Trypanosome Species Research Articles

Molecular detection and therapeutic study of Trypanosoma brucei evansi from naturally infected horses in Punjab, Pakistan.

Trypanosomiasis is one of the severe pathogenic infections, caused by several Trypanosoma species, affecting both animals and humans, causing substantial economic losses and severe illness. The objective of this study was to determine the molecular diagnosis and the risk factors associated with trypanosomiasis in District Jhang, Punjab, Pakistan. For this purpose, blood samples were randomly collected from 200 horses. A predesigned questionnaire was used to collect data on risk factors before the sample collection. The microscopy examination through Giemsa staining, formol gel test and PCR techniques were used to find the prevalence. The prevalence was recorded as 22.5% with microscopy examination, 21% through formol gel test and 15.5% with PCR based results. Analysis of risk factors associated with Trypanosoma brucei evansi occurrence was carried out using Chi-square test. It showed the prevalence of Trypanosoma brucei evansi was significantly (p⟨0.05) associated with sex, age, rearing purpose and body condition whereas non-significantly (p⟩0.05) with insects control practices. This study supports the idea that PCR is a sensitive, robust and more reliable technique to diagnose trypanosomiasis. It was concluded that Trypanosoma brucei evansi is widely prevalent in Jhang (Pakistan), highlighting a dire need to develop control strategies and education programmes to control this disease in developing countries.

Identification of Blood-sucking Flies of Bats in Lombok Island, Indonesia

Bats and blood-sucking bat flies have an important role in transmitting several hemoparasites. Bat flies have been identified as vectors transmitting hemoparasites from wild bats. The purpose of the present study was to identify bats and their blood-sucking flies as vectors of hemoparasites in bat caves located at Lombok Island, Indonesia. In the course of the study, a survey was conducted on three bat caves from September to December 2018. The bats were captured by a net trap and the species of bats and bat flies were identified. A total of 66 captured bats were identified as Hipposideros species (n = 28), Eonycteris spelaea (n = 23), and Taphozouss species (n = 15). The blood-sucking flies were identified as Eucampsipoda sundaica on Eonycteris spelaea, and Stylidia cf. euxesta, Brachytarsina species, Raymondia species, and Megastrebla nigriceps on Hipposideros species. The results showed that five species of blood-sucking flies were present in captured bats. The bat and blood-sucking flies can influence the transmission of Polychromophilus species, Babesia species, Plasmodium species, and Trypanosoma species to humans and other hosts.

Parasites and blood-meal hosts of the tsetse fly in Tanzania: a metagenomics study

BackgroundTsetse flies can transmit various Trypanosoma spp. that cause trypanosomiasis in humans, wild animals, and domestic animals. Amplicon deep sequencing of the 12S ribosomal RNA (rRNA) gene can be used to detect mammalian tsetse hosts, and the 18S rRNA gene can be used to detect all associated eukaryotic pathogens, including Trypanosoma spp.MethodsTsetse flies were collected from the Serengeti National Park (n = 48), Maswa Game Reserve (n = 42), and Tarangire National Park (n = 49) in Tanzania in 2012–13. Amplicon deep sequencing targeting mammal-specific 12S rRNA and 18S rRNA genes was performed to screen the blood-feeding sources of tsetse flies and eukaryotic parasites in tsetse flies, respectively.Results12S rRNA gene deep sequencing revealed that various mammals were blood-feeding sources of the tsetse flies, including humans, common warthogs, African buffalos, mice, giraffes, African elephants, waterbucks, and lions. Genes of humans were less frequently detected in Serengeti (P = 0.0024), whereas African buffaloes were detected more frequently as a blood-feeding source (P = 0.0010). 18S rRNA gene deep sequencing showed that six tsetse samples harbored the Trypanosoma gene, which was identified as Trypanosoma godfreyi and Trypanosoma simiae in subsequent ITS1 gene sequencing.ConclusionsThrough amplicon deep sequencing targeting the 12S rRNA and 18S rRNA genes, various mammalian animals were identified as blood-meal sources, and two Trypanosoma species were detected in tsetse flies collected from the Maswa Game Reserve, Serengeti National Park, and Tarangire National Park in Tanzania. This study illustrates the patterns of parasitism of tsetse fly, wild animals targeted by the fly, and Trypanosoma spp. carried by the fly in Tanzania. It may provide essential data for formulating better strategies to control African trypanosomes.Graphical

