Subgenus Megatrypanum Research Articles

Diagnóstico parasitológico e molecular de Trypanosoma theileri em bovinos amostrados na zona sul do município de São Paulo, SP, Brasil

OBJECTIVE: Trypanosoma theileri is the type species of the subgenus Megatrypanum, first registered in cattle in South Africa and East Africa. Cattle farms in the extreme south of the municipality of São Paulo and Embu-Guaçu, are characterized as subsistence, on small properties, and 500 head of cattle were registered in 2019. This work aimed to evaluate the occurrence of T. theileri in cattle from family farms in the extreme south of the city of São Paulo and in the municipality of Embu-Guaçu, SP, Brazil. METHODS: Parasitological and molecular diagnoses were carried out on samples taken from 68 cattle in small farms in the extreme south of São Paulo. RESULTS: The specific molecular diagnosis of T. theileri was 17.6% positive (12/68) and the parasitological diagnosis was 5.8% (4/68). CONCLUSION: Although considered non-pathogenic, its presence in the host may favor other parasitic and infectious diseases.

"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, 50years 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.

A new species of mammalian trypanosome, Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27–37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.

Molecular detection of Megatrypanum trypanosomes intabanid flies.

Trypanosomes of the subgenus Megatrypanum have been isolated from many mammalian hosts around the world. They are usually non-pathogenic, although they may confuse the parasitological diagnosis of trypanosomosis. Additionally, Trypanosoma theileri has been associated with disease in cattle. Megatrypanum trypanosomes are considered to be transmitted by different arthropods, including tabanids. However, little is known about the potential vectors of Megatrypanum trypanosomes in different parts of the world. The present study reports on the detection of Megatrypanum trypanosomes in Heamatopota pluvialis, Tabanus bromius, Tabanus maculicornis and Tabanus distinguendus in Poland. It also discusses the possible role of these tabanids in the transmission of Megatrypanum trypanosomes.

Molecular characterization of a new Trypanosoma (Megatrypanum) theileri isolate supports the two main phylogenetic lineages of this species in Japanese cattle.

Trypanosoma (Megatrypanum) theileri is a cosmopolitan, usually non-pathogenic, trypanosome of cattle transmitted by blood-sucking arthropods, mainly tabanid flies. Several T. theileri strains isolated from domestic and wild ruminants via co-culturing with mammalian feeder cells or blood cells have been characterized morphologically and genetically. Here, we cultured a new trypanosome isolate from a Holstein cow in Hokkaido, Japan, and performed morphological and molecular characterization studies. The new isolate (Obihiro strain) was co-cultivated with Madin-Darby bovine kidney (MDBK) cells in GIT medium supplemented with 10% fetal bovine serum. Trypomastigotes and epimastigotes, but not intracellular parasites, were identified in the culture. Analysis of the V7-V8 region of 18S rRNA sequences showed that the Obihiro strain is positioned within the subgenus Megatrypanum. A dendrogram based on whole internal transcribed spacer rDNA sequence showed that the Obihiro strain clustered in the lineage TthII together with the Japanese isolates of T. theileri, Esashi 9, and Esashi 12, and isolates from Zambia and the USA. T. theileri of the KM strain and a T. theileri-like trypanosome isolated from deer (TSD1 strain) clustered in the lineage TthI, separate from the Obihiro strain. Based on a partial cathepsin L-like protein gene analysis, the Obihiro strain clustered with isolates of the TthIIF genotype, which includes T. theileri from Vietnam, Sri Lanka, and Brazil. Our analyses of the T. theileri Obihiro strain provide relevant insights into its genetic diversity in Japanese cattle and corroborate the host specificity of cattle and deer trypanosomes of the subgenus Megatrypanum.

Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade

BackgroundBat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli.MethodsTrypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy.ResultsNew trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species.ConclusionPhylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca2+ transients and TGF-β-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.

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.

Trypanosoma (Megatrypanum) saloboensen. sp. (Kinetoplastida: Trypanosomatidae) parasite ofMonodelphis emiliae(Marsupiala: Didelphidae) from Amazonian Brazil

Trypanosoma (Megatrypanum) soloboense n. sp., is described in the Brazilian opossum Monodelphis emiliae (Thomas, 1912) from primary forest in the Salobo area of the Serra dos Carajás (6 degrees S, 50 degrees 18' W) Pará State, North Brazil. Two morphologically different trypomastigotes were noted. Slender forms, regarded as immature parasites, have a poorly developed undulating membrane adhering closely to the body: large, broad forms with a well developed membrane are considered to be the mature trypomastigotes and have a mean total length of 71.2 microm (62.4-76.2) and a width of 6.1 (5.0-8.0). Infections studied in two opossums were of very low parasitaemia. The large size of T. (M.) saloboense readily distinguishes it from the two previously described members of the subgenus Megatrypanum of neotropical marsupials, T. (M.) freitasi Régo et al., 1957 of Didelphis ozarae and D. marsupialis, and T. (M.) samueli Mello, 1977 of Monodelphis domesticus, which measure only 49.0-51.5 microm and 42.4 microm respectively. No infections were obtained in hamsters inoculated with triturated liver and spleen from one infected M. emiliae, or in laboratory mice inoculated with epimastigotes from a blood-agar culture. No division stages could be detected in the internal organs or the peripheral blood.

