Diversity and prevalence of intestinal parasites of zoonotic potential in animal hosts from different biomes in the central region of Brazil.

The Brazilian state of Mato Grosso comprises the biomes Cerrado, Pantanal, and Amazon rainforest. The animals that make up the biodiversity of these biomes have an ongoing relationship with domestic animals. This study aimed to describe the prevalence and diversity of intestinal parasites in different class of animal hosts, domestic and wild, from the three biomes of Brazil's central region, during 2019. We analyzed animal faecal samples by sedimentation tests and centrifugal flotation in sucrose. The Shannon index test to calculate diversity and equitability, was used. In the 280 samples analyzed, 79.64% was positive. Domestic animals showed positivity of 87% and wild animals of 51%, being mammals the most prevalent, with 81%. Blastocystis sp. was the most prevalent protozoan, and Ascaris sp. of the helminths. Shannon's indexes were higher for the Pantanal. Overall, pigs, cows, and chickens had high prevalence values and diversity and equitability of enteroparasites. Due to the great diversity of parasites found in pigs, cows and chickens, these animals can play an important role in the transmission and maintenance of the infection to other mammals, including humans.

Pan-American Trypanosoma (Megatrypanum) trinaperronei n. sp. in the white-tailed deer Odocoileus virginianus Zimmermann and its deer ked Lipoptena mazamae Rondani, 1878: morphological, developmental and phylogeographical characterisation.

BACKGROUND: The subgenus Megatrypanum Hoare, 1964 of Trypanosoma Gruby, 1843 comprises trypanosomes of cervids and bovids from around the world. Here, the white-tailed deer Odocoileus virginianus (Zimmermann) and its ectoparasite, the deer ked Lipoptena mazamae Rondani, 1878 (hippoboscid fly), were surveyed for trypanosomes in Venezuela. RESULTS: Haemoculturing unveiled 20% infected WTD, while 47% (7/15) of blood samples and 38% (11/29) of ked guts tested positive for the Megatrypanum-specific TthCATL-PCR. CATL and SSU rRNA sequences uncovered a single species of trypanosome. Phylogeny based on SSU rRNA and gGAPDH sequences tightly cluster WTD trypanosomes from Venezuela and the USA, which were strongly supported as geographical variants of the herein described Trypanosoma (Megatrypanum) trinaperronei n. sp. In our analyses, the new species was closest to Trypanosoma sp. D30 from fallow deer (Germany), both nested into TthII alongside other trypanosomes from cervids (North American elk and European fallow, red and sika deer), and bovids (cattle, antelopes and sheep). Insights into the life-cycle of T. trinaperronei n. sp. were obtained from early haemocultures of deer blood and co-culture with mammalian and insect cells showing flagellates resembling Megatrypanum trypanosomes previously reported in deer blood, and deer ked guts. For the first time, a trypanosome from a cervid was cultured and phylogenetically and morphologically (light and electron microscopy) characterised. CONCLUSIONS: In the analyses based on SSU rRNA, gGAPDH, CATL and ITS rDNA sequences, neither cervids nor bovids trypanosomes were monophyletic but intertwined within TthI and TthII major phylogenetic lineages. One host species can harbour more than one species/genotype of trypanosome, but each trypanosome species/genotype was found in a single host species or in phylogenetically closely related hosts. Molecular evidence that L. mazamae may transmit T. trinaperronei n. sp. suggests important evolutionary constraints making tight the tripartite T. trinaperronei-WTD-deer ked association. In a plausible evolutionary scenario, T. trinaperronei n. sp. entered South America with North American white-tailed deer at the Pliocene-Pleistocene boundary following the closure of the Panama Isthmus.

Expanding our knowledge on African trypanosomes of the subgenus Pycnomonas: A novel Trypanosoma suis-like in tsetse flies, livestock and wild ruminants sympatric with Trypanosoma suis in Mozambique.

