Molecular identification of new Trypanosoma evansi type non-A/B isolates from buffaloes and cattle in Indonesia.

Trypanosoma evansi is reportedly divided into two genotypes: types A and B. The type B is uncommon and reportedly limited to Africa: Kenya Sudan, and Ethiopia. In contrast, type A has been widely reported in Africa, South America, and Asia. However, Trypanosoma evansi type non-A/B has never been reported. Therefore, this study aims to determine the species and genotype of the Trypanozoon subgenus using a robust identification algorithm. Forty-three trypanosoma isolates from Indonesia were identified as Trypanosoma evansi using a molecular identification algorithm. Further identification showed that 39 isolates were type A and 4 isolates were possibly non-A/B types. The PML, AMN-SB1, and STENT3 isolates were likely non-A/B type Trypanosoma evansi isolated from buffalo, while the PDE isolates were isolated from cattle. Cladistic analysis revealed that Indonesian Trypanosoma evansi was divided into seven clusters based on the gRNA-kDNA minicircle gene. Clusters 6 and 7 are each divided into two sub-clusters. The areas with the highest genetic diversity are the provinces of Banten, Central Java (included Yogyakarta), and East Nusa Tenggara. The Central Java (including Yogyakarta) and East Nusa Tenggara provinces, each have four sub-clusters, while Banten has three.

Molecular diversity and polyparasitism of avian trypanosomes in the Brazilian Atlantic Rainforest.

The current study proposes to investigate the diversity and phylogeny of trypanosomes parasitizing wild birds from the Brazilian Atlantic Forest. Cytological examination was carried out by light microscopy of blood smears and positive birds were selected for amplification of the 18S rDNA sequence through PCR. The resulting amplicons were subjected to purification, cloning, and sequencing analysis. Phylogenetic reconstruction was conducted, including all avian trypanosomes representative's lineages. A total of ten bird samples from species of Turdus flavipes (N=1/12), T. albicollis (N=1/8), Tachyphonus coronatus (N=6/121), Thamnophilus caerulescens (N=1/22) and Synallaxis spixi (N=1/8) were positive for Trypanosoma spp. In the six specimens of T. coronatus, five distinct lineages of Trypanosoma spp. 18S-rRNA were observed in ninety sequences obtained, and using the strategy of cloning independent PCR, it was possible to observe that two of them were related to T. avium (JB01/JB02), and three were closed related to T. bennetti (JB03/ JB04/JB05). Addionaly, all fifteen sequences obtained from T. caerulescens/ S. spixi/T. flavipes/T. albicollis were identical. The present research is the first study to access molecular diversity and polyparasitism by avian trypanosomes in Brazil. The current research exhibits the wide genetic variability in avian trypanosomes and its non-specific relationship with its avian hosts.

Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat, India.

BACKGROUND OBJECTIVES: Vector-borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at the identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region in India using microscopy and Polymerase Chain Reaction (PCR). METHODS: A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. RESULTS: The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45 % sensitive and 100 % specific). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. INTERPRETATION CONCLUSION: Despite holding much significance to the animal sector, little work has been undertaken in regional parts of India regarding camel parasites. The present study offers first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

The Transmission of Animal African Trypanosomiasis in Two Districts in the Forest Zone of Ghana.

Animal African trypanosomiasis, also known as nagana, is caused by Trypanosoma species, which cause significant clinical diseases and lead to losses in animal production. We carried out a cross-sectional survey to investigate the composition of vectors and parasite diversity in two districts in the eastern region of Ghana where pigs and cattle were exposed to tsetse bites. We performed cytochrome c oxidase subunit 1 polymerase chain reaction (PCR) to identify tsetse species and internal transcribed spacer 1 PCR to identify Trypanosoma species. Also, we investigated the source of tsetse blood meal based on mitochondrial cytochrome b gene sequence analysis. A total of 229 tsetse, 65 pigs, and 20 cattle were investigated for trypanosomes. An overall vector density of 4.3 tsetse/trap/day was observed. A trypanosome prevalence of 58.9% (95% CI = 52.5-65.1%), 46.2% (95% CI = 34.6-58.1%), and 0.0% (95% CI = 0.0-16.1%) in tsetse, pigs, and cattle, respectively, was detected. Trypanosoma congolense was predominant, with a prevalence of 33.3% (95% CI = 73.3-86.5%) in tsetse. There was evidence of multiple infections in tsetse and pigs. Approximately 39% of the tsetse were positive for multiple infections of T. congolense and Trypanosoma simiae. Parasite prevalence in pigs across the communities was high, with significant differences associated between locations (χ2 = 28.06, 95% CI = 0.05-0.81, P = 0.0009). Tsetse blood meal analysis revealed feeding on domestic Sus scrofa domesticus (pigs) and Phacochoerus africanus (warthogs). Infective tsetse may transmit trypanosomes to livestock and humans in the communities studied.

