Evaluation of Lab-on-a-Disc Technique Performance for Soil-Transmitted Helminth Diagnosis in Animals in Tanzania.

Soil-transmitted helminth (STH) infections are caused by roundworms, hookworms, whipworms, and thread worms. Accurate diagnosis is essential for effective treatment, prevention, and control of these infections. This study evaluates a new diagnostic method called Single-image Parasite Quantification (SIMPAQ), which uses a lab-on-a-disc (LoD) technique to isolate STH eggs into a single imaging zone for digital analysis. The study evaluates the purification performance of the SIMPAQ technique for detecting STH eggs in animal samples. This was a cross-sectional study conducted among 237 pigs and 281 dogs in the Morogoro region in Tanzania. Faecal samples were collected and processed with the LoD technique, as well as flotation and McMaster (McM) methods for comparison purposes. The overall prevalence of STH infections was high as per the LoD technique (74%), followed by McM (65.44%) and flotation (65.04%). Moreover, the overall performance of the LoD technique, using McM as the gold standard, was 93.51% (sensitivity), 60.89% (specificity), 81.91% (PPV), and 83.21% (NPV). The LoD technique exhibited high prevalence, sensitivity, and NPV, which demonstrates its value for STH egg detection and its crucial role in the era of accurate STH diagnosis, promoting proper management of the infection.

Genetic Diversity of Trypanosomes Infesting Cattle from Savannah District in North of Côte d'Ivoire Using Conserved Genomic Signatures: rRNA, ITS1 and gGAPDH.

The potential danger to livestock from African animal trypanosomiasis is well known. However, the trypanosome species circulating in cattle and their genetics are poorly understood. After different alignments according to three regions (ITS1, gGAPDH and rRNA gene) of the trypanosome genome, phylogenetic analyses were used to show the genetic diversity of the different species that were circulating in the cattle in three regions (Bagoue, Poro and Tchologo) of Côte d'Ivoire. These analyses were performed by alignment of ITS1; by alignment of partial 18S, ITS1, 5.8S, ITS2 and partial 28S rRNA genes; and by alignment of gGAPDH gene with sequences of Trypanosomes found in GenBank. Three species were identified (T. vivax, T. theileri and T. congolense) in the cattle in the three northern regions of Côte d'Ivoire. T. vivax and T. theileri were the most abundant species in the present study. Contrary to the other primers used in this study, the ITS1 primers were not able to amplify T. theileri. We observed mixed infections between T. theileri and the other two species identified (T. vivax and T. congolense). As far as primers are concerned, in some cases, rRNA was able to identify the same species of trypanosomes that the ITS1 and gGAPDH primers were able to identify. Two main distinct groups of T. theileri complex were identified. The T. congolense and T. vivax strains were close to African strains, such as those from Kenya, Nigeria and Cameroon, unlike the T. theileri strain. Three trypanosome species (T. vivax, T. theileri and T. congolense) circulate in cattle in the Savannah district of Côte d'Ivoire. The genetic diversity of the trypanosome species encountered in this study cannot be classified as intraspecies according to geographical area and breed of cattle they infect.

Seasonal variation in tsetse fly apparent density and Trypanosoma spp. infection rate and occurrence of drug-resistant trypanosomes in Lambwe, Kenya.

