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The Indonesian island of Sulawesi is a globally significant biodiversity hotspot with substantial undescribed biota, particularly blood-borne parasites of endemic wildlife. Documenting the blood parasites of Sulawesi's murine rodents is the first fundamental step towards the discovery of pathogens likely to be of concern for the health and conservation of Sulawesi's endemic murines. We screened liver samples from 441 specimens belonging to 20 different species of murine rodents from 2 mountain ranges on Sulawesi, using polymerase chin reaction (PCR) primers targeting the conserved 18S rDNA region across the protozoan class Kinetoplastea. We detected infections in 156 specimens (10 host species) with a mean prevalence of 35.4% (95% confidence interval [CI] = 30.9-39.8%). Sequences from these samples identified 4 infections to the genus Parabodo, 1 to Blechomonas, and the remaining 151 to the genus Trypanosoma. Within Trypanosoma, we recovered 17 haplotypes nested within the Trypanosoma theileri clade infecting 117 specimens (8 host species) and 4 haplotypes nested within the Trypanosoma lewisi clade infecting 34 specimens (6 host species). Haplotypes within the T. theileri clade were related to regional Indo-Australian endemic trypanosomes, displayed geographic structuring but with evidence of long-term connectivity between mountains, and had substantial phylogenetic diversity. These results suggest T. theileri clade parasites are native to Sulawesi. Conversely, T. lewisi clade haplotypes were recovered from both endemic and introduced rodents, demonstrated complete geographic separation between clades, and had low genetic diversity. These results suggest that the T. lewisi clade parasites invaded Sulawesi recently and likely in 2 separate invasion events. Our results provide the first records of metakinetoplastids in Sulawesi's rodents and highlight the need for more extensive sampling for pathogens in this biodiversity hotspot., (© American Society of Parasitologists 2020.)

Trypanosoma evansi is the most widespread of the pathogenic salivarian trypanosomes; it causes a serious disease called surra that affects domestic animals such as camels, horses, and dogs, and often leads to reduced productivity and economic losses. Therefore, the objectives of the present study were to determine the prevalence rates of trypanosomiasis using 3 parasitological tests (wet blood film, Giemsa staining, and microhematocrit centrifugation technique) and polymerase chain reaction (PCR) among stray dogs from Riyadh Province, Saudi Arabia. In the current study, 117 dog blood samples collected from certain districts of Riyadh Province showed that 5 of 117 dogs (4.3%) were positive for the genus Trypanosoma. In addition, the findings indicated no effect of dog gender or age on parasite infection. For a more specific diagnosis, PCR amplification of the RoTat 1.2 VSG gene in 5 internal transcribed spacer1-positive samples diagnosed with Trypanosoma indicated that 2 were positive for RoTat 1.2 T. evansi. The absence of the RoTat 1.2 VSG gene in 3 of the 5 T. evansi-positive samples could be explained by the circulation of T. evansi type B in dogs from Saudi Arabia. Thus, this is the first study demonstrating T. evansi type B outside of Africa.

Fish trypanosomes are widely distributed in commercially important fish, with high prevalence in some Brazilian species. This study provides the first record of the isolation and in vitro maintenance of trypanosomes from Brazilian fish. We produced 49 trypanosome isolates from naturally infected catfish (Hypostomus affinis and Hypostomus luetkeni), using 9 different culture media (out of 31 tested). Trypanosomes were maintained in culture for at least 15 mo and were successfully cryopreserved. Culture forms-epimastigotes and short trypomastigotes-were capable of dividing in vitro. Our study is an important step in the investigation of ultrastructure, taxonomy, and phylogeny of trypanosomes from commercially important Brazilian fish.

Trypanosoma caninum constitutes the most recent trypanosomatid species infecting dogs in Brazil. Due to the limited data available about this parasite, this study aims to disclose clinical and laboratory findings from 14 dogs naturally infected. The dogs were diagnosed during a cross-sectional survey in Cuiabá (Mato Grosso, Brazil) and followed up at an interval of 3, 6, and 12 mo in order to evaluate the clinical evolution and to investigate the parasite, the DNA, or both in different biological samples (intact skin, cutaneous scar, blood, bone marrow, and lymph node aspirate) by parasitological (culture and smear exam) and molecular (DNA-based tests) methods. Specific anti-T. caninum and anti-Leishmania antibody production was also evaluated. Ten of 14 dogs infected by T. caninum showed a good general state at the time of diagnosis, and this status did not vary during the follow-up. Anti-T. caninum and anti-Leishmania IgG antibodies were detected by IFAT in 10 and 2 animals, respectively. Concomitant infection by Leishmania chagasi was confirmed in 2 dogs, indicating an overlap of endemic areas in Cuiabá. Trypanosoma caninum (parasite or DNA) was found only in the intact skin in all animals examined. Our results suggest that T. caninum infection can be manifested as an asymptomatic case with low humoral immune response.