Adipose and skin distribution of African trypanosomes in natural animal infections.

BACKGROUND: Animal African trypanosomiasis, which is caused by different species of African trypanosomes, is a deadly disease in livestock. Although African trypanosomes are often described as blood-borne parasites, there have been recent reappraisals of the ability of these parasites to reside in a wide range of tissues. However, the majority of those studies were conducted on non-natural hosts infected with only one species of trypanosome, and it is unclear whether a similar phenomenon occurs during natural animal infections, where multiple species of these parasites may be present. METHODS: The infective trypanosome species in the blood and other tissues (adipose and skin) of a natural host (cows, goats and sheep) were determined using a polymerase chain reaction-based diagnostic. RESULTS: The animals were found to harbour multiple species of trypanosomes. Different patterns of distribution were observed within the host tissues; for instance, in some animals, the blood was positive for the DNA of one species of trypanosome and the skin and adipose were positive for the DNA of another species. Moreover, the rate of detection of trypanosome DNA was highest for skin adipose and lowest for the blood. CONCLUSIONS: The findings reported here emphasise the complexity of trypanosome infections in a natural setting, and may indicate different tissue tropisms between the different parasite species. The results also highlight the need to include adipose and skin tissues in future diagnostic and treatment strategies.

Morphological and molecular characteristics of a Trypanosoma sp. from triatomines (Triatoma rubrofasciata) in China.

BACKGROUND: Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. METHODS: The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. RESULTS: Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was -4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. CONCLUSIONS: Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated.

The prevalence of selected vector-borne diseases in dromedary camels (Camelus dromedarius) in the United Arab Emirates.

Vector-borne diseases (VBDs) affecting dromedary camels (Camelus dromedarius) have considerable importance in the United Arab Emirates (UAE) because of the consequences associated with production decline and economic losses. Our study aimed to determine the prevalence of selected VBDs in camels in the UAE and identify risk factors. This research is currently affected by the low number of epidemiological molecular surveys addressing this issue. Blood samples were obtained from 425 dromedary camels from different locations across the UAE. Whole genomic DNA was isolated, and PCR screening was done to detect piroplasmids (Babesia/Theileria spp.), Trypanosoma spp., and Anaplasmataceae spp. (Anaplasma, Ehrlichia, Neorickettsia and Wolbachia spp.). Amplicons were sequenced, and phylogenetic trees were constructed. Trypanosoma sequences were identified as T. brucei evansi, whereas Anaplasmataceae sequences were identified as A. platys-like. All camels were negative for Babesia/Theileria spp. (0%); however, 18 camels were positive for T. b. evansi (4%) and 52 were positive for A. platys-like (12%). Mixed infection with T. b. evansi and A. platys-like was found in one camel. Statistical analyses revealed that camels with a brown coat colour were significantly more prone to acquire the A. platys-like strain compared with those having a clearer coat. A similar finding was observed when comparing urban moving camels with desert indoor and urban indoor camels. Continuous disease surveillance is required to ensure and maintain the good health status of the camels in the UAE. Nonetheless, the risk of disease outbreak remains if the misuse of drugs continues.

Trypanosomatids Associated in the Alimentary Canal of Bagrada hilaris (Hemiptera: Pentatomidae).