Isolation and in vitro cultivation of Trypanosoma evansi Thai strains

Trypanosoma evansi is a flagellate protozoan parasite responsible for “surra”. To generate T. evansi antigens for serodiagnosis, parasites are generally propagated in laboratory animals before isolation. The alternation of animal models using axenic cultivation systems to produce trypomastigotes of various Trypanosoma species is currently available but has never been applied in Thailand. The isolation protocol for separation of live T. evansi trypomastigotes from animal blood components before in vitro cultivation has not been clearly documented. This study focused on validation of trypomastigote isolation method, in vitro cultivation of T. evansi Thai strains, and its virulence ability in vivo. In this study, two strains of T. evansi collected from Thailand were used. Trypanosoma evansi trypomastigotes were propagated in mice, and three different isolation methods, including: low-speed centrifugation, high-speed centrifugation, and ion exchange chromatography using diethylaminoethyl (DEAE) cellulose (or DE52), were compared. Four solutions of in vitro cultivation media, two different in vitro cultivation containers, and different trypomastigote densities for initiation of in vitro culture were compared. Virulence test using in vitro-adapted parasite for 100 days was conducted in vivo. The results showed that the DE52 isolation method was suitable for separation of live T. evansi trypomastigotes from animal blood components before conducting in vitro cultivation. Trypanosoma evansi Thai strains were successfully cultivated and multiplied in HMI-9 Solution I using 25 cm2 flasks and 12-well plates. The parasite was growing slowly at the initiation of in vitro culture for 15–16 days, and then rapidly increased to 10, 20, 50, 100, and 200 folds, approximately. The doubling times were varied from 11.95 ± 8 h to 41.18 ± 4.29 h in vitro. The maximum densities have reached from 0.14 × 106 to 4.63 × 106 trypomastigotes/ml. Virulence test showed that the in vitro-cultivated T. evansi was virulent in mice. In conclusion, T. evansi Thai strains were successfully isolated and cultivated in vitro for the first time. The isolation and in vitro cultivation protocols were clearly provided. The benefit of using the in vitro cultivation system helps in the production of T. evansi antigen, and replacing the use of experimental animals. It is also useful for the development of diagnostic tests in the future.

Spatial Distribution of Tsetse Flies and Trypanosome Infection Status in a Vector Genetic Transition Zone in Northern Uganda.

Background Tsetse flies are vectors of the genus Trypanosoma that cause African trypanosomiasis, a serious parasitic disease of people and animals. Reliable data on the vector distribution and the trypanosome species they carry is pertinent for planning sustainable control strategies. This study was carried out to estimate the spatial distribution, apparent density, and trypanosome infection rates of tsetse flies in two districts that fall within a vector genetic transition zone in northern Uganda. Materials and Methods Capturing of tsetse flies was done using biconical traps deployed in eight villages in Oyam and Otuke, two districts that fall within the vector genetic transition zone in northern Uganda. Trapped tsetse flies were sexed and morphologically identified to species level and subsequently analyzed for detection of trypanosome DNA. Trypanosome DNA was detected using a nested PCR protocol based on primers amplifying the internal transcribed spacer (ITS) region of ribosomal DNA. Results A total of 717 flies (406 females; 311 males) were caught, all belonging to the Glossina fuscipes fuscipes species. The overall average flies/trap/day (FTD) was 2.20 ± 0.3527 (mean ± SE). Out of the 477 (201 male; 276 females) flies analyzed, 7.13% (34/477) were positive for one or more trypanosome species. Three species of bovine trypanosomes were detected, namely, Trypanosoma vivax, 61.76% (21/34), T. congolense, 26.47% (9/34), and T. brucei brucei, 5.88% (2/34), and two cases of mixed infection of T. congolense and T. brucei brucei, 5.88% (2/34). The infection rate was not significantly associated with the sex of the fly (generalized linear model (GLM), χ2 = 0.051, p = 0.821, df = 1, n = 477) and district of origin (χ2 = 0.611, p = 0.434, df = 1, n = 477). However, trypanosome infection was highly significantly associated with the fly's age based on wing fray category (χ2 = 7.56, p = 0.006, df = 1, n = 477), being higher among the very old than the young. Conclusion The relatively high tsetse density and trypanosome infection rate indicate that the transition zone is a high-risk area for perpetuating animal trypanosomiasis. Therefore, appropriate mitigation measures should be instituted targeting tsetse and other biting flies that may play a role as disease vectors, given the predominance of T. vivax in the tsetse samples.

