Multiple and frequent trypanosomatid co-infections of insects: the Cuban case study.

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

Occurrence of Nosema ceranae, Ascosphaera apis and trypanosomatids in Vespa orientalis linneus 1771.

Vespa orientalis is spreading across the Italian and European territories leading to new interactions among species, which could lead to the transmission of pathogens between species. Detection of honey bee viruses in V. orientalis has already been revealed in both adults and larvae, while no information is available regarding parasitic occurrence. Sixty adult hornets collected across apiaries in the South of Italy were subjected to cytological, histopathological and biomolecular examination to evaluate the occurrence of Nosema ceranae, Ascosphaera apis, Lotmaria passim, Crithidia mellificae, and Crithidia bombi. Cytological examination revealed the presence of Nosema spores in 38.33% of individuals while histopathological analysis showed the presence of L. passim-like elements in the rectum of two examined specimens and the presence of fungal hyphae in the small intestine of another hornet. Biomolecular investigation revealed that N. ceranae was the most prevalent pathogen (50.0%), followed by A. apis (6.66%), L. passim (6.66%) and C. bombi (6.0%).

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Analysis of honey environmental DNA indicates that the honey bee (Apis mellifera L.) trypanosome parasite Lotmaria passim is widespread in the apiaries of the North of Italy.

Lotmaria passim is a trypanosomatid that infects honey bees. In this study, we established an axenic culture of L. passim from Italian isolates and then used its DNA as a control in subsequent analyses that investigated environmental DNA (eDNA) to detect this trypasonosomatid. The source of eDNA was honey, which has been already demonstrated to be useful to detect honey bee parasites. DNA from a total of 164 honey samples collected in the North of Italy was amplified with three L. passim specific PCR primers and 78% of the analysed samples gave positive results. These results indicated a high prevalence rate of this trypanosomatid in the North of Italy, where it might be considered another threat to honey bee health.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Noninvasive Biological Samples to Detect and Diagnose Infections due to Trypanosomatidae Parasites: A Systematic Review and Meta-Analysis.

Unicellular eukaryotes of the Trypanosomatidae family include human and animal pathogens that belong to the Trypanosoma and Leishmania genera. Diagnosis of the diseases they cause requires the sampling of body fluids (e.g., blood, lymph, peritoneal fluid, cerebrospinal fluid) or organ biopsies (e.g., bone marrow, spleen), which are mostly obtained through invasive methods. Body fluids or appendages can be alternatives to these invasive biopsies but appropriateness remains poorly studied. To further address this question, we perform a systematic review on clues evidencing the presence of parasites, genetic material, antibodies, and antigens in body secretions, appendages, or the organs or proximal tissues that produce these materials. Paper selection was based on searches in PubMed, Web of Science, WorldWideScience, SciELO, Embase, and Google. The information of each selected article (n = 333) was classified into different sections and data were extracted from 77 papers. The presence of Trypanosomatidae parasites has been tracked in most of organs or proximal tissues that produce body secretions or appendages, in naturally or experimentally infected hosts. The meta-analysis highlights the paucity of studies on human African trypanosomiasis and an absence on animal trypanosomiasis. Among the collected data high heterogeneity in terms of the I2 statistic (100%) is recorded. A high positivity is recorded for antibody and genetic material detection in urine of patients and dogs suffering leishmaniasis, and of antigens for leishmaniasis and Chagas disease. Data on conjunctival swabs can be analyzed with molecular methods solely for dogs suffering canine visceral leishmaniasis. Saliva and hair/bristles showed a pretty good positivity that support their potential to be used for leishmaniasis diagnosis. In conclusion, our study pinpoints significant gaps that need to be filled in order to properly address the interest of body secretion and hair or bristles for the diagnosis of infections caused by Leishmania and by other Trypanosomatidae parasites.

High Prevalence and Endemism of Trypanosomatids on a Small Caribbean Island.