Among the subgenera of African tsetse-transmitted trypanosomes pathogenic to livestock, the least known is the subgenus Pycnomonas, which contains a single species, Trypanosoma suis (TSU), a pathogen of domestic pigs first reported in 1905 and recently rediscovered in Tanzania and Mozambique. Analysis by Fluorescent Fragment Length Barcoding (FFLB) revealed an infection rate of 20.3% (108 out of 530 tsetse flies) in a recent study in the Gorongosa and Niassa wildlife reserves in Mozambique, and demonstrated two groups of Pycnomonas trypanosomes: one (14.1%, 75 flies) showing an FFLB profile identical to the reference TSU from Tanzania, and the other (6.2%, 33 flies) differing slightly from reference TSU and designated Trypanosoma suis-like (TSU-L). Phylogenetic analyses tightly clustered TSU and TSU-L from Mozambique with TSU from Tanzania forming the clade Pycnomonas positioned between the subgenera Trypanozoon and Nannomonas. Our preliminarily exploration of host ranges of Pycnomonas trypanosomes revealed TSU exclusively in warthogs while TSU-L was identified, for the first time for a member of the subgenus Pycnomonas, in ruminants (antelopes, Cape buffalo, and in domestic cattle and goats). The preferential blood meal sources of tsetse flies harbouring TSU and TSU-L were wild suids, and most of these flies concomitantly harboured the porcine trypanosomes T. simiae, T. simiae Tsavo, and T. godfreyi. Therefore, our findings support the link of TSU with suids while TSU-L remains to be comprehensively investigated in these hosts. Our results greatly expand our knowledge of the diversity, hosts, vectors, and epidemiology of Pycnomonas trypanosomes. Due to shortcomings of available molecular diagnostic methods, a relevant cohort of trypanosomes transmitted by tsetse flies to ungulates, especially suids, has been neglected or most likely misidentified. The method employed in the present study enables an accurate discrimination of trypanosome species and genotypes and, hence, a re-evaluation of the "lost" subgenus Pycnomonas and of porcine trypanosomes in general, the most neglected group of African trypanosomes pathogenic to ungulates.

Zoonotic Trypanosomes in Rats and Fleas of Venezuelan Slums.

Rattus spp. are reservoirs of many human zoonoses, but their role in domestic transmission cycles of human trypanosomiasis is underestimated. In this study, we report trypanosome-infected Rattus norvegicus and Rattus rattus in human dwellings in slums neighboring Maracay, a large city near Caracas, the capital of Venezuela. Blood samples of R. norvegicus and R. rattus examined by PCR and FFLB (fluorescent fragment length barcoding) revealed a prevalence of 6.3% / 31.1% for Trypanosoma lewisi (agent of rat- and flea-borne human emergent zoonosis), and 10.5% / 24.6% for Trypanosoma cruzi (agent of Chagas disease). Detection in flea guts of T. lewisi (76%) and, unexpectedly, T. cruzi (21.3%) highlighted the role of fleas as carriers and vectors of these trypanosomes. A high prevalence of rats infected with T. lewisi and T. cruzi and respective flea and triatomine vectors poses a serious risk of human trypanosomiasis in Venezuelan slums. Anthropogenic activities responsible for growing rat and triatomine populations within human dwellings drastically increased human exposure to trypanosomes. This scenario has allowed for the reemergence of Chagas disease as an urban zoonosis in Venezuela and can propitiate the emergence of atypical T. lewisi infection in humans.

Serological evidence of Coxiella burnetii infection in cattle and farm workers: is Q fever an underreported zoonotic disease in Ecuador?

Background: Q fever is an underreported zoonotic disease of cattle and men in most countries of the world. Very little information about the prevalence of Coxiella burnetii infection in animals and humans comes from South and Central America and systematic studies are lacking. Methods: A seroprevalence survey for Q fever amongst cattle, farm workers and students was conducted in Ecuador using a commercial ELISA kit. Results: Survey results showed an unexpectedly high prevalence of Coxiella burnetii antibodies in dairy cattle (43%) and in farm workers (34%). In addition, a clinical case in a human of acute Q fever in the convalescent stage was detected. Conclusion: We conclude that the disease is endemic in Ecuador but is overlooked by medical and laboratory personnel. Q fever should be considered a public health issue in Ecuador and further research into the clinical relevance of this infection is recommended.

Shared species of crocodilian trypanosomes carried by tabanid flies in Africa and South America, including the description of a new species from caimans, Trypanosoma kaiowa n. sp.

BACKGROUND: The genus Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown. RESULTS: Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade (terrestrial lineage) forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi (trypanosomes from caimans, African and Brazilian tabanids and tsetse flies); Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp. CONCLUSIONS: Prior to this description of Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.