Detection of Trypanosoma Infection in Dromedary Camels by Using Different Diagnostic Techniques in Northern Oman.

Simple SummaryThe camel is a salient character of the pastoral economy around the world. Apropos of this title role, the camel plays a particularly crucial role in arid and semi-arid areas of the world. The camel population is growing despite much urbanization around the world. The importance of the camel concerns the acquisition of milk, meat and other byproducts. Due to emerging health issues, people mainly rely on camel milk and meat due to their remedial purposes, hence why it is held in high regard. Therefore, it is crucial to take into consideration the camels’ health, risks, associated diseases and diagnoses. In this investigation, an effort was made to detect trypanosomoses in dromedary camels by using different diagnostic techniques in Northern Oman.Camel trypanosomoses is considered a devastating disease with severe health consequences that can be caused by different hemoprotozoan parasites. Camel samples (388) from the five regions in Northern Oman were assessed using a thin blood film. In addition, 95 seropositive samples were analyzed using various primers of mechanically transmitted trypanosomes. Out of the 388 blood smears examined, 0.8% (CI 95%, 2/388) were found to be positive for Trypanosoma sp. using a microscope. The parasitologically positive cases were detected in samples from females. The overall molecular prevalences were as follows: TBR was 78/95, 77% (CI 73.1–89.2%); ITS was 30/95, 31.6% (CI 73.1–89.2%); and T. evansi type A (RoTat 1.2) was 8/95, 8.4% (CI 4.0–16.0%). There were two species of trypanosomes that were observed in the camels.

Environmental mutations in the Campo focus challenge elimination of sleeping sickness transmission in Cameroon.

Sleeping sickness is still prevalent in Campo, southern Cameroon, despite the efforts of World Health Organization and the National Control Programme in screening and treating cases. Reducing disease incidence still further may need the control of tsetse vectors. We update entomological and parasitological parameters necessary to guide tsetse control in Campo. Tsetse flies were trapped, their apparent densities were evaluated as the number of flies captured per trap per day and mapped using GIS tools. Polymerase chain reaction based methods were used to identify their trypanosome infection rates. Glossina palpalis palpalis was the dominant vector species representing 93.42% and 92.85% of flies captured respectively during the heavy and light dry seasons. This species presented high densities, that is, 3.87, 95% CI [3.84-3.91], and 2.51, 95% CI [2.49-2.53] flies/trap/day in the two seasons. Moreover, 16.79% (of 1054) and 20.23% (of 1132 flies) were found infected with at least 1 trypanosome species for the 2 seasons respectively, Trypanosoma congolense being the most prevalent species, and Trypanosoma. brucei gambiense identified in 4 samples. Tsetse flies are abundant in Campo and present high trypanosome infection rates. The detection of tsetse infected with human trypanosomes near the newly created palm grove show workers' exposition. Tsetse densities maps built will guide vector control with 'Tiny Targets'.

African Animal Trypanosomiasis: A Systematic Review on Prevalence, Risk Factors and Drug Resistance in Sub-Saharan Africa.

