrient inputs in winter from nearby estuaries may be contributing to golden tides on the shoreline. These findings are useful in developing strategies aimed at controlling and managing golden tides globally, as well as using them as bioindicators of ecological status in coastal environments.

African trypanosomes are vector-borne protozoa, which cause significant human and animal disease across sub-Saharan Africa, and animal disease across Asia and South America. In humans, infection is caused by variants of Trypanosoma brucei, and is characterized by varying rate of progression to neurological disease, caused by parasites exiting the vasculature and entering the brain. Animal disease is caused by multiple species of trypanosome, primarily T. congolense, T. vivax, and T. brucei. These trypanosomes also infect multiple species of mammalian host, and this complexity of trypanosome and host diversity is reflected in the spectrum of severity of disease in animal trypanosomiasis, ranging from hyperacute infections associated with mortality to long-term chronic infections, and is also a main reason why designing interventions for animal trypanosomiasis is so challenging. In this review, we will provide an overview of the current understanding of trypanosome determinants of infection progression and severity, covering laboratory models of disease, as well as human and livestock disease. We will also highlight gaps in knowledge and capabilities, which represent opportunities to both further our fundamental understanding of how trypanosomes cause disease, as well as facilitating the development of the novel interventions that are so badly needed to reduce the burden of disease caused by these important pathogens.

"The Centre for Tropical Livestock Genetics and Health at the Royal (Dick) School of Veterinary Studies, University of Edinburgh: improving livestock genetics to enhance animal welfare and production for African smallholder farmers" published on 01 Jan 2023 by American Veterinary Medical Association.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy–light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy–light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.