Genetic diversity of trypanosomes pathogenic to livestock in tsetse flies from the Nech Sar National Park in Ethiopia: A concern for tsetse suppressed area in Southern Rift Valley?

In Ethiopia, home to the largest African herd of cattle, animal trypanosomiasis is a major constraint to the efforts made for food self-sufficiency. We searched for trypanosomes in tsetse flies caught in the Nech Sar National Park (NSNP), Southern Rifty Valley, Ethiopia, at the district of Arba Minch where intensive tsetse control is successfully improving cattle productivity. Despite narrow geographical and temporal scales of our survey, we found a remarkable diversity of trypanosomes using the sensitive and discriminative method of fluorescent fragment length barcoding. We also found a high density of Glossina pallidipes (47.8 flies/trap/day) showing relevant cytochrome oxidase I gene variability. The identification of blood meal sources through cytochrome b gene sequences revealed cattle and warthog as preferential ungulate hosts of tsetse flies in the study area. Our survey identified trypanosomes in 38% of the 287 flies examined (42% of proboscises and 32% of guts), and the following infection rates for each species: Trypanosoma vivax 23%, T. simiae 23%, T. congolense 22%, T. theileri 19.9%, T. (Trypanozoon) spp. 10.5%, T. godfreyi 9.4%, T. simiae Tsavo 6.3%, and mixed infections in proboscises (30%) and guts (61%). Phylogenetic analysis revealed T. vivax of the "West African-South American" genotype, T. congolense of Savannah (16.7%), Kilifi (3.5%) and Forest (2.1%) lineages, and new genotypes of T. simiae. To our knowledge, this is the first survey of trypanosomes in the NSNP, and the most comprehensive molecular characterisation of trypanosomes in tsetse flies of Ethiopia, including the comparison with samples from West and other East African countries. Our results support the diversification of T. vivax in East Africa, and the dispersion of the genotype herein identified in Ethiopia across West Africa and then in South America. Altogether, tsetse density and infection rate, repertoire of trypanosomes and feeding behavior indicate a high risk of transmission of trypanosomes pathogenic to ungulates by tsetse flies from the NSNP, a hotspot of tsetse infestation and trypanosome diversity. Our findings reinforce the need for constant surveillance, and the reliance on community efforts to prevent reinvasion of tsetse and animal trypanosomiasis in suppressed areas of Southern Rift Valley.

Shared species of crocodilian trypanosomes carried by tabanid flies in Africa and South America, including the description of a new species from caimans, Trypanosoma kaiowa n. sp.

Background The genus Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown. Results Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade (terrestrial lineage) forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi (trypanosomes from caimans, African and Brazilian tabanids and tsetse flies); Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp. Conclusions Prior to this description of Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.

Shared species of crocodilian trypanosomes carried by tabanid flies in Africa and South America, including the description of a new species from caimans, Trypanosoma kaiowa n. sp.

Background The genus Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown. Results Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade (terrestrial lineage) forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi (trypanosomes from caimans, African and Brazilian tabanids and tsetse flies); Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp. Conclusions Prior to this description of Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.

Remarkable richness of trypanosomes in tsetse flies (Glossina morsitans morsitans and Glossina pallidipes) from the Gorongosa National Park and Niassa National Reserve of Mozambique revealed by fluorescent fragment length barcoding (FFLB).