African animal trypanosomiasis (AAT) a parasitic disease of livestock in sub-Saharan Africa causing tremendous loses. Sub-Saharan continental estimation of mean prevalence in both large and small domestic animals, risk factors, tsetse and non-tsetse prevalence and drug resistance is lacking. A review and meta-analysis was done to better comprehend changes in AAT prevalence and drug resistance. Publish/Perish software was used to search and extract peer-reviewed articles in Google scholar, PubMed and CrossRef. In addition, ResearchGate and African Journals Online (AJOL) were used. Screening and selection of articles from 2000-2021 was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Articles 304 were retrieved; on domestic animals 192, tsetse and non-tsetse vectors 44, risk factors 49 and trypanocidal drug resistance 30. Prevalence varied by, host animals in different countries, diagnostic methods and species of Trypanosoma. Cattle had the highest prevalence with Ethiopia and Nigeria leading, T. congolense (11.80-13.40%) and T. vivax (10.50-18.80%) being detected most. This was followed by camels and pigs. Common diagnostic method used was buffy coat microscopy. However; polymerase chain reaction (PCR), CATT and ELISA had higher detection rates. G. pallidipes caused most infections in Eastern regions while G. palpalis followed by G. mortisans in Western Africa. Eastern Africa reported more non-tsetse biting flies with Stomoxys leading. Common risk factors were, body conditions, breed type, age, sex and seasons. Ethiopia and Nigeria had the highest trypanocidal resistance 30.00-35.00% and highest AAT prevalence. Isometamidium and diminazene showed more resistance with T. congolense being most resistant species 11.00-83.00%.

In vivo analysis of trypanocidal drug resistance in sahelian goats infected by Trypanosoma vivax strains collected in northern Togo

Trypanosoma (T.) vivax is one of the animal trypanosomes species causing calf mortality and economic losses in Togo. Despite its importance as the most widely distributed trypanosome species, T. vivax has received little attention because it is difficult to cultivate most field isolates in rodents. No molecular diagnostic tools for the identification of drug-resistant in T. vivax are currently available. Herein, four field isolates of T. vivax from Togo were cryopreserved and assessed for susceptibility to diminazene aceturate (DA) and isometamidium chloride (ISM) in goats. For field isolate preparation, 1 ml of blood from an infected goat was diluted in 111 µl of phosphate-buffered-saline and stored in liquid nitrogen. The in vivo experiment drug test was performed using twenty Sahelian goats with six-month of age and weighing 14.5 ± 1.6 kg. These experimental goats were purchased from a tsetse free-area Dori, a Sahelian region of Burkina Faso. The cryopreserved T. vivax isolates with unknowns, DA, and ISM sensitivity was inoculated to five goats and one goat was used as control. Each animal was inoculated by intravenously route 1 × 105 trypanosomes from the donor goat. Relapses were earlier in the first phase of treatment (14.85 ± 1.08 days) compared with the second phase (20 ± 3.39 days). The overall mean PCV of the control group decreased from 32% to 17% at day-60 (P-value < 0.001). Three isolates were phenotypically resistant to 0.5 mg per kg body weight (BW) ISM and one for 3.5 mg per kg BW of DA. There were no relapses with the 7 mg per kg BW dose DA. This study shows the resistance of T. vivax to two main trypanocidal drugs in different villages of Mango. The results suggest the extension of surveillance strategies to remote villages in Togo and will guide the veterinarian or herder in choosing a mass treatment strategy. Further studies will be needed to better understand the molecular basis of the observed resistance.

Nucleoside analogues for the treatment of animal trypanosomiasis

Animal trypanosomiasis (AT) is a parasitic disease with high socio-economic impact. Given the limited therapeutic options and problems of toxicity and drug resistance, this study assessed redirecting our previously identified antitrypanosomal nucleosides for the treatment of AT. Promising hits were identified with excellent in vitro activity across all important animal trypanosome species. Compound 7, an inosine analogue, and our previously described lead compound, 3′-deoxytubercidin (8), showed broad spectrum anti-AT activity, metabolic stability in the target host species and absence of toxicity, but with variable efficacy ranging from limited activity to full cure in mouse models of Trypanosoma congolense and T. vivax infection. Several compounds show promise against T. evansi (surra) and T. equiperdum (dourine). Given the preferred target product profile for a broad-spectrum compound against AT, this study emphasizes the need to include T. vivax in the screening cascade given its divergent susceptibility profile and provides a basis for lead optimization towards such broad spectrum anti-AT compound.