In the order Chiroptera, more than 30 trypanosome species belonging to the subgenera Herpetosoma, Schizotrypanum, Megatrypanum, and Trypanozoon have been described. The species Trypanosoma cruzi , Trypanosoma cruzi marinkellei, and Trypanosoma dionisii are the most common in bats and belong to the Schizotrypanum subgenus. Bats from 2 different biomes, Pantanal and Amazonia/Cerrado in the state of Mato Grosso, Brazil, were evaluated according to the presence of trypanosome parasites by means of hemoculture and PCR in primary samples (blood samples). A total of 211 bats from 20 different species were caught and the trypanosome prevalence, evaluated through hemoculture, was 9.0% (19), 15.5% (13), and 4.8% (6) in the municipalities of Confresa (Amazonia/Cerrado biome) and Poconé (Pantanal biome). Among the 123 primary samples obtained from the bats, only 3 (2.4%) were positive. Phylogenetic analysis using trypanosomatid barcoding (V7V8 region of SSU rDNA) identified all the isolates and primary samples as T. c. marinkellei. The sequences of the isolates were segregated according to the bat host genus or species and suggest that co-evolutionary patterns exist between hosts and parasites. Further studies in different Brazilian regions and biomes need to be conducted in order to gain real understanding of the diversity of trypanosomes in bats.

The purpose of this study was to verify the in vitro development of Trypanosoma sp. isolated from Leptodactylus ocellatus frogs under a new protocol using a biphasic medium composed of Novy, McNeal, and Nicolle (NNN) blood agar medium as a solid phase and liver infusion, brain heart infusion, and tryptose (LIBHIT) medium as a liquid phase. Blood forms, collected by cardiac puncture or after the maceration of different organs, were inoculated in culture tubes containing the biphasic medium composed by NNN and LIBHIT. Trypanosomes were observed 4 days postinoculation; most bloodstream trypomastigotes had differentiated into epimastigotes and amastigotes by this time. Trypomastigotes were again observed in older cultures (7 days). Parasites were successfully subcultured for 8 mo in this medium and successfully cryopreserved. The present study provides a new protocol medium for the isolation and culture of anuran trypanosomes.

Inferences about the evolution of host-parasitic relationships are often made based on the prevalence of avian malaria, which is usually estimated in a large sample of birds using either microscopic or molecular screening of blood samples. However, different techniques often have variable accuracy; thus, screening methodology can raise issues about statistical bias if method sensitivity varies systematically across parasites or hosts. To examine this possibility, published information was collected on the prevalence of species in 4 genera of avian blood parasites ( Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma) from various sources that used different tools. The data were tested to determine if the application of different methods provided different estimates for the same hosts. In these comparisons between the main methodologies, the PCR-based molecular methods were generally found to provide higher estimates for Plasmodium spp. prevalence than microscopic tools, while there was no significant tendency for such a trend in species of Haemoproteus and Leucocytozoon. When analyzing intraspecific variance of prevalence within molecular studies, some studies provided consistently higher estimates for Haemoproteus spp. prevalence than others, indicating that differences between studies can affect detected estimates. Within microscopic studies, surveys that examined more microscopic fields were more likely to report higher prevalence for Plasmodium spp. than those relying on fewer microscopic fields. Consequently, studies making comparisons across parasite genera and/or host species from different sources need to consider several types of bias originating from variation in method sensitivity.

In a survey of blood parasites in Ugandan frogs, 30 (17%) of 180 frogs were infected with at least 1 species of Hepatozoon or Trypanosoma, or with microfilariae. There were significant differences in the prevalence of parasitism among species, with parasitemia detected in only 3 of 9 species. The intensity of blood parasite infection ranged from 1 to 1,080 infected cells per 5,000 cells examined. Seasonal changes in the prevalence and intensity of parasitemia were not observed, nor was there any association between parasitemia and infection with the pandemic fungus Batrachochytrium dendrobatidis.