Trypanosomes of African wild ungulates transmitted by tsetse flies can cause human and livestock diseases. However, trypanosome diversity in wild tsetse flies remains greatly underestimated. We employed FFLB (fluorescent fragment length barcoding) for surveys of trypanosomes in tsetse flies (3086) from the Gorongosa National Park (GNP) and Niassa National Reserve (NNR) in Mozambique (MZ), identified as Glossina morsitans morsitans (GNP/NNR=77.6%/90.5%) and Glossina pallidipes (22.4%/9.5%). Trypanosomes were microscopically detected in 8.3% of tsetse guts. FFLB of gut samples revealed (GNP/NNR): Trypanosoma congolense of Savannah (27%/63%), Kilifi (16.7%/29.7%) and Forest (1.0%/0.3%) genetic groups; T. simiae Tsavo (36.5%/6.1%); T. simiae (22.2%/17.7%); T. godfreyi (18.2%/7.0%); subgenus Trypanozoon (20.2%/25.7%); T. vivax/T. vivax-like (1.5%/5.2%); T. suis/T. suis-like (9.4%/11.9%). Tsetse proboscises exhibited similar species composition, but most prevalent species were (GNP/NNR): T. simiae (21.9%/28%), T. b. brucei (19.2%/31.7%), and T. vivax/T. vivax-like (19.2%/28.6%). Flies harboring mixtures of trypanosomes were common (~ 64%), and combinations of more than four trypanosomes were especially abundant in the pristine NNR. The non-pathogenic T. theileri was found in 2.5% while FFLB profiles of unknown species were detected in 19% of flies examined. This is the first report on molecular diversity of tsetse flies and their trypanosomes in MZ; all trypanosomes pathogenic for ungulates were detected, but no human pathogens were detected. Overall, two species of tsetse flies harbor 12 species/genotypes of trypanosomes. This notable species richness was likely uncovered because flies were captured in wildlife reserves and surveyed using the method of FFLB able to identify, with high sensitivity and accuracy, known and novel trypanosomes. Our findings importantly improve the knowledge on trypanosome diversity in tsetse flies, revealed the greatest species richness so far reported in tsetse fly of any African country, and indicate the existence of a hidden trypanosome diversity to be discovered in African wildlife protected areas.

Diagnosis and genetic analysis of the worldwide distributed Rattus-borne Trypanosoma (Herpetosoma) lewisi and its allied species in blood and fleas of rodents.

Trypanosoma (Herpetosoma) lewisi is a cosmopolitan parasite of rodents strongly linked to the human dispersal of Rattus spp. from Asia to the rest of the world. This species is highly phylogenetically related to trypanosomes from other rodents (T. lewisi-like), and sporadically infects other mammals. T. lewisi may opportunistically infect humans, and has been considered an emergent rat-borne zoonosis associated to poverty. We developed the THeCATL-PCR based on Cathepsin L (CATL) sequences to specifically detect T. (Herpetosoma) spp., and assess their genetic diversity. This method exhibited high sensitivity using blood samples, and is the first molecular method employed to search for T. lewisi in its flea vectors. THeCATL-PCR surveys using simple DNA preparation from blood preserved in ethanol or filter paper detected T. lewisi in Rattus spp. from human dwellings in South America (Brazil and Venezuela), East Africa (Mozambique), and Southeast Asia (Thailand, Cambodia and Lao PDR). In addition, native rodents captured in anthropogenic and nearby human settlements in natural habitats harbored T. (Herpetosoma) spp. PCR-amplified CATL gene fragments (253bp) distinguish T. lewisi and T. lewisi-like from other trypanosomes, and allow for assessment of genetic diversity and relationships among T. (Herpetosoma) spp. Our molecular surveys corroborated worldwide high prevalence of T. lewisi, incriminating Mastomys natalensis as an important carrier of this species in Africa, and supported its spillover from invader Rattus spp. to native rodents in Brazil and Mozambique. THeCATL-PCR provided new insights on the accurate diagnosis and genetic repertoire of T. (Herpetosoma) spp. in rodent and non-rodent hosts, revealing a novel species of this subgenus in an African gerbil. Phylogenetic analysis based on CATL sequences from T. (Herpetosoma) spp. and other trypanosomes (amplified using pan-trypanosome primers) uncovered rodents harboring, beyond mammal trypanosomes of different subgenera, some species that clustered in the lizard-snake clade of trypanosomes.

New insights from Gorongosa National Park and Niassa National Reserve of Mozambique increasing the genetic diversity of Trypanosoma vivax and Trypanosoma vivax-like in tsetse flies, wild ungulates and livestock from East Africa.