Tsetse flies are major arthropod vectors of trypanosomes that cause debilitating African animal trypanosomiasis. The emergence of drug-resistant trypanosomes is a common problem in sub-Saharan Africa. This study aimed to identify tsetse flies' seasonal variation in apparent densities and their infection rates and the occurrence of drug-resistant trypanosomes. Tsetse flies were collected from Lambwe, Kenya, during May and September 2021. Genomic DNA was extracted from them, and the ITS1 gene was amplified to detect Trypanosoma infection with subsequent species determination. Transporter genes DMT, E6M6, TbAT/P2, and TcoAde2 were targeted to detect polymorphisms associated with drug-resistance, using sequencing and comparison to drug-sensitive trypanosome species referenced in Genbank. A total of 498 tsetse flies and 29 non-tsetse flies were collected. The apparent density of flies was higher in wet season 6.2 fly per trap per density (FTD) than in the dry season 2.3 FTD (P = 0.001), with n = 386 and n = 141 flies caught in each season, respectively. Male tsetse flies (n = 311) were more numerous than females (n = 187) (P = 0.001). Non-tsetse flies included Tabanids and Stomoxys spp. Overall, Trypanosoma infection rate in tsetse was 5% (25/498) whereby Trypanosoma vivax was 4% (11/25), Trypanosoma congolense 36% (9/25), and Trypanosoma brucei 20% (5/25) (P = 0.186 for the distribution of the species), with infections being higher in females (P = 0.019) and during the wet season (P < 0.001). Numerous polymorphisms and insertions associated with drug resistance were detected in DMT and E6M6 genes in two T. congolense isolates while some isolates lacked these genes. T. brucei lacked TbAT/P2 genes. TcoAde2 sequences in three T. congolense isolates were related to those observed in trypanosomes from cattle blood in our previous study, supporting tsetse fly involvement in transmission in the region. We report Trypanosoma associated with trypanocidal drug-resistance in tsetse flies from Lambwe, Kenya. Female tsetse flies harbored more Trypanosoma infections than males. Tsetse transmission of trypanosomes is common in Lambwe. Risk of trypanosome infection would seem higher in the wet season, when tsetse flies and Trypanosoma infections are more prevalent than during the dry season. More efforts to control animal trypanosome vectors in the region are needed, with particular focus on wet seasons.

Molecular epidemiological survey of pathogenic trypanosomes in naturally infected cattle in northern Côte d'ivoire.

Bovine trypanosomiasis is a significant health concern for livestock intensification in Côte d'Ivoire. This study aimed to determine the prevalence and distribution of pathogenic trypanosomes and identify the most infected cattle breed in northern Côte d'Ivoire. We examined 700 cattle and found that polymerase chain reaction (PCR) was more sensitive (12.3%) than microscopic observation (5.6%). Among the trypanosome species detected in naturally infected cattle, Trypanosoma vivax was 7.3%, Trypanosoma simiae tsavo was 6.7%, and Trypanosoma congolense was 0.4%. The overall prevalence of trypanosome infection in all cattle breeds was 12.3%, while the prevalence in individual breeds was 14.8%, 7.3%, 10.6%, and 12.3% for N'Dama, Baoule, Zebu, and Mere breed, respectively. The infected animals had low packed cell volume, influencing the prevalence. Our findings indicate that bovine trypanosomes are prevalent in Côte d'Ivoire, and their prevalence varies by region and breed. These pathogens include T. vivax, T. simiae tsavo, and T. congolense.

Prevalence of bovine trypanosomiasis in Côte d'Ivoire: Systematic review and meta-analysis.

Bovine trypanosomiasis is a parasitic disease caused by protozoans of the genus Trypanosoma. The disease cause economic losses in livestock production. In order to determine the status of research on this disease in Côte d'Ivoire, we used the systematic review method and meta-analysis. Three electronics databases, namely Google Scholar, PubMed and CrossRef were used to search for publications on trypanosomiasis prevalence that met our inclusion criteria. Twenty five articles were identified, 11 of which met the inclusion criteria. Bovine trypanosomiasis prevalence of 2.99% (95% confidence interval [CI]: 2.96% - 3.01%) to 25.28% (95% CI: 25.17% - 25.38%) were recorded between 1960 and 2021. The analyses showed that the most infected regions were the Bagoue 11.26% (95% CI: 11.25% - 11.27%), Bounkani 14.94% (95% CI: 14.93% - 14.95%), Gbeke 10.34% (95% CI: 10.33% - 10.35%), Marahoue 13.79% (95% CI: 13.78% - 13.80%), Poro 8.50% (95% CI: 8.49% - 8.51%), and Tchologo 11.83% (95% CI: 11.82% - 11.84%).The most sensitive diagnostic method used was the polymerase chain reaction (PCR). The species of trypanosomes diagnosed were Typanosoma vivax 4.99% (95% CI: 4.97% - 5.01%), T. congolense 1.51% (95% CI: 1.49% - 1.52%), and T. brucei 0.61% (95% CI: 0.59% - 0.62%). Despite some variation, the prevalence of bovine trypanosomiasis in Côte d'Ivoire caused mainly by T. vivax has increased in the years between 1977 and 2017. Efforts to control tsetse and other mechanical vectors should also be put in place to minimize its transmission.Contribution: The authors studied the prevalence of bovine trypanosomiasis using the systematic review method and MA in order to determine the status of research on this disease in Côte d'Ivoire.