Bagrada hilaris (Burmeister) is an invasive pest of economically important crops in the United States. During physiological investigations of B. hilaris, a flagellated protozoan was discovered in the alimentary canal of many specimens. This manuscript characterizes the morphology and molecular identification of the trypanosomatid, which appears similar to trypanosomatids identified in other stink bug species. It has been identified as a species in the Blastocrithidia genus based on morphological characteristics and molecular analyses.

Molecular characterization of Trypanosoma evansi, T. vivax and T. congolense in camels (Camelus dromedarius) of KSA.

BACKGROUND: Trypanosoma evansi is the leading infectious Trypanosoma spp. in camels (Camelus dromedarius) present in the Kingdom of Saudi Arabia (KSA) that could lead to extensive economic losses. The present study was aimed to assess the prevalence rate of T. evansi in Taif governorate, Makkah province, KSA using parasitological and molecular evaluations, and analyze their genetic relationship targeting internal transcribed spacer 1 (ITS1) and variable surface glycoprotein (VSG) genes. For evaluation, we have used 102 blood samples of camels obtained from three different regions in Taif. RESULTS: Results show a considerable prevalence rate of trypanosomosis 2/102 (2.0%) according to Giemsa-stained buffy coat smear, and 16/102 (15.7%) according to touchdown PCR. T. evansi (n = 10/102, 9.8%) was the main infectious species found in camels then T. vivax (n = 3/102, 2.9%). Mixed infections were detected in three camels with T. evansi, T. vivax, and T. congolense (n = 3/102, 2.9%). Regarding gender, the results indicate that female camels (11/66, 16.7%) show higher prevalence of Trypanosoma than males (5/36, 13.9%). Sequencing and phylogenetic analyses of ITS1 and VSG showed their relationships with T. evansi in other hosts from different countries. CONCLUSIONS: In our peer knowledge, it is the first time to report a research-based prevalence of trypanosomosis in the camels of Taif governorate, Makkah province, KSA.

A distinct complex of PRP19-related and trypanosomatid-specific proteins is required for pre-mRNA splicing in trypanosomes.

The pre-mRNA splicing factor PRP19 is recruited into the spliceosome after forming the PRP19/CDC5L complex in humans and the Nineteen complex in yeast. Additionally, 'PRP19-related' proteins enter the spliceosome individually or in pre-assemblies that differ in these systems. The protistan family Trypanosomatidae, which harbors parasites such as Trypanosoma brucei, diverged early during evolution from opisthokonts. While introns are rare in these organisms, spliced leader trans splicing is an obligatory step in mRNA maturation. So far, ∼70 proteins have been identified as homologs of human and yeast splicing factors. Moreover, few proteins of unknown function have recurrently co-purified with splicing proteins. Here we silenced the gene of one of these proteins, termed PRC5, and found it to be essential for cell viability and pre-mRNA splicing. Purification of PRC5 combined with sucrose gradient sedimentation revealed a complex of PRC5 with a second trypanosomatid-specific protein, PRC3, and PRP19-related proteins SYF1, SYF3 and ISY1, which we named PRP19-related complex (PRC). Importantly, PRC and the previously described PRP19 complex are distinct from each other because PRC, unlike PRP19, co-precipitates U4 snRNA, which indicates that PRC enters the spliceosome prior to PRP19 and uncovers a unique pre-organization of these proteins in trypanosomes.

Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei.

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.

Occurrence, diversity and distribution of Trypanosoma infections in cattle around the Akagera National Park, Rwanda.