BACKGROUND: Trypanosoma (Duttonella) vivax is a major pathogen of livestock in Africa and South America (SA), and genetic studies limited to small sampling suggest greater diversity in East Africa (EA) compared to both West Africa (WA) and SA. METHODS: Multidimensional scaling and phylogenetic analyses of 112 sequences of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) gene and 263 sequences of the internal transcribed spacer of rDNA (ITS rDNA) were performed to compare trypanosomes from tsetse flies from Gorongosa National Park and Niassa National Reserve of Mozambique (MZ), wild ungulates and livestock from EA, and livestock isolates from WA and SA. RESULTS: Multidimensional scaling (MDS) supported Tvv (T. vivax) and TvL (T. vivax-like) evolutionary lineages: 1) Tvv comprises two main groups, TvvA/B (all SA and WA isolates plus some isolates from EA) and TvvC/D (exclusively from EA). The network revealed five ITS-genotypes within Tvv: Tvv1 (WA/EA isolates), Tvv2 (SA) and Tvv3-5 (EA). EA genotypes of Tvv ranged from highly related to largely different from WA/SA genotypes. 2) TvL comprises two gGAPDH-groups formed exclusively by EA sequences, TvLA (Tanzania/Kenya) and TvLB-D (MZ). This lineage contains more than 11 ITS-genotypes, seven forming the lineage TvL-Gorongosa that diverged from T. vivax Y486 enough to be identified as another species of the subgenus Duttonella. While gGAPDH sequences were fundamental for classification at the subgenus, major evolutionary lineages and species levels, ITS rDNA sequences permitted identification of known and novel genotypes. CONCLUSIONS: Our results corroborate a remarkable diversity of Duttonella trypanosomes in EA, especially in wildlife conservation areas, compared to the moderate diversity in WA. Surveys in wilderness areas in WA may reveal greater diversity. Biogeographical and phylogenetic data point to EA as the place of origin, diversification and spread of Duttonella trypanosomes across Africa, providing relevant insights towards the understanding of T. vivax evolutionary history.

Trypanosoma vivax in water buffalo of the Venezuelan Llanos: An unusual outbreak of wasting disease in an endemic area of typically asymptomatic infections.

Trypanosoma vivax has been associated with asymptomatic infections in African and South American buffalo. In this study, T. vivax was analyzed in water buffalo (Bubalus bubalis) from Venezuela in a molecular survey involving 293 blood samples collected from 2006 to 2015 across the Llanos region. Results demonstrated constant infections (average 23%) during the years analyzed. In general, animals were healthy carriers of T. vivax with low levels of parasitemia and were diagnosed exclusively by TviCATL-PCR. However, an outbreak of severe acute infections mostly in dairy animals was reported during a prolonged drought affecting 30.4% of a buffalo herd (115 animals examined). During the outbreak, animals exhibiting anemia and neurological disorders developed fatal infections, and 7% of the herd died within nine months before treatment against trypanosomosis. Microsatellite locus genotyping (MLG) of T. vivax samples before and during the outbreak revealed similar genotypes, but outbreak isolates exhibited the most divergent MLG. Venezuelan samples from symptomless and sick buffalo did not share the MLGs previously detected in asymptomatic Brazilian buffalo. Trypanosoma evansi was not detected in the herd examined during the outbreak. However, as expected Babesia sp. (62.6%) and Anaplasma sp. (55.6%) infections were highly prevalent in asymptomatic buffalo in the studied areas. This is the first South American outbreak of highly lethal acute T. vivax infections in water buffalo. Our results suggest that chronically infected and asymptomatic buffalo living in areas of enzootic equilibrium can develop symptomatic/lethal disease triggered by stressful scarcity of green forage and water during long droughts, inappropriate management of herds and likely concomitant anaplasmosis and babesiosis. Altogether, these factors weaken buffalo immune defenses, allowing T. vivax to proliferate and, consequently, allowing for progression to wasting disease.

Field and experimental symptomless infections support wandering donkeys as healthy carriers of Trypanosoma vivax in the Brazilian Semiarid, a region of outbreaks of high mortality in cattle and sheep.