Trypanosoma Congolense Resistant to Trypanocidal Drugs Homidium and Diminazene and their Molecular Characterization in Lambwe, Kenya.

PURPOSE: African animal trypanosomiasis (AAT) is a disease affecting livestock in sub-Saharan Africa. The use of trypanocidal agents is common practice to control AAT. This study aimed to identify drug-resistant Trypanosoma congolense in Lambwe, Kenya, and assess if molecular test backed with mice tests is reliable in detecting drug sensitivity. METHODS: Blood samples were collected from cattle, in Lambwe, subjected to buffy coat extraction and Trypanosoma spp. detected under a microscope. Field and archived isolates were subjected to molecular characterization. Species-specific T. congolense and TcoAde2 genes were amplified using PCR to detect polymorphisms. Phylogenetic analysis were performed. Four T. congolense isolates were evaluated individually in 24 test mice per isolate. Test mice were then grouped (n=6) per treatement with diminazene, homidium, isometamidium, and controls. Mice were subsequently assessed for packed cell volume (PCV) and relapses using microscopy. RESULTS: Of 454 samples, microscopy detected 11 T. congolense spp, eight had TcoAde2 gene, six showed polymorphisms in molecular assay. Phylogenetic analysis grouped isolates into five. Two archived isolates were homidium resistant, one was also diminazene resistant in mice. Two additional isolates were sensitive to all the drugs. Interestingly, one sensitive isolate lacked polymorphisms, while the second lacked TcoAde2, indicating the gene is not involved in drug sensitivity. Decline in PCV was pronounced in relapsed isolates. CONCLUSION: T. congolense associated with homidium and diminazene resistance exist in Lambwe. The impact can be their spread and AAT increase. Polymorphisms are present in Lambwe strains. TcoAde2 is unlikely involved in drug sensitivity. Molecular combined with mice tests is reliable drug sensitivity test and can be applied to other genes. Decline in PCV in infected-treated host could suggest drug resistance.

Prevalence and Associated Risk Factors of African Animal Trypanosomiasis in Cattle in Lambwe, Kenya.