BACKGROUND: African Trypanosomiases threaten the life of both humans and animals. Trypanosomes are transmitted by tsetse and other biting flies. In Rwanda, the African Animal Trypanosomiasis (AAT) endemic area is mainly around the tsetse-infested Akagera National Park (NP). The study aimed to identify Trypanosoma species circulating in cattle, their genetic diversity and distribution around the Akagera NP. METHODOLOGY: A cross-sectional study was carried out in four districts, where 1,037 cattle blood samples were collected. The presence of trypanosomes was determined by microscopy, immunological rapid test VerY Diag and PCR coupled with High-Resolution Melt (HRM) analysis. A parametric test (ANOVA) was used to compare the mean Packed cell Volume (PCV) and trypanosomes occurrence. The Cohen Kappa test was used to compare the level of agreement between the diagnostic methods. FINDINGS: The overall prevalence of trypanosome infections was 5.6%, 7.1% and 18.7% by thin smear, Buffy coat technique and PCR/HRM respectively. Microscopy showed a low sensitivity while a low specificity was shown by the rapid test (VerY Diag). Trypanosoma (T.) congolense was found at a prevalence of 10.7%, T. vivax 5.2%, T. brucei brucei 2% and T. evansi 0.7% by PCR/HRM. This is the first report of T.evansi in cattle in Rwanda. The non-pathogenic T. theileri was also detected. Lower trypanosome infections were observed in Ankole x Friesian breeds than indigenous Ankole. No human-infective T. brucei rhodesiense was detected. There was no significant difference between the mean PCV of infected and non-infected animals (p>0.162). CONCLUSIONS: Our study sheds light on the species of animal infective trypanosomes around the Akagera NP, including both pathogenic and non-pathogenic trypanosomes. The PCV estimation is not always an indication of trypanosome infection and the mechanical transmission should not be overlooked. The study confirms that the area around the Akagera NP is affected by AAT, and should, therefore, be targeted by the control activities. AAT impact assessment on cattle production and information on the use of trypanocides are needed to help policymakers prioritise target areas and optimize intervention strategies. Ultimately, these studies will allow Rwanda to advance in the Progressive Control Pathway (PCP) to reduce or eliminate the burden of AAT.

Apparent density, trypanosome infection rates and host preference of tsetse flies in the sleeping sickness endemic focus of northwestern Uganda.

BACKGROUND: African trypanosomiasis, caused by protozoa of the genus Trypanosoma and transmitted by the tsetse fly, is a serious parasitic disease of humans and animals. Reliable data on the vector distribution, feeding preference and the trypanosome species they carry is pertinent to planning sustainable control strategies. METHODOLOGY: We deployed 109 biconical traps in 10 villages in two districts of northwestern Uganda to obtain information on the apparent density, trypanosome infection status and blood meal sources of tsetse flies. A subset (272) of the collected samples was analyzed for detection of trypanosomes species and sub-species using a nested PCR protocol based on primers amplifying the Internal Transcribed Spacer (ITS) region of ribosomal DNA. 34 blood-engorged adult tsetse midguts were analyzed for blood meal sources by sequencing of the mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) genes. RESULTS: We captured a total of 622 Glossina fuscipes fuscipes tsetse flies (269 males and 353 females) in the two districts with apparent density (AD) ranging from 0.6 to 3.7 flies/trap/day (FTD). 10.7% (29/272) of the flies were infected with one or more trypanosome species. Infection rate was not significantly associated with district of origin (Generalized linear model (GLM), χ2 = 0.018, P = 0.895, df = 1, n = 272) and sex of the fly (χ2 = 1.723, P = 0.189, df = 1, n = 272). However, trypanosome infection was highly significantly associated with the fly's age based on wing fray category (χ2 = 22.374, P < 0.001, df = 1, n = 272), being higher among the very old than the young tsetse. Nested PCR revealed several species of trypanosomes: T. vivax (6.62%), T. congolense (2.57%), T. brucei and T. simiae each at 0.73%. Blood meal analyses revealed five principal vertebrate hosts, namely, cattle (Bos taurus), humans (Homo sapiens), Nile monitor lizard (Varanus niloticus), African mud turtle (Pelusios chapini) and the African Savanna elephant (Loxodonta africana). CONCLUSION: We found an infection rate of 10.8% in the tsetse sampled, with all infections attributed to trypanosome species that are causative agents for AAT. However, more verification of this finding using large-scale passive and active screening of human and tsetse samples should be done. Cattle and humans appear to be the most important tsetse hosts in the region and should be considered in the design of control interventions.

Development of an Amplicon-Based Next-Generation Sequencing Protocol to Identify Leishmania Species and Other Trypanosomatids in Leishmaniasis Endemic Areas.