Peripheral blood from a ball python (Python reginus) imported from Ghana was cultured in Barbour-Stoenner-Kelly (BSK) medium for Borrelia spp. isolation, resulting in the prominent appearance of free, and clusters of, trypanosomes in a variety of morphological forms. The molecular phylogenetic characterization of these cultured trypanosomes, using the small subunit rDNA, indicated that this python was infected with a species closely related to Trypanosoma varani Wenyon, 1908, originally described in the Nile monitor lizard (Varanus niloticus) from Sudan. Furthermore, nucleotide sequences of glycosomal glyceraldehyde-3-phosphate dehydrogenase gene of both isolates showed few differences. Giemsa-stained blood smears, prepared from the infected python 8 mo after the initial observation of trypanosomes in hemoculture, contained trypomastigotes with a broad body and a short, free flagellum; these most closely resembled the original description of T. varani, or T. voltariae Macfie, 1919 recorded in a black-necked spitting cobra (Naja nigricollis) from Ghana. It is highly possible that lizards and snakes could naturally share an identical trypanosome species. Alternatively, lizards and snakes in the same region might have closely related, but distinct, Trypanosoma species as a result of sympatric speciation. From multiple viewpoints, including molecular phylogenetic analyses, reappraisal of trypanosome species from a wide range of reptiles in Africa is needed to clarify the relationship of recorded species, or to unmask unrecorded species.

The present study provides the first record of Trypanosoma chattoni Mathis and Leger, 1911, in a new host, Leptodactylus fuscus Schneider, 1799 (Anura, Leptodactylidae), and the occurrence of Trypanosoma rotatorium-like species in Leptodactylus chaquensis Cei, 1950. The anurans were captured in the State of Mato Grosso, Brazil. Blood samples were obtained by cardiac puncture, and blood smears were examined for the presence of hemoparasites. The Trypanosoma rotatorium-like species in this study refers to a short-bodied trypomastigote that has a conspicuous undulating membrane but lacks a free flagellum; T. chattoni refers to a monomorphic parasite that has a rounded body, a kinetoplast adjacent to the nucleus, and a short flagellum.

Of 2,122 marine fishes representing 36 species collected in the northeastern Pacific Ocean in the vicinity of Newport, Oregon from 1971 to 1973, 541 individuals (25.5%) representing 8 species (22.2%) were infected with hemoflagellates. Four morphologically distinct trypanosomes and 3 distinct trypanoplasms were found in fishes collected offshore, but no hemoflagellates were observed in fishes from Yaquina Bay estuary. Trypanosoma pacifica was found in English sole Parophrys vetulus, Pacific sanddab Citharichthys sordidus, and slender sole Lyopsetta exilis, and survived in 5 other species after intraperitoneal injection. Trypanosoma gargantua was found in big skate Raja binoculata, and the leech Orientobdella confluens was able to transmit the trypanosome in experimental conditions. Trypanosoma khani n. sp. occurred in P. vetulus, petrale sole Eopsetta jordani, and Dover sole Microstomus pacificus. Trypanosoma murmanense was found in L. exilis collected from 200 m, but not in L. exilis collected from 80 m. Trypanoplasma beckeri parasitized the cabezon Scorpaenichthys marmoratus. Trypanoplasma bobolsoni n. sp. was found in E. jordani, L. exilis, and P. vetulus, and survived in 2 other species after intraperitoneal injection. A distinct, but unnamed trypanoplasm, was found in P. vetulus.

To test the hypothesis that migrants infected with blood parasites arrive on the northern coast of the Gulf of Mexico in poorer condition than uninfected birds, we examined 1705 migrant passerine birds representing 54 species of 11 families from 2 Gulf Coast sites for blood parasites. Three hundred and sixty (21.1%) were infected with 1 or more species of 4 genera of blood parasites. The prevalence of parasites was as follows: Haemoproteus spp. (11.7%), Plasmodium spp. (6.7%), Leucocytozoon spp. (1.3%), and Trypanosoma spp. (1.2%). Both prevalence and density of Haemoproteus spp. infection varied among species. We found no relationship of gender or age with the prevalence of Haemoproteus spp. infection or Plasmodium spp. infection, with the exception of the orchard oriole (Icterus spurius) for which older birds were more likely to be infected with Haemoproteus spp. than younger birds. We also found that scarlet tanagers and summer tanagers infected with species of Haemoproteus have lower fat scores than uninfected individuals and that rose-breasted grosbeaks and Baltimore orioles infected with Haemoproteus spp. have a smaller mean body mass than uninfected individuals. Blood parasites do seem to pose a physiological cost for Neotropical migrant passerines and may be important components of the ecology of these species.