The prevalence of trypanosome infections in goats at Niassa National Reserve, Mozambique

African animal trypanosomosis (AAT), a debilitating tsetse-transmitted parasitic disease, is a major constraint to livestock ruminant production in Sub-Saharan Africa. In the southern Africa region, the main Trypanosoma species causing AAT include T. brucei, T. vivax and T. congolense. All three species have been described in acute, subacute, chronic or subclinical cases of AAT in goats. In the tsetse belt region, goat infections are estimated at 40 million per year. Despite this, the role of these small ruminants in the epidemiology of AAT is yet to be fully understood. To determine trypanosome prevalence and contribute to the understanding of the role of these small ruminants in the epidemiology of trypanosomosis, at Niassa National Reserve (NNR), a cross-sectional survey was conducted between July and September 2018 in five districts located inside the NNR (Sanga, Mavago, Majune, Marrupa and Mecula), Niassa Province, Mozambique. A total of 416 goat blood samples were collected and screened for trypanosomes using microscopy and Polymerase Chain Reaction (PCR). The overall prevalence was 0.96%, out of which, 0.72% corresponded to T. congolense and 0.24% to T. vivax. Mecula was the district with the highest prevalence (0.48%), followed by Sanga and Majune (both with 0.24%). Considering the aforementioned results, the role of goats in the epidemiology of the disease, within the NNR, may be considered negligible.

A longitudinal two-year survey of the prevalence of trypanosomes in domestic cattle in Ghana by massively parallel sequencing of barcoded amplicons.

BackgroundAnimal African Trypanosomiasis (AAT) is one of the most economically important diseases affecting livestock productivity in sub-Saharan Africa. The disease is caused by a broad range of Trypanosoma spp., infecting both wild and domesticated animals through cyclical and mechanical transmission. This study aimed to characterize trypanosomes present in cattle at regular intervals over two years in an AAT endemic and a non-endemic region of Ghana.Methodology/Principal findingsGroups of cattle at Accra and Adidome were selected based on their geographical location, tsetse fly density, prevalence of trypanosomiasis and the breed of cattle available. Blood for DNA extraction was collected at approximately four to five-week intervals over a two-year period. Trypanosome DNA were detected by a sensitive nested PCR targeting the tubulin gene array and massively parallel sequencing of barcoded amplicons. Analysis of the data was a semi-quantitative estimation of infection levels using read counts obtained from the sequencing as a proxy for infection levels. Majority of the cattle were infected with multiple species most of the time [190/259 (73%) at Adidome and 191/324 (59%) at Accra], with T. vivax being the most abundant. The level of infection and in particular T. vivax, was higher in Adidome, the location with a high density of tsetse flies. The infection level varied over the time course, the timings of this variation were not consistent and in Adidome it appeared to be independent of prophylactic treatment for trypanosome infection. Effect of gender or breed on infection levels was insignificant.Conclusions/SignificanceMost cattle were infected with low levels of several trypanosome species at both study sites, with T. vivax being the most abundant. The measurements of infection over time provided insight to the importance of the approach in identifying cattle that could suppress trypanosome infection over an extended time and may serve as reservoir.

Chemotherapeutic potentials of β-ionone against Trypanosoma congolense infection: Inhibitionofparasite proliferation, anemia development, trans-sialidase (TconTS3 and TconTS4) gene expressions, and phospholipase A2.