We describe the monoxenous trypanosomatids parasitizing true bugs and flies on the island of Curaçao. Out of 248 examined true bugs belonging to 17 species, 93 individuals were found to be infected (overall 38% prevalence) by at least one trypanosomatid species (referred to as typing units; TUs). Out of 80 flies, six were infected. All detected trypanosomatids were compared based on their 18S rRNA sequences with TUs parasitizing bugs and flies described from mainland South America, allowing us to assess their diversity and distribution. Besides Leptomonas pyrrhocoris and Leptomonas seymouri, two known species of the subfamily Leishmaniinae, our analysis revealed six new TUs falling into the groups 'jaculum', Blastocrithidia and Herpetomonas. Moreover, two new members of the genus Phytomonas and three new TUs belonging to the monophyletic group designated as 'new clade II' sensu Mol. Phylogenet. Evol, 69, 255 (2013) were isolated. The detected trypanosomatids were characterized by moderate diversity (13 TUs) species richness. Out of nine and four TUs from the heteropteran and dipteran hosts, respectively, 11 TUs have not been encountered before. Although a sampling bias may partially affect the comparison between trypanosomatid communities on Curaçao and the mainland, the high proportion of unique TUs from the former location suggests that the prominent role of islands in increasing the global diversity of macroscopic organisms may also extend to their protistan parasites.

Correlates of blood parasitism in a threatened marshland passerine: infection by kinetoplastids of the genus Trypanosoma is related to landscape metrics of habitat edge.

In birds, vector-borne parasites invading the bloodstream are important agents of disease, affect fitness and shape population viability, thus being of conservation interest. Here, we molecularly identified protozoan blood parasites in two populations of the threatened Aquatic Warbler Acrocephalus paludicola, a migratory passerine nesting in open marsh. We explored whether prevalence and lineage diversity of the parasites vary by population and whether infection status is explained by landscape metrics of habitat edge and individual traits (body mass, fat score, wing length and sex). Aquatic Warblers were infected by genera Plasmodium, Leucocytozoon and Trypanosoma, with seven, one and four lineages, and 29.9, 0.7 and 12.5% prevalence, respectively. No Haemoproteus infections were detected. Prevalence did not vary between the populations, but lineage diversity was higher in Polesie than in Biebrza for all the lineages pooled and for Plasmodium. Infection by Trypanosoma decreased with patch core area and increased with density of habitat edge. Infection status was not predicted by the individual traits. Our study is the first to show an association between edge-related landscape features and blood parasitism in an open habitat bird. This finding will support informed conservation measures for avian species of the globally shrinking marshland and other treeless habitats.

A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus.

Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

BACKGROUND: Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES: We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS: The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS: In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS: This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Triplex real-time PCR for detection of Crithidia mellificae and Lotmaria passim in honey bees.

Currently, light microscopic examination of cell morphology cannot discriminate Crithidia mellificae and Lotmaria passim with 100% certainty. Here, a minor groove-binding (MGB) probe-based multiplex real-time PCR assay was developed for the simultaneous and quantitative detection of C. mellificae and L. passim in honey bees. A conserved Hymenoptera 18S rRNA gene was built in as an internal control that allows accurate detection of PCR inhibition and failure of DNA extraction. The newly developed assay was also applied to field samples. Of 21 honey bee colonies (446 bees) sampled from six counties in both central and eastern Massachusetts, 3 colonies (14.29%) and 8 bees (1.79%) were infected with L. passim, and 1 colony (4.76%) and 1 bee (0.22%) with C. mellificae. Our data showed a low rate of trypanosomatid infection, and L. passim was more prevalent than C. mellificae in honey bee samples in Massachusetts.

Leptomonas seymouri narna-like virus 1 and not leishmaniaviruses detected in kala-azar samples from India.

The great majority of kala-azar/visceral leishmaniasis (VL) cases, which are caused by Leishmania donovani (LD), are reported in Asia. We investigated whether leishmaniaviruses (LRVs) are present in LD isolates. These dsRNA viruses contribute to hyperpathogenicity, as observed in the case of other members of the genus Leishmania. However, LRVs could not be detected in 22 Indian LD isolates tested in the present study, while 70% of these original LD isolates harboured a virus that was not of LD but instead of Leptomonas seymouri (LS) origin. LS is another protozoon that parasitizes the sandfly vector of LD. Historically, LD clinical isolates from India often showed high incidence of LS coinfection. LS was detected in 20 out of the 22 (91%) above-mentioned LD isolates. Leptomonas seymouri narna-like virus 1 (Lepsey NLV1) was identified by whole-genome sequencing in an LD-LS coinfected sample, and its presence was confirmed by PCR and sequencing in 15 (75%) of the 20 LD-LS coinfected samples. The LS-negative LD samples were also virus negative by PCR. That the human host is exposed to an RNA virus in LS, another coinfecting parasite with LD, i.e., the "LD-LS-Lepsey NLV1 triple pathogen" phenomenon, unveils a new paradigm of research towards revisiting the mysteries of Indian leishmaniasis pathogenesis and management.