During May 1997, specimens of 7 species of anurans, that included 5 Phrynohyas venulosa Laurenti, 5 Rana forreri Boulenger, 7 Rana vaillanti Brucchi, 6 Eleutherodactylus fitzingeri Schimdt, 4 Smilisca baudinii Duméril and Bibron, 1 Leptodactylus melanonotus, and 3 Bufo marinus Linneaus, from the Guanacaste Conservation Area, Costa Rica were examined for blood parasites. Their hematozoan fauna included intraerythrocytic and intraleukocytic icosahedral viruses, a rickettsia (Aegyptianella sp.), 2 species of Hepatozoon, Lankesterella minima, 2 unknown species of apicomplexans, 9 morphologically distinct types of trypanosomes, and 2 species of microfilariae. Rana vaillanti, the most aquatic species of frog, harbored the most species of parasites. Recent evidence indicates that morphological changes in the highly pleomorphic trypanosomes of anurans from different geographical regions have not kept pace with biochemical (isozyme) and molecular (DNA sequence) changes. Describing new species based solely on bloodstream trypomastigotes is discouraged. Additional criteria described herein should be applied when naming new species of anuran trypanosomes.

A survey of the blood parasites of 20 Liomys salvini revealed 3 types of parasites. Sixty percent of the mice harbored Trypanosoma zeledoni, 5% an unnamed species of Hepatozoon, and 20% of the mice were infected with a species of Haemobartonella.

We examined 30 Sorex cinereus, 5 Sorex fumeus, and 21 Blarina brevicauda collected from Pennsylvania in 1995 for blood parasites. Trypomastigotes of Trypanosoma sp. were visible in 13% of the S. cinereus. Ten percent of S. cinereus, 20% of S. fumeus, and 14% of B. brevicauda were infected with Bartonella sp. (or spp.). In S. cinereus, we detected no concurrent Trypanosoma and Bartonella infections.

Seven Trypanosoma spp. isolates obtained from bats (Eptesicus sp.) were characterized using experimental infection in mice, triatomines, and culicines; complement lysis; indirect fluorescence assays; as well as isoenzyme and random-amplified polymorphic DNA (RAPD) profiles. The Trypanosoma sp. isolates were compared with Trypanosoma cruzi, Trypanosoma rangeli. and 2 other bat trypanosomes species, Trypanosoma vespertilionis and Trypanosoma hastatus. Trypanosoma sp. isolates were different from the other species in all experiments, except in complement lysis. Experimental infection of triatomines and culicines with Trypanosoma sp. proved to be transitory. These parasites were noninfective for both normal and immunosuppressed mice. Isoenzyme and RAPD profiles obtained for Trypanosoma sp. were quite distinct from T. cruzi and T. rangeli and closely related to T. vespertilionis and T. hastatus. No cross-reaction was observed between sera from mice infected with Trypanosoma sp. and the other trypanosomatids and vice-versa. Trypanosoma sp. induced no protection against T. cruzi infection in mice. The very low, or nonsimilarity between Trypanosoma sp. isolates and the other species used in this study suggests that they might be members of a distinct bat trypanosome species. However, further studies should be done to prove their affinities with Trypanosoma cruzi-marinkellei, another trypanosome species from bats.

Ticks and blood samples were collected from wild birds mist-netted on St. Catherine's Island, Georgia, and at the Wedge Plantation in coastal South Carolina in 1994 and 1995. Immature stages of 5 species of ixodid ticks were recovered from 10 of 148 (7%) birds belonging to 6 species in Georgia, whereas 6 ixodid species were recovered from 45 of 259 (17%) birds representing 10 avian species in South Carolina. Borrelia burgdorferi sensu lato was isolated from 27 of 120 (23%) screened ticks (Ixodes scapularis and Ixodes minor) recovered from South Carolina birds, but from none of 16 screened ticks removed from Georgia birds. This spirochete was also isolated from 1 of 97 (1%) birds in South Carolina. In 1995, neither eastern equine encephalitis (EEE) virus nor St. Louis encephalitis (SLE) virus was isolated from any of 218 bird sera screened, but serum neutralizing antibodies were found to EEE virus in 4 of 121 (3%) sera and to SLE virus in 2 of 121 (2%) sera from South Carolina. No antibody to either virus was detected in 51 avian sera screened from Georgia. Trypanosomes (probably Trypanosoma avium) were isolated from 1 of 51 (2%) birds from Georgia and from 13 of 97 (13%) birds from South Carolina. Our data suggest that some wild birds may be reservoir hosts for the Lyme disease spirochete and for encephalitis viruses in coastal Georgia and South Carolina and that migrating birds can disperse immature ticks infected with B. burgdorferi.