Infection rates and genotypes of Trypanosoma rangeli and T. cruzi infecting free-ranging Saguinus bicolor (Callitrichidae), a critically endangered primate of the Amazon Rainforest

Parasites of wild primates are important for conservation biology and human health due to their high potential to infect humans. In the Amazon region, non-human primates are commonly infected by Trypanosoma cruzi and T. rangeli, which are also infective to man and several mammals. This is the first survey of trypanosomiasis in a critically endangered species of tamarin, Saguinus bicolor (Callitrichidae), from the Brazilian Amazon Rainforest. Of the 96 free-ranging specimens of S. bicolor examined 45 (46.8%) yielded blood smears positive for trypanosomes. T. rangeli was detected in blood smears of 38 monkeys (39.6%) whereas T. cruzi was never detected. Seven animals (7.3%) presented trypanosomes of the subgenus Megatrypanum. Hemocultures detected 84 positive tamarins (87.5%). Seventy-two of 84 (85.7%) were morphologically diagnosed as T. rangeli and 3 (3.1%) as T. cruzi. Nine tamarins (9.4%) yielded mixed cultures of these two species, which after successive passages generated six cultures exclusively of T. cruzi and two of T. rangeli, with only one culture remaining mixed. Of the 72 cultures positive for T. rangeli, 62 remained as established cultures and were genotyped: 8 were assigned to phylogenetic lineage A (12.9%) and 54 to lineage B (87.1%). Ten established cultures of T. cruzi were genotyped as TCI lineage (100%). Transmission of both trypanosome species, their potential risk to this endangered species and the role of wild primates as reservoirs for trypanosomes infective to humans are discussed.

Trypanosoma manulis n. sp. from the Russian Pallas cat Felis manul.

The morphology of Trypanosoma manulis n. sp. is described from living and stained specimens obtained from the blood of a Pallas cat, Felis manul, from Kazakhstan. The cat was also infected with a Hepatozoon sp. and feline immunodeficiency virus. The morphology of the trypanosome most closely resembles that of Trypanosoma mpapuense Reichenow and Trypanosoma heybergi Rodhain found in bats. Trypanosoma manulis does not grow well in conventional media, but co-culture with African green monkey kidney cells in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum at approximately 27 degrees C resulted in luxuriant growth of trypanosomes. Under these growth conditions, epimastigotes adhered to the surface of the culture flask and to African green monkey kidney cells, as well as forming large rosettes. At 37 degrees C, although growth was poor, transformation of the epimastigotes into the bloodstream forms occurred. This represents the first report of a trypanosome of the subgenus Megatrypanum in a felid.

Lipoptena cervi (Diptera), a potential vector ofMegatrypanum trypanosomes of deer (Cervidae)

In three different areas in northern Germany, hippoboscids were collected from red deer (Cervus elaphus). Typanosomatids were demonstrated in the midgut and hindgut of 9/37 Lipoptena cervi. The trypanosomatids were morphologically similar to vector forms of the subgenus Megatrypanum that had been found in tabanids at the same location 5 years previously. Hippoboscids are therefore potential vectors of Trypanosoma (Megatrypanum) spp. that infect red deer.

Trypanosomes of the subgenus Megatrypanum from armadillos (Xenarthra: Dasypodidae)

A new species of trypanosome, Trypanosoma (Megatrypanum) peba, is described from the peripheral blood of the armadillo Euphractus sexcinctus setosus from Bahia State, Brazil. Ten out of 29 specimens of the armadillo Dasypus novemcinctus from Pará State were found to have trypanosomes, including epimastigote forms, in impression smears of subcutaneous lymph nodes. The trypanosomes from D. novemcinctus are illustrated and were identified as belonging to the subgenus Megatrypanum on the basis of their general appearance, although they failed to multiply in blood-agar culture medium and no bloodstream forms were seen. This is the first published record of trypanosomes of this subgenus from armadillos and the first demonstration of epimastigote trypanosomes in the mammalian host other than in the bloodstream, or in the anal glands of opossums.

Trypanosoma (Megatrypanum) megachiropterorum sp. n. from the Flying Fox, Pteropus tonganus Quoy & Gaimard

SYNOPSIS. Trypanosoma (Megatrypanum) megachiropterorum sp. n. from the flying fox, Pteropus tonganus Quoy & Gaimard, from the Kingdom of Tonga, is described. The trypomastigotes measure 32 ± 1.74 (here and below S.D.) (27–37.5) × 3.6 ± 0.23 (3–4.5) μm and has a flagellum averaging in length 6.4 ± 0.92 (4.5–10) μm. Their nuclear index is 1.2 ± 0.16 (0.7–1.6) and their kinetoplastic index equals 6.2 ± 0.32 (5.5–7.1). This is the first report of a member of the subgenus Megatrypanum from bats outside the American and African continents and also the first record of a trypanosome from bats in the Pacific area.

Trypanosoma (Megatrypanum) lizae n. sp.