Trypanosomatid infections are an important public health threat affecting many low-income countries across the tropics, particularly in the Americas. Trypanosomatids can infect many vertebrate, invertebrate, and plant species and play an important role as human pathogens. Among these clinically relevant pathogens are species from the genera Leishmania and Trypanosoma. Mixed trypanosomatid infections remain a largely unexplored phenomenon. Herein, we describe the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatid species in mammalian reservoirs, human patients, and sand fly vectors throughout regions of Leishmania endemicity. Sixty-five samples from different departments of Colombia, including two samples from Venezuela, were analyzed: 49 samples from cutaneous leishmaniasis (CL) patients, 8 from sand flies, 2 from domestic reservoirs (Canis familiaris), and 6 from wild reservoirs (Phyllostomus hastatus). DNA from each sample served to identify the presence of trypanosomatids through conventional PCR using heat shock protein 70 (HSP70) gene as the target. PCR products underwent sequencing by Sanger sequencing and NGS, and trypanosomatid species were identified by using BLASTn against a reference database built from trypanosomatid-derived HSP70 sequences. The alpha and beta diversity indexes of amplicon sequence variants were calculated for each group. The results revealed the presence of mixed infections with more than two Leishmania species in 34% of CL samples analyzed. Trypanosoma cruzi was identified in samples from wild reservoirs, as well as in sand fly vectors. Coinfection events with three different Leishmania species were identified in domestic reservoirs. These findings depose the traditional paradigm of leishmaniasis as being a single-species-driven infection and redraw the choreography of host-pathogen interaction in the context of multiparasitism. Further research is needed to decipher how coinfections may influence disease progression. This knowledge is key to developing an integrated approach for diagnosis and treatment. IMPORTANCE Traditionally, there has been a frequent, yet incorrect assumption that phlebotomine vectors, animal reservoirs, and human hosts are susceptible to Leishmania infection by a single parasite species. However, current evidence supports that these new vector-parasite-reservoir associations lend vectors and reservoirs greater permissiveness to certain Leishmania species, thus promoting the appearance of coinfection events, particularly in disease-endemic regions. The present study describes the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatid species in mammalian reservoirs, human patients, and sand fly vectors from regions of endemicity for leishmaniasis. This changes our understanding of the clinical course of leishmaniasis in areas of endemicity.

Molecular identification and prevalence of trypanosomes in cattle distributed within the Jebba axis of the River Niger, Kwara state, Nigeria.

BACKGROUND: Trypanosomiasis is a fatal disease that threatens the economy of at least 37 countries in sub-Saharan Africa, particularly with regard to livestock farming. In this study, we investigated the prevalence of trypanosome infection in cattle, and molecularly identified the species of trypanosomes in infected cattle and the spatial distribution of trypanosome-infected herds along the Jebba axis of the River Niger. METHODS: A randomized cross-sectional study was conducted along the Jebba axis of the River Niger by screening cattle from 36 herd clusters by nested PCR using ITS-1 generic primers. Data generated were analysed using the Chi-square test at a 95% confidence interval. RESULTS: Microscopic examination revealed three infected cattle out of 398 examined, representing 0.8% prevalence. Twelve animals (3.0%) were positive by PCR. Our results showed a decline in the packed cell volume of infected animals (24.7%). The infection rates were categorized as single infection in 11/12 (91.7%) and mixed infection in 1/12 (8.3%). Animals were most frequently infected by Trypanosoma congolense (50.0%), with T. congolense Savannah being the most prevalent subspecies (71.4%). Aside from the infection rate by age (10.0%) and relative distance of animals from the River Niger (56.2%), statistical differences in every other parameter tested were based on mere probabilistic chance. Spatial data showed that the disease was prevalent among herds located less than 3 km from the River Niger. CONCLUSIONS: Six species of trypanosomes were identified in cattle herds along the Jebba axis of the River Niger, with T. congolense being the most prevalent. Age and relative distance of herds from the River Niger may be risk factors for trypanosome infection in cattle herds in this area.