BACKGROUND: The Brazilian Semiarid is the home of the largest herd of donkeys in South America and of outbreaks of Trypanosoma vivax infection of high mortality in dairy cattle and sheep. For a comprehensive understanding of the underlying mechanisms of these outbreaks and epidemiological role of donkeys, we surveyed for T. vivax in wandering donkeys and follow the experimental infection of donkeys and sheep with a highly virulent isolate from the Semiarid. METHODS: Blood samples from 180 randomly selected wandering donkeys from the Brazilian Semiarid region were employed for PCV and parasitemia assessments and tested using the T. vivax-specific TviCATL-PCR assay. PCR-amplifed Cathepsin L (CATL) sequences were employed for genotyping and phylogenetic analysis. Four wandering donkeys were experimentally infected with a T. vivax isolate obtained during an outbreak of high mortality in the Semiarid; the control group consisted of two non-inoculated donkeys. RESULTS: We detected T. vivax in 30 of 180 wandering donkeys (16.6 %) using TviCATL-PCR. The prevalence was higher during the dry (15.5 %) than the wet season (1.1 %) and more females (23.1 %) than males (8.9 %) were infected. All the PCR-positive donkeys lacked patent parasitemia and showed normal values of body condition score (BCS) and packed cell volume (PCV). To evaluate the probable tolerance of donkeys to T. vivax, we inoculated five donkeys with a highly virulent isolate (TviBrRp) from the Semiarid. All inoculated donkeys became PCR-positive, but their parasitemia was always subpatent. A control goat inoculated with TviBrRp showed increasing parasitemia concurrently with fever, declining PCV, tachycardia, mucous membrane pallor, enlarged lymph nodes and anorexia. None of these signs were observed in donkeys. However, T. vivax from wandering donkeys shared identical or highly similar genotypes (identified by Cathepsin L sequences) with isolates from cattle and sheep outbreaks of acute disease in the Semiarid. CONCLUSIONS: This is the first report of T. vivax in donkeys in Brazil and, to our knowledge, the first experimental infection of donkeys with T. vivax. The symptomless field and experimental infections corroborated that donkeys are more tolerant to T. vivax than other livestock species as shown in African countries. Therefore, farmers, veterinaries and control programmes should be aware of healthy carrier donkeys as a possible source of T. vivax for susceptible livestock species in the Brazilian Semiarid.

Microsatellite analysis supports clonal propagation and reduced divergence of Trypanosoma vivax from asymptomatic to fatally infected livestock in South America compared to West Africa.

BACKGROUND: Mechanical transmission of the major livestock pathogen Trypanosoma vivax by other biting flies than tsetse allows its spread from Africa to the New World. Genetic studies are restricted to a small number of isolates and based on molecular markers that evolve too slowly to resolve the relationships between American and West African populations and, thus, unable us to uncover the recent history of T. vivax in the New World. METHODS: T. vivax genetic diversity, population structure and the source of outbreaks was investigated through the microsatellite multiloci (7 loci) genotype (MLGs) analysis in South America (47isolates from Brazil, Venezuela and French Guiana) and West Africa (12 isolates from The Gambia, Burkina Faso, Ghana, Benin and Nigeria). Relationships among MLGs were explored using phylogenetic, principal component and STRUCTURE analyses. RESULTS: Although closely phylogenetically related, for the first time, genetic differences were detected between T. vivax isolates from South America (11 genotypes/47 isolates) and West Africa (12 genotypes/12 isolates) with no MLGs in common. Diversity was far greater across West Africa than in South America, where genotypes from Brazil (MLG1-6), Venezuela (MLG7-10) and French Guiana (MLG11) shared similar but not identical allele composition. No MLG was exclusive to asymptomatic (endemic areas) or sick (outbreaks in non-endemic areas) animals, but only MLGs1, 2 and 3 were responsible for severe haematological and neurological disorders. CONCLUSIONS: Our results revealed closely related genotypes of T. vivax in Brazil and Venezuela, regardless of endemicity and clinical conditions of the infected livestock. The MLGs analysis from T. vivax across SA and WA support clonal propagation, and is consistent with the hypothesis that the SA populations examined here derived from common ancestors recently introduced from West Africa. The molecular markers defined here are valuable to assess the genetic diversity, to track the source and dispersion of outbreaks, and to explore the epidemiological and pathological significance of T. vivax genotypes.

Congopain genes diverged to become specific to Savannah, Forest and Kilifi subgroups of Trypanosoma congolense, and are valuable for diagnosis, genotyping and phylogenetic inferences.