Trypanosoma congolense is a pathogenic African animal trypanosome species causing devastating conditions leading to death of an infected host. The drawbacks of the existing trypanocidal drugs have led to the search for new drug candidates. In this study, β-ionone at 15 and 30mg/kg body weight (BW) was orally administered to T.congolense infected rats for 14days followed by an assessment of anemia, organ damages, and the expression of T.congolense trans-sialidase gene variants. A significant decrease in parasitemia (p<.05) was observed in the animals treated with 15mg/kg BW β-ionone besides increased animal survival rate. A trypanosome-induced decrease in packed cell volume (PCV) and histopathological changes across tissues was significantly (p<.05) ameliorated following treatment with both doses of β-ionone. This is in addition to reversing the parasite-induced upsurge in free serum sialic acid (FSA) and expression of T.congolense trans-sialidase gene variants (TconTS1, TconTS3, and TconTS4). Correlation analysis revealed a positive correlation (p>.05) between FSA with the TconTS gene expressions. In addition, the compound inhibited partially purified T.congolense sialidase and phospholipase A2 via mixed inhibition pattern with inhibition binding constants of 25.325 and 4.550µM, respectively, while molecular docking predicted binding energies of -5.6kcal/mol for both enzymes. In conclusion, treatment with β-ionone suppressed T.congolense proliferation and protected the animals against some of the parasite-induced pathologies whilst the effect on anemia development might be due to inhibition of sialidase and PLA2 activities as well as the expression levels of TconTS3 and TconTS4.

Molecular detection of Trypanosoma species in sheep and goats in Mosul city

In this study, we examined blood samples of 385 sheep and goats of different ages, sexes, and sources under routine microscopic examination of the blood smear (wet, thin, thick, buffy coat layer smears) to detect Trypanosoma. Results show that 81 samples were positive. These samples are succumbed to the molecular detection of Trypanosoma and other species by the extraction of parasitic DNA this parasitic DNA is detected in samples using KIN1, KIN2, and AITSF, AITSR primers. After that, conventional polymerase chain reaction was applied, and the results showed that 81 samples had a positive reaction in using KIN1 and KIN2 primers, while the positive samples were 76 when using AITSF, AITSR primers. Moreover, results showed a high rate of infection in sheep as compared with goats using both pairs of primers and two species of Trypanosoma in sheep and goats. Molecular was recorded, which include T. conglense and T. vivax. Animals more than 1-2 years old group showed a high rate of infection as compared with other ages group, and females have recorded a high rate of infection as compared with males. According to the source of animals, imported animals showed a high infective rate compared to native ones. This study is the first recorded Trypanosoma species in small ruminants in Mosul city.

Atypical human trypanosomosis: Potentially emerging disease with lack of understanding.

Trypanosomes are the hemoflagellate kinetoplastid protozoan parasites affecting a wide range of vertebrate hosts having insufficient host specificity. Climatic change, deforestation, globalization, trade agreements, close association and genetic selection in links with environmental, vector, reservoir and potential susceptible hosts' parameters have led to emergence of atypical human trypanosomosis (a-HT). Poor recording of such neglected tropical disease, low awareness in health professions and farming community has approached a serious intimidation for mankind. Reports of animal Trypanosoma species are now gradually increasing in humans, and lack of any compiled literature has diluted the issue. In the present review, global reports of livestock and rodent trypanosomes reported from human beings are assembled and discrepancies with the available literature are discussed along with morphological features of Trypanosoma species. We have described 21 human cases from the published information. Majority of cases 10 (47%) are due to T.lewisi, followed by 5 (24%) cases of T.evansi, 4 (19%) cases of T.brucei and 1 (5%) case each of T.vivax and T.congolense. Indian subcontinent witnessed 13 cases of a-HT, of which 9 cases are reported from India, which includes 7 cases of T.lewisi and 2 cases of T.evansi. Apart from, a-HT case reports, epidemiological investigation and treatment aspects are also discussed. An attempt has been made to provide an overview of the current situation of atypical human trypanosomosis caused by salivarian animal Trypanosoma globally. The probable role of Trypanosoma lytic factors (TLF) present in normal human serum (NHS) in providing innate immunity against salivarian animal Trypanosoma species and the existing paradox in medical science after the finding on intact functional apolipoprotein L1 (ApoL1) in Vietnam T.evansi Type A case is also discussed to provide an update on all aspects of a-HT. Insufficient data and poor reporting in Asian and African countries are the major hurdle resulting in under-reporting of a-HT, which is a potential emerging threat. Therefore, concerted efforts must be directed to address attentiveness, preparedness and regular surveillance in suspected areas with training of field technicians, medical health professionals and veterinarians. Enhancing a one health approach is specifically important in case of trypanosomosis.