Prevalence and risk factors for trypanosome infection in cattle from communities surrounding the Murchison Falls National Park, Uganda.

BACKGROUND: Bovine trypanosomosis transmitted by tsetse flies is a major constraint to cattle health and productivity in all sub-Saharan countries, including Uganda. The objectives of this study were to determine the prevalence of bovine trypanosomosis and identify its associated risk factors and the species of trypanosomes associated with the disease. METHODOLOGY: A cross-sectional study was conducted around Murchison Falls National Park, Uganda from January 2020 to April 2020. Trypanosomes were detected in blood samples by PCR analysis targeting the internal transcribed spacer 1 (ITS-PCR assays), and trypanosomes in positive blood samples were sequenced. RESULTS: Of 460 blood samples collected and tested, 136 (29.6%) were positive for trypanosome infections and 324 (70.4%) were negative. The overall trypanosome prevalence was 29.6% (95% confidence interval 25.4-33.8%), attributed to three trypanosome species. Of these three species, Trypanosoma vivax was the most prevalent (n = 130, 28.3%) while the others were detected as mixed infections: T. vivax + Trypanosoma congolense (n = 2, 0.4%) and T. vivax + Trypanosoma evansi (n = 1, 0.2%). There were significant differences in trypanosome prevalence according to sex (χ2 = 62, df = 1, P < 0.05), age (χ2 = 6.28, df = 2, P = 0.0043) and cattle breed (χ2 = 10.61, df = 1, P = 0.001). CONCLUSIONS: Trypanosomosis remains a major limitation to cattle production around Murchison Falls National Park and interventions are urgently needed. In our study, the prevalence of trypanosome infections was high, with T. vivax identified as the most prevalent species. Age, sex and breed of cattle were risk factors for trypanosome infection.

A New Model Trypanosomatid, Novymonas esmeraldas: Genomic Perception of Its "Candidatus Pandoraea novymonadis" Endosymbiont.

The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.

Monomorphic Trypanozoon: towards reconciling phylogeny and pathologies.

Trypanosoma brucei evansi and T. brucei equiperdum are animal infective trypanosomes conventionally classified by their clinical disease presentation, mode of transmission, host range, kinetoplast DNA (kDNA) composition and geographical distribution. Unlike other members of the subgenus Trypanozoon, they are non-tsetse transmitted and predominantly morphologically uniform (monomorphic) in their mammalian host. Their classification as independent species or subspecies has been long debated and genomic studies have found that isolates within T. brucei evansi and T. brucei equiperdum have polyphyletic origins. Since current taxonomy does not fully acknowledge these polyphyletic relationships, we re-analysed publicly available genomic data to carefully define each clade of monomorphic trypanosome. This allowed us to identify, and account for, lineage-specific variation. We included a recently published isolate, IVM-t1, which was originally isolated from the genital mucosa of a horse with dourine and typed as T. equiperdum. Our analyses corroborate previous studies in identifying at least four distinct monomorphic T. brucei clades. We also found clear lineage-specific variation in the selection efficacy and heterozygosity of the monomorphic lineages, supporting their distinct evolutionary histories. The inferred evolutionary position of IVM-t1 suggests its reassignment to the T. brucei evansi type B clade, challenging the relationship between the Trypanozoon species, the infected host, mode of transmission and the associated pathological phenotype. The analysis of IVM-t1 also provides, to our knowledge, the first evidence of the expansion of T. brucei evansi type B, or a fifth monomorphic lineage represented by IVM-t1, outside of Africa, with important possible implications for disease diagnosis.

Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro, Southern Chad-A matter of concern for zoonotic potential?