Cultures of Trypanosoma ambystomae were initiated and maintained in hypo-osmotic biphasic blood-agar medium. Epimastigotes and trypomastigotes occurred in culture and although they were similar in size, epimastigotes predominated with about 70% of all stages. Epimastigotes divided by equal binary fission and occasionally formed rosettes. Cultured trypomastigotes did not divide. Cultures isolated 118 days earlier were infective to all 3 laboratory-reared Ambystoma jeffersonianum and to 2 of 3 Ambystoma mexicanum; the latter is a new host record. Trypanosomes were morphometrically similar in both experimental hosts. Specimens from 20-day-old experimental infections were smaller than those from natural infections. Dividing bloodstream forms were detected in experimental infections. Standard culture conditions should be adopted if the morphology of culture forms is to have taxonomic significance.

Trypanosoma catostomi was found in 36.2% of 558 white suckers (Catostomus commersoni) from Ontario, Canada. The abundance of Actinobdella inequiannulata was 35% (68 leeches/197 suckers examined for leeches). The susceptibility of 3 species of leeches (Hemiclepsis marginata, Desserobdella phalera, and A. inequiannulata) and 7 species of fishes (C. commersoni, Amia calva, Anguilla rostrata, Ictalurus nebulosus, Oncorhynchus mykiss, Perca flavescens, and Esox lucius) to infection with T. catostomi was examined. Metatrypanosomes were found in the crop and proboscis sheath of 13 of 21 A. inequiannulata and in the crop of 10 of 12 H. marginata and 1 of 21 D. phalera. Only flagellates from A. inequiannulata were infective to C. commersoni. Cultured T. catostomi infected C. commersoni and A. calva but not any other fish species. Laboratory-reared C. commersoni were more susceptible than wild-caught specimens. Cultured Trypanosoma phaleri did not infect its natural host, A. calva. Host specificity should be established experimentally before a specific diagnosis is made. Cultures may be useful in simulating factors that affect development in the vector.

Cattle (from 5 sites in Louisiana) were examined by blood culture for the presence of Trypanosoma theileri. A prevalence of 81% was found in the 291 cattle examined. A greater number of beef cattle (93%) than dairy cattle (73%) was infected. Differences in prevalence in cattle from different regions of the state were not noted.

Trypanosoma evansi was demonstrated by an immunohistochemical technique in formalin-fixed paraffin-embedded tissues of experimentally infected rats. Trypanosoma evansi was visible readily, nuclei were stained darkly, the cytoplasm was stained moderately, and the cell membranes were delineated clearly. The parasites were present in small- to large-sized blood vessels of all organs, in extravascular spaces of ventricles and neuropil of the brain, and in interstitial tissues of the lung and testes. This method also stained nuclei but not cytoplasm or cell membranes of Trypanosoma congolense, and did not stain Trypanosoma theileri. In a water buffalo (Bubalus bubalis) with nonsuppurative meningoencephalitis, the presence of T. evansi could not be demonstrated by conventional histological stains. However, the trypanosomes were recognized readily in the Virchow-Robin spaces and neuropil of the brain by the immunohistochemical method.

To examine the influence of an established infection on subsequent challenge with another unrelated trypanosome serodeme, cattle were subjected to two challenges, 5 to 6 wk apart, with unrelated isolates of Trypanosoma congolense. The primary infection inhibited the establishment of the second infection despite the initial absence of detectable antibody to the trypanosomes used for the second challenge. This was true whether the second challenge consisted of bloodstream forms of the parasite or metacyclics from infected Glossina m. morsitans. Rechallenge with bloodstream forms resulted in a slight antibody response, which was only detectable by immunofluorescence and was much less than in the challenge controls. Although animals subjected to the second challenge by tsetse flies showed no appreciable increase in parasitemia and, in most instances, no chancre reaction at the site where the tsetse bit, they developed readily detectable neutralizing antibody to the metacyclic trypanosomes. That this interference effect was not the result of specific immunity and required an active infection was confirmed by the finding that when infected animals were treated with Berenil prior to rechallenge, they were fully susceptible to the infection.

Trypanosoma (Herpetosoma) leeuwenhoeki, originally described in Panamanian sloths, was isolated from Didelphis marsupialis (Marsupialia) and Choloepus hoffmanni (Edentata) inhabiting the Pacific coast of Colombia. Trypanosomes were characterized by their large blood forms (total length 51-53 microns), poor infectivity for mice, and lack of development in Rhodnius prolixus. Isoenzyme studies, with either strains or clones, revealed homogeneous profiles clearly distinct from Trypanosoma cruzi and Trypanosoma rangeli reference strains. The present report extends the geographical distribution of T. leeuwenhoeki to South America and broadens its known host range to another order of mammals.