Diversity of Trypanosomatids in Cockroaches and the Description of Herpetomonas tarakana sp. n.

In this study, we surveyed six species of cockroaches, two synanthropic (i.e. ecologically associated with humans) and four wild, for intestinal trypanosomatid infections. Only the wild cockroach species were found to be infected, with flagellates of the genus Herpetomonas. Two distinct genotypes were documented, one of which was described as a new species, Herpetomonas tarakana sp. n. We also propose a revision of the genus Herpetomonas and creation of a new subfamily, Phytomonadinae, to include Herpetomonas, Phytomonas, and a newly described genus Lafontella n. gen. (type species Lafontella mariadeanei comb. n.), which can be distinguished from others by morphological and molecular traits.

PCR-specific detection of recently described Lotmaria passim (Trypanosomatidae) in Chilean apiaries.

The recently described trypanosome Lotmaria passim is currently considered the most predominant trypanosomatid in honey bees worldwide and could be a factor in honey bee declines. For a specific and quick detection of this pathogen, we developed primers based on the SSU rRNA and gGAPDH genes for the detection of L. passim in Chilean honey beehives. PCR products amplified and sequenced for these primers shared 99-100% identity with other sequences of L. passim. The designed primers were specific and we were able to detect a high prevalence (40-90%) of L. passim in bee hives distributed throughout Chile. Our described PCR-based method offers a feasible and specific detection of L. passim in any honey bee samples.

Implications of co-infection of Leptomonas in visceral leishmaniasis in India.

Protozoan parasites Leishmania donovani (family: Trypanosomatidae) cause fatal visceral leishmaniasis (VL) and the infection relapses in apparently cured population as post kala-azar dermal leishmaniasis (PKDL) in the Indian subcontinent. In recent years co-infection of another Trypanosomatid parasite Leptomonas with L. donovani during VL/PKDL in this region has become prominent. The observation of clinically lesser-known insect parasite, Leptomonas in leishmaniasis is intriguing to researchers. The presence of Leishmania look alike Leptomonas in the cultures of clinical isolates of Leishmania has been worrisome to those, who prefer to work with pure Leishmania cultures for drug and vaccine development or immune response studies. The exact implications of such a co-habitation, which might lead to a delay in the diagnostics of VL and elevate mortality, need a thorough investigation. Also whether Leptomonas is involved in leishmaniasis manifestation needs to be ascertained. Thus we are currently witnessing a new paradigm of a parasitic co-infection in VL/PKDL cases in India and this review outlines various opportunities for further research in understanding such emerging co-infection.

Identification of Avian and Hemoparasite DNA in Blood-Engorged Abdomens of Culex pipiens (Diptera; Culicidae) from a West Nile Virus Epidemic region in Suburban Chicago, Illinois.

Multiple mosquito-borne parasites cocirculate in nature and potentially interact. To understand the community of parasites cocirculating with West Nile virus (WNV), we screened the bloodmeal content of Culex pipiens L. mosquitoes for three common types of hemoparasites. Blood-fed Cx. pipiens were collected from a WNV-epidemic area in suburban Chicago, IL, from May to September 2005 through 2010. DNA was extracted from dissected abdomens and subject to PCR and direct sequencing to identify the vertebrate host. RNA was extracted from the head or thorax and screened for WNV using quantitative reverse transcriptase PCR. Seventy-nine engorged females with avian host origin were screened using PCR and amplicon sequencing for filarioid nematodes, Haemosporida, and trypanosomatids. Filarioid nematodes were identified in 3.8% of the blooded abdomens, Plasmodium sp. in 8.9%, Haemoproteus in 31.6%, and Trypanosoma sp. in 6.3%. The sequences from these hemoparasite lineages were highly similar to sequences from birds in prior studies in suburban Chicago. Overall, 50.6% of blood-fed Culex pipiens contained hemoparasite DNA in their abdomen, presumably from current or prior bloodmeals. Additionally, we detected hemoparasite DNA in the blooded abdomen of three of 10 Cx. pipiens infected with WNV.