Trypanosoma congolense is the most important agent of nagana, a wasting livestock trypanosomosis in sub-Saharan Africa. This species is a complex of three subgroups (Savannah, Forest and Kilifi) that differ in virulence, pathogenicity, drug resistance, vectors, and geographical distribution. Congopain, the major Cathepsin L-like cysteine protease (CP2) of T. congolense, has been extensively investigated as a pathogenic factor and target for drugs and vaccines, but knowledge about this enzyme is mostly restricted to the reference strain IL3000, which belongs to the Savannah subgroup. In this work we compared sequences of congopain genes from IL3000 genome database and isolates of the three subgroups of T. congolense. Results demonstrated that the congopain genes diverged into three subclades consistent with the three subgroups within T. congolense. Laboratory and field isolates of Savannah exhibited a highly polymorphic repertoire both inter- and intra-isolates: sequences sharing the archetypical catalytic triad clustered into SAV1-SAV3 groups, whereas polymorphic sequences that, in general, exhibited unusual catalytic triad (variants) assigned to SAV4 or not assigned to any group. Congopain homologous genes from Forest and Kilifi isolates showed, respectively, moderate and limited diversity. In the phylogenetic tree based on congopain and homologues, Savannah was closer to Forest than to Kilifi. All T. congolense subgroup nested into a single clade, which together with the sister clade formed by homologues from Trypanosoma simiae and Trypanosoma godfreyi formed a clade supporting the subgenus Nannomonas. A single PCR targeting congopain sequences was developed for the diagnosis of T. congolense isolates of the three subgroups. Our findings demonstrated that congopain genes are valuable targets for the diagnosis, genotyping, and phylogenetic and taxonomic inferences among T. congolense isolates and other members of the subgenus Nannomonas.

Prevalencia de trypanosoma spp. mediante ELISA e inmunofluorescencia indirecta en tres rebaños de búfalos de agua del estado Cojedes, Venezuela / Prevalence of trypanosoma spp. by ELISA and indirect fluorescent antibody test in three water buffaloes herds from the State of Cojedes, Venezuela

Animal trypanosomiasis is a disease caused by parasites of the genus Trypanosome. This malady is widely distributed in many countries, located in tropical and subtropical areas of the world where blood-sucking flies are present. Water buffaloes are important domestic animals used for meat and milk production, and draught power. Buffalo herds are raised in areas where trypanosomiasis is endemic. In Venezuela, the buffalo industry is becoming a very important and common livestock. However, animals imported from non-endemic areas may suffer severe infections. The development of methods which ensure an efficient epidemiological surveillance against this disease is of great relevance. The immunological tests are of great importance for this purpose, because of the low sensitivity of the current parasitological methods, due to the low parasite burden that occur in subclinical and chronic infections caused by trypanosomes. To estimate the serological prevalence of trypanosome in water buffaloes, an enzyme-linked immunosorbent assay (ELISA) was used in buffalo samples of healthy animals from the municipalities of Rómulo Gallegos, Ricaurte and Girardot, in the State of Cojedes, Venezuela. Additionally, samples were also assessed with the indirect fluorescence antibody test (IFAT) and the microhematocrit test (MHCT). A total of 180 blood samples, none of which had an active parasitemia by TMC, were assessed. The prevalence determined by ELISA was 45.56%, which was significantly (P<0.05) higher than that obtained by IFAT (28.89%). The results of the experiments showed a moderate Kappa index of concordance of 0.45 (95% CI: 0.31-0.58); whereas the concordance value for both tests was 73.33%. Both the sensitivity and specificity of ELISA, compared to the IFAT, was 82.69% and 69.53%, respectively. The predictive positive and negative values were 52.44% and 90.82%, respectively. The findings suggest an endemic condition, with moderate infection values caused by Trypanosoma spp. in buffaloes from these regions of Venezuela and show, for the first time, the usefulness of ELISA for epidemiological studies of trypanosomiasis.

The phylogeography of trypanosomes from South American alligatorids and African crocodilids is consistent with the geological history of South American river basins and the transoceanic dispersal of Crocodylus at the Miocene.