Background: African animal trypanosomiasis (AAT) affects livestock productivity in sub-Saharan Africa. This study aimed to determine cattle AAT's prevalence and associated risk factors in Lambwe Valley, Kenya. Methods: In a cross-sectional survey, livestock owners were recruited from four villages of Lambwe in Homa Bay, Kenya. Blood samples were collected from the jugular veins of cattle, and buffy coat smears were examined under a microscope. Parasites were further detected using polymerase chain reaction (PCR). Using a semistructured questionnaire, livestock owners were interviewed on their knowledge of AAT and control practices. Chi-square and multilevel models were used for the analysis. Results: The overall prevalence was 15.63% (71/454). Trypanosoma vivax 10.31% and T. congolense Savannah 6.01% were the common species and subspecies. A total of 61 livestock keepers were involved in the study. Of these, 91.80% (56/61) knew AAT, and 90.16% (55/61) could describe the symptoms well and knew tsetse fly bite as transmission mode. Self-treatment (54.09%; 33/61) was common, with up to 50.00% of the farmers using drugs frequently. Isometamidium (72.13%; 44/61) and diminazene (54.09%; 33/61) were drugs frequently used. Although 16.39% (10/61) of the farmers claimed to use chemoprophylactic treatment, 6/10 did not use the right drugs. Animals (92.1%; 58/63) with clinical signs had positive infections. Villages closer to the national park recorded a higher prevalence. Infections were higher in cattle owned by those self-treating (27.23%; 58/213), those using drug treatment without vector control (27.62%; 50/181), those using single-drug therapy, and those practicing communal grazing (20.00%; 59/295). Clinical signs strongly associate with positive infections under multilevel modeling. Conclusion: Cattle trypanosomiasis is prevalent in the Lambwe region of Kenya. This is influenced by inappropriate control practices, communal grazing, and the proximity of farms to the national park. In addition, clinical signs of the disease have a strong association with infections.

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%.

"Visiting old, learn new": taxonomical overview of chiropteran trypanosomes from the morphology to the genes.

Bats (the order Chiroptera) account for more than 20% of all mammalian species in the world; remarkably, they are the only mammals capable of true and sustained flight using their wing-like forelimbs. Since the beginning of the twentieth century, various morphotypes (or genotypes in the last decade) of haemoflagellates in the genus Trypanosoma (Euglenozoa: Kinetoplastea: Trypanosomatidae) have been reported worldwide in the blood of bats. Of note, the latent nature of chiropteran trypanosome infection with low levels of parasitaemia, together with the apparent morphological variation of the bloodstream forms related to phenotypical plasticity and the morphological resemblance of different parasite species, has hampered the taxonomic classification of bat trypanosomes based on morphological criteria. This said, 50 years ago, Hoare (1972) provisionally divided bat trypanosomes into two major morphotypes: the megadermae group (corresponding to the subgenus Megatrypanum in the traditional taxonomic system; 8 species) and the vespertilionis group (similar to the subgenus Schizotrypanum; 5 species). Importantly, the biological and biochemical analyses of bat trypanosomes isolated by haemoculture, together with the molecular genetic characterisation using various gene markers, allowed the establishment of clear phylogenetic and taxonomic relationships of various isolates from different continents in the last two decades. Here, we review the historical taxonomic approaches used to define chiropteran trypanosomes, as well as the ones currently employed to shed light on the diversity and evolutional tracks of the globally distributed chiropteran trypanosomes.

Integrative taxonomic approach of trypanosomes in the blood of rodents and soricids in Asian countries, with the description of three new species.

Trypanosoma lewisi (Kinetoplastea: Trypanosomatida: Trypanosomatidae) with a cosmopolitan distribution is the type species of the subgenus Herpetosoma, which includes ca. 50 nominal species isolated mainly from rodents. Since members of Herpetosoma in different host species have an almost identical morphology of bloodstream forms, these trypanosomes are referred to as 'T. lewisi-like', and the molecular genetic characterization of each species is necessary to verify their taxonomy. In the present study, we collected blood samples from 89 murid rodents of 15 species and 11 soricids of four species in Indonesia, Philippines, Vietnam, Taiwan, and mainland China for the detection of hemoprotozoan infection. T. lewisi and T. lewisi-like trypanosomes were found in the blood smears of 10 murid animals, which included Bandicota indica (two rats), Rattus argentiventer (one rat), and Rattus tiomanicus (two rats) in Indonesia; Rattus rattus (one rat) in the Philippines; and Niviventer confucianus (four rats) in mainland China. Furthermore, large- or medium-sized non-T. lewisi-like trypanosomes were detected in two soricids, Crocidura dracula in Vietnam and Anourosorex yamashinai in Taiwan, respectively. Molecular genetic characterization of the small subunit (SSU) ribosomal RNA gene (rDNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene indicated that the trypanosomes from all the murid hosts had identical SSU rDNA or gGAPDH gene nucleotide sequences except for those in N. confucianus in mainland China. These N. confucianus-infecting trypanosomes also showed several unique morphological features such as smaller bodies, anteriorly positioned nuclei, and larger rod-shaped kinetoplasts when compared with T. lewisi trypomastigotes. Trypanosoma (Herpetosoma) niviventerae n. sp. is erected for this new species. Similarly, based on morphological and molecular genetic characterization, Trypanosoma sapaensis n. sp. and Trypanosoma anourosoricis n. sp. are proposed for the trypanosomes in C. dracula in Vietnam and A. yamashinai in Taiwan, respectively. More effort directed toward the morphological and molecular genetic characterization of the trypanosomes of rodents and soricids is required to fully understand the real biodiversity of their hemoflagellates.