Development of two species of the Trypanosoma theileri complex in tabanids

BackgroundTrypanosoma theileri species complex includes parasites of Bovidae (cattle, sheep, goat, etc.) and Cervidae (deer) transmitted mainly by Tabanidae (horse flies and deerflies) and keds (Hippoboscidae). While morphological discrimination of species is challenging, two big clades, TthI and TthII, each containing parasites isolated from bovids and cervids, have been identified phylogenetically. To date, the development in the vector has been studied in detail only for the ked-transmitted sheep parasite T. melophagium (TthII), while the fate of trypanosomes in tabanids was described only briefly by light microscopy.MethodsWe collected infected tabanids of various species and identified trypanosomes by molecular phylogenetic analysis. The morphology and development of trypanosomes was studied using the combination of statistical analyses as well as light and electron microscopy.ResultsTwo trypanosome species belonging to both TthI and TthII clades of the T. theileri complex were identified. The phylogenetic position of these two trypanosomes suggests that they parasitize deer. Both species were indiscernible by morphology in the vector and showed the same development in its intestine. In contrast to the previously described development of T. melophagium, both trypanosomes of tabanids only transiently infected midgut and settled mainly in the ileum, while pylorus and rectum were neglected. Meanwhile, the flagellates developing in the tabanid ileum (pyriform epimastigotes and metacyclic trypomastigotes) showed similarities to the corresponding stages in T. melophagium by morphology, mode of attachment to the host cuticle and formation of the fibrillar matrix surrounding the mass of developing parasites. In addition, for the first time to our knowledge we documented extraintestinal stages in these trypanosomes, located in the space between the epithelium and circular muscles.ConclusionsThe development of different species of flagellates of the T. theileri complex in their insect vectors shows many similarities, which can be explained not only by their common origin, but also the same transmission mode, i.e. contamination of the oral mucosa with the gut content released after squashing the insect either by tongue or teeth. The observed differences (concerning primarily the distribution of developmental stages in the intestine) are associated rather with the identity of vectors than the phylogenetic position of parasites.Graphical

A novel metabarcoded deep amplicon sequencing tool for disease surveillance and determining the species composition of Trypanosoma in cattle and other farm animals

The World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have developed strategies to control trypanosomiasis in humans and livestock in endemic areas. These require a better understanding of the distribution of different Trypanosoma species and improved predictions of where they might appear in the future, based on accurate diagnosis and robust surveillance systems. Here, we describe a metabarcoding deep amplicon sequencing method to identify and determine the Trypanosoma species in co-infecting communities. First, four morphological verified Trypanosoma species (T. brucei, T. congolense, T. vivax and T. theileri) were used to prepare test DNA pools derived from different numbers of parasites to evaluate the method's detection threshold for each of the four species and to assess the accuracy of their proportional quantification. Having demonstrated the accurate determination of species composition in Trypanosoma communities, the method was applied to determine its detection threshold using blood samples collected from cattle with confirmed Trypanosoma infections based on a PCR assay. Each sample showed a different Trypanosoma species composition based on the proportion of MiSeq reads. Finally, we applied the assay to field samples to develop new insight into the species composition of Trypanosoma communities in cattle, camels, buffalo, horses, sheep, and goat in endemically infected regions of Pakistan. We confirmed that Trypanosoma evansi is the major species in Pakistan and for the first time showed the presence of Trypanosoma theileri. The metabarcoding deep amplicon sequencing method and bioinformatics pathway have several potential applications in animal and human research, including evaluation of drug treatment responses, understanding of the emergence and spread of drug resistance, and description of species interactions during co-infections and determination of host and geographic distribution of trypanosomiasis in humans and livestock.