Trypanosoma (Megatrypanum) lizae n. sp.: trypanosome with giant forms reaching a length of 1400 micrometers from the Microchiroptera Hipposideros cyclops in Gabon. Because of the peculiar morphology of giant forms and the characteristics of small individuals it is placed in a new species within the subgenus Megatrypanum.

Trypanosoma (Megatrypanum) samueli n. sp., a Trypanosomatidae isolated from Monodelphis domesticus (Wagner, 1842) (Marsupiala).

In the present paper the description of a new Trypanosome species of the subgenus Megatrypanum is presented. The proposed name is Trypanosoma (Megatrypanum ) samueli.

Morphology of typanosomes from white-tailed deer and wapiti in Michigan.

Trypanosomes were isolated from a wapiti (Cervus canadensis) and 72 white-tailed deer (Odocoileus virginianus) from several locations in Michigan. Although significantly fewer fawns were infected, there were no significant differences in rate of infection between sexes or among geographic areas. From appearance of the trypomastigote, the trypanosome from white-tailed deer belongs in the genus Trypanosoma and the subgenus Megatrypanum. It was morphometrically similar to the common trypanosome of cattle, Trypanosoma theileri.

First european record of Trypanosoma (Megatrypanum) sp. of bats.

PREVIOUS records of Trypanosoma from European bats (Chiroptera) have all been of species belonging to the subgenus Schizotrypanum1,2. The subgenus Megatrypanum has been reported from bats in Africa and South America2. I have seen trypanosomes belonging to the latter subgenus in the blood of 1 of 7 Pipistrellus pipistrellus (Schreber) trapped in Rollesby, Norfolk, England, during July 1972. Dimensions and indices2 of ten parasites are given in Table 1 and one organism is illustrated (Fig. 1).

A Review of the Role Played by the Hippoboscidae (Diptera) as Vectors of Endoparasites

Members of the Hippoboscidae, a family of pupiparous Diptera, are known to act as vectors of two species of Trypanosoma (T. melophagium and T. theodori) belonging to the subgenus Megatrypanum, and of one species (T. avium) parasitizing birds. They also transmit three species of Haemoproteus (H. columbae, H. lophortyx, H. palumbis) with avian hosts and, very probably, Dipetalonema dracunculoides of certain Camivora in Africa. There is some evidence that two other species of Trypanosoma, and three other species of Haemoproteus, are transmitted by flies of this family. The life cycles of the protozoan parasites known to be transmitted by Hippoboscidae are summarized, and the history of the discovery of the role of these insects as vectors is briefly discussed. It is suggested that other parasitic Protozoa and helminths may prove to be transmitted by Hippoboscidae. The Hippoboscidae (louse-flies) are a family of cyclorrhaphous Diptera, sometimes included with the families Nycteribiidae and Streblidae in a separate section, the Pupipara. However, Bequaert (1954-1957) regards this grouping as an unnatural one. In all Hippoboscidae (as well as in the other two families just mentioned and the Glossinidae) the eggs (levelop singly and are retained within the body of the female instead of being laid. The egg hatches, and larval development commences within the mother's uterus, the larva being nourished from special milk glands. It is deposited only after it has reached the third instar. The larva pupates almost immediately after parturition and its integument hardens into a protective shell or puparium. Both sexes of hippoboscids feed only on blood. They are fairly long-lived insects, the adults of many species probably surviving for several (about 4) months. Larval development, within the female, takes about 1 to 2 weeks, in those species in which it has been studied, while the duration of the pupal period ranges from about 3 weeks in Melophagus ovinus up to some 10 months in Ornithomyia avicularia. Members of the family show a tendency to reduction of the power of flight. Many, such Received for publication 10 October 19'66. * This paper was contributed to a symposium on Arthropod Vectors of Parasites of Wildlife at a meeting of the Wildlife Disease Association at the University of Maryland, 14-19 August 1966. The author's attendance at the meeting was made possible by a grant from the Wellcome Trust, receipt of which is gratefully acknowledged. as the genera Pseudolynchia and Ornithomyia, have functional wings on both sexes, but even this group are not active fliers, tending more to dart from host to host rather than flying long distances. They do not readily leave a living host, but run about with a crab-like motion beneath its hair or feathers. Other genera of the same subfamily (Omithomyiinae) have reduced, functionless wings throughout their adult life (e.g., Stenepteryx, Crataerhina, and Myophthiria) and can become airborne only as passengers upon their avian hosts. Again, some members of another subfamily, the Melophaginae (e.g., Lipoptena) emerge from the puparium with functional wings which are shed when they settle on a host. Finally, another member (Melophagus) of this subfamily is totally devoid of wings (and halteres) at all stages of its life. A comprehensive monograph on the family has been published by Bequaert (1953 and 1954-1957); the British species have been dealt with by Edwards, Oldroyd, and Smart (1939) and, in more detail, by Thompson (1953-1955). It is from these works that the above rather sketchy introduction has been largely drawn. Those species which parasitize game animals in the U. S. A. have been discussed by Herman (1945), while the British and northern European species of Ornithomyia have recently been revised by Hill (1962a, b,