First record of Trypanosoma dionisii of the T. cruzi clade from the Eastern bent-winged bat (Miniopterus fuliginosus) in the Far East.

Chiropteran mammals worldwide harbour trypanosomes (Euglenozoa: Kinetoplastea: Trypanosomatida) of the subgenus 'Schizotrypanum' in the classical sense. Latterly, these trypanosomes have been referred to as members of the 'Trypanosoma cruzi clade' as their phylogenetic relationships, structure and life cycle conform to T. cruzi, parasitising various terrestrial mammals as well as humans in Latin America. Little is known, however, about the trypanosome species in Asian bats. During a survey on Borrelia spp. in the Eastern bent-winged bat (Miniopterus fuliginosus) living in a cave in Wakayama Prefecture, Japan, incidental proliferation of trypanosomes was detected in two of 94 haemocultures. Squat or slender trypanosomes that proliferated in the cultures were 7.5-20.5 µm in length between both body ends and 1.0-3.8 µm in width with/without free flagella up to 14.5 µm (n = 29). The nucleotide sequences of the small subunit ribosomal RNA gene (SSU rDNA; 2176 bp), large subunit ribosomal RNA gene (1365 bp) and glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH; 843 bp) of the present isolates were characterized to clarify their molecular phylogenetic position in T. cruzi-like trypanosomes. The newly obtained SSU rDNA and gGAPDH nucleotide sequences showed the highest identities with Brazilian and European isolates of Trypanosoma dionisii of the T. cruzi clade, ranging between 99.4 and 99.7% or between 95.6 and 99.3% identities, respectively. Although multiple T. dionisii isolates from the North and South American continents showed the closest molecular genetic relatedness to the present Far East isolates, only short SSU rDNA segments of the former isolates were deposited. Therefore, a definitive conclusion cannot be made until full nucleotide sequencing of at least the American isolates' SSU rDNA is available. This is the first confirmation of a Far East distribution of T. dionisii, demonstrating a wide geographical distribution of the species in the Eurasian and American continents with a limited nucleotide variation.

New host records of monacanthid fish for three Kudoa spp. (K. septempunctata, K. thyrsites, and K. shiomitsui) prevalent in the olive flounder (Paralichthys olivaceus), with the description of K. parathyrsites n. sp. from a black scraper (Thamnaconus modestus).