Differences in Transporters Rather than Drug Targets Are the Principal Determinants of the Different Innate Sensitivities of Trypanosoma congolense and Trypanozoon Subgenus Trypanosomes to Diamidines and Melaminophenyl Arsenicals

The animal trypanosomiases are infections in a wide range of (domesticated) animals with any species of African trypanosome, such as Trypanosoma brucei, T. evansi, T. congolense, T. equiperdum and T. vivax. Symptoms differ between host and infective species and stage of infection and are treated with a small set of decades-old trypanocides. A complication is that not all trypanosome species are equally sensitive to all drugs and the reasons are at best partially understood. Here, we investigate whether drug transporters, mostly identified in T. b. brucei, determine the different drug sensitivities. We report that homologues of the aminopurine transporter TbAT1 and the aquaporin TbAQP2 are absent in T. congolense, while their introduction greatly sensitises this species to diamidine (pentamidine, diminazene) and melaminophenyl (melarsomine) drugs. Accumulation of these drugs in the transgenic lines was much more rapid. T. congolense is also inherently less sensitive to suramin than T. brucei, despite accumulating it faster. Expression of a proposed suramin transporter, located in T. brucei lysosomes, in T. congolense, did not alter its suramin sensitivity. We conclude that for several of the most important classes of trypanocides the presence of specific transporters, rather than drug targets, is the determining factor of drug efficacy.

Seasonal Patterns: Bovine Trypanosomosis, Glossina pallidipes Density, and Infection in Rift Valleys of Gamo Zone, Southern Ethiopia.

Bovine trypanosomosis is a parasitic disease causing serious economic losses in livestock productivity and agricultural development. The disease has been reported in different parts of Ethiopia. However, seasonal pattern of trypanosomosis, tsetse fly apparent density, and infection are very limited in the southern rift valley of the country, particularly in Gamo Zone. Therefore, the objective of this cross-sectional study design was to estimate seasonal prevalence of bovine trypanosomosis, assessing tsetse fly apparent density and its infection by trypanosomes. For the parasitological study, a total of 600 cattle (300 in each season) were sampled and assayed using the buffy coat technique. A total of 80 standard NGU traps were deployed around the watering and grazing areas for the entomological survey. An overall prevalence of trypanosomosis was 10.17% (61/600), of which 7.33% (22/300) and 13% (39/300) accounted for the dry and wet seasons, respectively. The prevalence of trypanosomosis was significantly higher during the wet season (OR = 2.47; p < 0.05), in black coat color (OR = 7.2, p < 0.05), and poor body-conditioned (OR = 3.15; p < 0.05) animals. Two species of trypanosomes, Trypanosoma congolense, 68.85% (42/61), and Trypanosoma vivax, 31.15% (19/61), were circulating in the area. The mean PCV value in infected animals (22.56 ± 4.61) was significantly lower than in non-infected animals (25.3 ± 4.75). Entomological result indicated that Glossina pallidipes (G. pallidipes) was the only species of tsetse found in the study area. Totally, 3,789 flies were caught of which 81.42% (3,085/3,789) belong to G. pallidipes and 18.58% (704/3,789) were other biting flies. The overall apparent density of G. pallidipes was 12.85 flies/trap/day (FTD). Relatively higher G. pallidipes/trap/day were caught in the wet season (13.64 F/T/D) than in the dry season (12.07F/T/D). Of the flies caught, 342 G. pallidipes were randomly selected and dissected. The overall proportion of G. pallidipes infection was 18.42% (63/342) of which 12.28% (21/171) and 24.56% (42/171) were accounted in the dry and wet seasons, respectively. Infection in G. pallidipes was significantly higher during the wet season (OR = 2.32; p < 0.05) and in park grazing areas (OR = 2.45; p < 0.05). In conclusion, trypanosomosis is the major challenge for cattle productivity in the district. So this study warrants the need for strengthening the vector and parasite control interventions in the area.

Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.