Trypanosomatids in ornithophilic bloodsucking Diptera.

Trypanosomes are known as widespread blood parasites of birds; however, knowledge of their prevalences in vectors and their overall biodiversity is rather limited. To assess the prevalences in potential vectors, we have microscopically examined ornithophilic bloodsucking Diptera (Culicidae, Simuliidae and Hippoboscidae) for the presence of trypanosomatids in their guts. In total, 3270 specimens were dissected, namely Culex pipiens Linnaeus, 1758 (n = 898), C. modestus Ficalbi, 1890 (136), Simulium vernum (Macquart, 1838) (1455), S. angustipes Edwards, 1915 (221) and Ornithomyia avicularia (Linnaeus, 1758) (560). All insect species were found to be infected with trypanosomatids, and the prevalence ranged from 4 to 8% but reached 60% in S. vernum. Blackflies and hippoboscids exclusively harboured trypanosomes (both T. cf. avium s.s. Danilewsky, 1885; T. corvi/culicavium group in hippoboscids). Mosquitoes were infected with T. culicavium Votypka, 2012 and T. avium s. l. but also with monoxenous parasites, namely Crithidia brevicula Frolov and Malysheva, 1989, and Paratrypanosoma confusum Votypka and Lukes, 2013. Only 4% of the isolated parasite strains were monoxenous whereas the majority were avian trypanosomes, confirming the vectorial status of the studied insects.

Molecular evidence of pathogens and endosymbionts in the black horse fly Osca lata (Diptera: Tabanidae) in Southern Chile.

Little is known about the role of horse flies in potential pathogen transmission in Chile. This study provides evidence of the molecular detection of microorganisms in southern Chile. In the present study, adult Osca lata horse flies were trapped from Punucapa (39°45'06"S/73°16'08"W, Región de Los Ríos) and Puyehue (40°39'10"S/72°10'57"W, Región de Los Lagos), Chile. Among the 95 samples analyzed by PCR using specific primers, microorganisms were detected in 23.2% (n = 22) of the samples. Rickettsia spp. DNA was detected in 15.8% (n = 15) of the samples, Trypanosomatidae DNA in 5.3% (n = 5) of the samples, and filarial DNA in 2.1% (n = 2) of the samples. This study found that horse flies in the region are capable of carrying a variety of both parasites and endosymbionts. Further research is needed to understand the specific impact of horse flies as mechanical or biological vectors and develop effective control measures to prevent the spread of any microorganisms associated with disease.

Coinfection of Leptomonas seymouri and Leishmania donovani in Indian leishmaniasis.

Leishmania donovani is considered the causative organism of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). Testing of 4/29 DNA samples from VL and PKDL patients as well as 2/7 field isolates showed an aberrant internal transcribed spacer 1 (ITS1) restriction fragment length polymorphism (RFLP) pattern, which upon sequencing strongly matched Leptomonas seymouri, thus confirming its presence in Indian leishmaniasis.

New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae.

Several new species of trypanosomatids (Euglenozoa, Kinetoplastea, Trypanosomatidae), isolated from the intestines of Neotropical insects (Heteroptera), were genotyped on the basis of spliced leader RNA, and also defined phylogenetically using gene sequences of small subunit ribosomal RNA and glycosomal glyceraldehyde phosphate dehydrogenase. The taxonomic descriptions also included characterization using morphometry and electron microscopy. Our phylogenetic analyses placed the new species within the clade, previously designated "SE" for "Slowly Evolving" sequences of ribosomal RNA genes, a clade that also includes numerous monoxenous parasites of insects from the genera Crithidia, Leptomonas, and Wallaceina, as well as the dixenous genus Leishmania. Based on the high phylogenetic support for this clade, which is consistently recovered in all recent phylogenetic reconstructions, a proposal is put forward to recognize this natural taxon as a new subfamily, Leishmaniinae, within the family Trypanosomatidae.