BACKGROUND: African trypanosomes are parasites mainly transmitted by tsetse flies. They cause trypanosomiasis in humans (HAT) and animals (AAT). In Chad, HAT/AAT are endemic. This study investigates the diversity and distribution of trypanosomes in Mandoul, an isolated area where a tsetse control campaign is ongoing, and Maro, an area bordering the Central African Republic (CAR) where the control had not started. METHODS: 717 human and 540 cattle blood samples were collected, and 177 tsetse flies were caught. Trypanosomal DNA was detected using PCR targeting internal transcribed spacer 1 (ITS1) and glycosomal glyceraldehyde-3 phosphate dehydrogenase (gGAPDH), followed by amplicon sequencing. RESULTS: Trypanosomal DNA was identified in 14 human samples, 227 cattle samples, and in tsetse. Besides T. b. gambiense, T. congolense was detected in human in Maro. In Mandoul, DNA from an unknown Trypanosoma sp.-129-H was detected in a human with a history of a cured HAT infection and persisting symptoms. In cattle and tsetse samples from Maro, T. godfreyi and T. grayi were detected besides the known animal pathogens, in addition to T. theileri (in cattle) and T. simiae (in tsetse). Furthermore, in Maro, evidence for additional unknown trypanosomes was obtained in tsetse. In contrast, in the Mandoul area, only T. theileri, T. simiae, and T. vivax DNA was identified in cattle. Genetic diversity was most prominent in T. vivax and T. theileri. CONCLUSION: Tsetse control activities in Mandoul reduced the tsetse population and thus the pathogenic parasites. Nevertheless, T. theileri, T. vivax, and T. simiae are frequent in cattle suggesting transmission by other insect vectors. In contrast, in Maro, transhumance to/from Central African Republic and no tsetse control may have led to the high diversity and frequency of trypanosomes observed including HAT/AAT pathogenic species. Active HAT infections stress the need to enforce monitoring and control campaigns. Additionally, the diverse trypanosome species in humans and cattle indicate the necessity to investigate the infectivity of the unknown trypanosomes regarding their zoonotic potential. Finally, this study should be widened to other trypanosome hosts to capture the whole diversity of circulating trypanosomes.

Diversity of tsetse flies and trypanosome species circulating in the area of Lake Iro in southeastern Chad.

BACKGROUND: African trypanosomiases are vector-borne diseases that affect humans and livestock in sub-Saharan Africa. Although data have been collected on tsetse fauna as well as trypanosome infections in tsetse flies and mammals in foci of sleeping sickness in Chad, the situation of tsetse fly-transmitted trypanosomes remains unknown in several tsetse-infested areas of Chad. This study was designed to fill this epidemiological knowledge gap by determining the tsetse fauna as well as the trypanosomes infecting tsetse flies in the area of Lake Iro in southeastern Chad. METHODS: Tsetse flies were trapped along the Salamat River using biconical traps. The proboscis and tsetse body were removed from each fly. DNA was extracted from the proboscis using proteinase K and phosphate buffer and from the tsetse body using Chelex 5%. Tsetse flies were identified by amplifying and sequencing the cytochrome c oxydase I gene of each tsetse fly. Trypanosome species were detected by amplifying and sequencing the internal transcribed spacer 1 of infecting trypanosomes. RESULTS: A total of 617 tsetse flies were trapped; the apparent density of flies per trap per day was 2. 6. Of the trapped flies, 359 were randomly selected for the molecular identification and for the detection of infecting trypanosomes. Glossina morsitans submorsitans (96.1%) was the dominant tsetse fly species followed by G. fuscipes fuscipes (3.1%) and G. tachinoides (0.8%). Four trypanosome species, including Trypanosoma vivax, T. simiae, T. godfreyi and T. congolense savannah, were detected. Both single infection (56.7%) and mixed infections of trypanosomes (4.6%) were detected in G. m. submorsitans. The single infection included T. simiae (20.5%), T. congolense savannah (16.43%), T. vivax (11.7%) and T. godfreyi (9.8%). The trypanosome infection rate was 61.4% in G. m. submorsitans, 72.7% in G. f. fuscipes and 66.6% in G. tachinoides. Trypanosome infections were more prevalent in tsetse bodies (40.6%) than in the proboscis (16.3%). CONCLUSION: This study revealed the presence of different tsetse species and a diversity of trypanosomes pathogenic to livestock in the area of Lake Iro. The results highlight the risks and constraints that animal African trypanosomiasis pose to livestock breeding and the importance of assessing trypanosome infections in livestock in this area.