Kudoa septempunctata (Myxosporean: Multivalvulida) is known as a cause of foodborne disease associated with consumption of raw flesh of the olive flounder (Paralichthys olivaceus). Knowledge of its life cycle, particularly alternate annelid hosts and reservoirs or susceptible fish hosts in natural waters, may facilitate disease control in aquaculture farms. Our recent survey of myxosporean infection in monacanthid fish in natural waters around Japan revealed infection with three kudoid species prevalent in the olive flounder, i.e., K. septempunctata, Kudoa thyrsites, and Kudoa shiomitsui. Of the 51 black scrapers (Thamnaconus modestus) examined, five fish were infected: two fish with K. septempunctata and three with K. thyrsites. One of the fish infected with K. septempunctata was also infected with a K. thyrsites-like species. One of the 17 threadsail filefish (Stephanolepis cirrhifer) and two of four unicorn leatherjackets (Aluterus monoceros) were parasitized with K. shiomitsui. Three modest filefish (Thamnaconus modestoides) had no kudoid infection. K. septempunctata from a black scraper fished in the Inland Sea of Japan off Yamaguchi had 6-8 (predominantly 7) shell valves/polar capsules, whereas K. septempunctata found in another black scraper from the Sea of Japan off Tottori had 5 or 6 (predominantly 6). However, the two isolates displayed identical 18S and 28S ribosomal RNA gene (rDNA) nucleotide sequences, which were also identical to the isolates from the olive flounder. K. thyrsites from the Inland Sea of Japan off Yamaguchi and Sea of Japan off Tottori and K. shiomitsui from the Sea of Japan off Shimane and western Pacific Ocean off Kochi were also morphologically and genetically characterized. They were found to be coincident with the previous reports from olive flounders. Furthermore, the K. thyrsites-like species found in a black scraper from the Inland Sea of Japan off Yamaguchi was morphologically and genetically characterized; a new species, Kudoa parathyrsites n. sp., is erected for this species. The relationships of the new species with K. thyrsites and related species as well as those of K. shiomitsui with Kudoa pericardialis and related species parasitizing the pericardium are briefly discussed.

Morphological and molecular genetic characterization of two Kudoa spp., K. musculoliquefaciens, and K. pleurogrammi n. sp. (Myxosporea: Multivalvulida), causing myoliquefaction of commercial marine fish.

Genetic characterization of myxosporean species, including members of the genus Kudoa, has advanced dramatically throughout the last decade. This is in stark contrast to those species described further back in time. Kudoa musculoliquefaciens described from the jellied muscle of swordfish, Xiphias gladius, in the western Pacific Ocean off the Sanriku Coast, northern Japan, is one such species. In the present study, multiple pseudocysts (0.66-1.35 mm average length and 0.06-0.10 mm average width) containing K. musculoliquefaciens spores were collected from three host groups: muscle blocks of swordfish caught in the western Pacific Ocean off the Sanriku Coast, or the northern Indian Ocean, and Indo-Pacific sailfish, Istiophorus platypterus, in the western Pacific Ocean off Kochi, western Japan. Subspherical K. musculoliquefaciens spores, 8.0-10.3 µm in width, 7.3-10.0 µm in thickness, 6.4-7.9 µm in sutural thickness, and 5.5-8.1 µm in length, had four subspherical polar capsules, 2.8-4.0 µm in length by 2.2-3.2 µm in width. The kudoid spores found in the different host groups showed morphometric variations to some extent but had essentially identical nucleotide sequences of the ribosomal RNA gene (rDNA), closest to those of Kudoa hemiscylli or Kudoa carcharhini recorded from elasmobranchs in the Indo-Pacific Ocean. Another kudoid species, Kudoa pleurogrammi n. sp., was recorded from the jellied and normal muscles of Atka mackerel, Pleurogrammus monopterygius and Pleurogrammus azonus, fished in the northern Pacific Ocean or northern Sea of Japan. Subquadrate spores found in round-ended pseudocysts (1.15-3.85 mm in length and 0.11-0.26 mm in width) in myofibers were 8.2-9.1 µm in width, 7.1-8.2 µm in thickness, 5.4-7.7 µm in sutural thickness, and 5.6-6.8 µm in length, with four ovoid polar capsules, 2.7-2.9 µm in length by 1.4-2.0 µm in width. Kudoid spores from both jellied and normal muscles or different host fish species had identical 18S or 28S rDNA nucleotide sequences. Thus, molecular genetic characterization of kudoid species with the potential to induce post-mortem myoliquefaction will facilitate the reliable and specific identification of myxosporeans found in either jellied or normal muscles of important commercial fish.