BACKGROUND: Little is known about the diversity, phylogenetic relationships, and biogeography of trypanosomes infecting non-mammalian hosts. In this study, we investigated the influence of host species and biogeography on shaping the genetic diversity, phylogenetic relationship, and distribution of trypanosomes from South American alligatorids and African crocodilids. METHODS: Small Subunit rRNA (SSU rRNA) and glycosomal Glyceraldehyde Phosphate Dehydrogenase (gGAPDH) genes were employed for phylogenetic inferences. Trypanosomes from crocodilians were obtained by haemoculturing. Growth behaviour, morphology, and ultrastructural features complement the molecular description of two new species strongly supported by phylogenetic analyses. RESULTS: The inferred phylogenies disclosed a strongly supported crocodilian-restricted clade comprising three subclades. The subclade T. grayi comprised the African Trypanosoma grayi from Crocodylus niloticus and tsetse flies. The subclade T. ralphi comprised alligatorid trypanosomes represented by Trypanosoma ralphi n. sp. from Melanosuchus niger, Caiman crocodilus and Caiman yacare from Brazilian river basins. T. grayi and T. ralphi were sister subclades. The basal subclade T. terena comprised alligatorid trypanosomes represented by Trypanosoma terena n. sp. from Ca. yacare sharing hosts and basins with the distantly genetic related T. ralphi. This subclade also included the trypanosome from Ca. crocodilus from the Orinoco basin in Venezuela and, unexpectedly, a trypanosome from the African crocodilian Osteolaemus tetraspis. CONCLUSION: The close relationship between South American and African trypanosomes is consistent with paleontological evidence of recent transoceanic dispersal of Crocodylus at the Miocene/Pliocene boundaries (4-5 mya), and host-switching of trypanosomes throughout the geological configuration of South American hydrographical basins shaping the evolutionary histories of the crocodilians and their trypanosomes.

Follicular degeneration in the ovaries of goats experimentally infected with Trypanosoma vivax from the Brazilian semi-arid region.

Infection by Trypanosoma vivax and other African trypanosomes plays an important role in reproductive disorders in male and female livestock. Outbreaks of T. vivax in the semi-arid region of northeastern Brazil are characterized by wasting disease in cattle, sheep and goats with hematological, cardiac and nervous compromises in addition to reproductive failures. Similar to reports from Africa, we previously observed a reduction in fertility rates and severe testicular degeneration and epididymitis in male sheep infected with T. vivax from this region. Although anestrus is frequently reported in goats and sheep infected with T. vivax, the effects of this infection on the female reproductive organs need clarification. In this study, we addressed this issue through a histopathological evaluation of ovarian follicular morphology and classification in goats experimentally infected with a T. vivax isolate from the Brazilian semi-arid region. The infected animals presented typical clinical signs of trypanosomosis by T. vivax, including anemia, hyperthermia, pallor of the mucous membranes, enlarged lymph nodes, and progressive loss of weight. All the infected goats remained anestrus throughout the experimental period and exhibited important disturbances in the ovaries, evidenced by reduced size and a smooth surface without follicles or corpora lutea, and abnormal follicular development. In addition, through PCR, we detected T. vivax DNA in the ovarian tissues of the infected goats. Our findings contributed to understand the female reproductive failure associated with trypanosomosis caused by T. vivax.

Trypanosoma (Megatrypanum) melophagium in the sheep ked Melophagus ovinus from organic farms in Croatia: phylogenetic inferences support restriction to sheep and sheep keds and close relationship with trypanosomes from other ruminant species.

Trypanosoma (Megatrypanum) melophagium is a parasite of sheep transmitted by sheep keds, the sheep-restricted ectoparasite Melophagus ovinus (Diptera: Hippoboscidae). Sheep keds were 100% prevalent in sheep from five organic farms in Croatia, Southeastern Europe, whereas trypanosomes morphologically compatible with T. melophagium were 86% prevalent in the guts of the sheep keds. Multilocus phylogenetic analyses using sequences of small subunit rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase, spliced leader, and internal transcribed spacer 1 of the rDNA distinguished T. melophagium from all allied trypanosomes from other ruminant species and placed the trypanosome in the subgenus Megatrypanum. Trypanosomes from sheep keds from Croatia and Scotland, the only available isolates for comparison, shared identical sequences. All biologic and phylogenetic inferences support the restriction of T. melophagium to sheep and, especially, to the sheep keds. The comparison of trypanosomes from sheep, cattle, and deer from the same country, which was never achieved before this work, strongly supported the host-restricted specificity of trypanosomes of the subgenus Megatrypanum. Our findings indicate that with the expansion of organic farms, both sheep keds and T. melophagium may re-emerge as parasitic infections of sheep.