Extracellular vesicles released by anaerobic protozoan parasites: Current situation.

Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. Parasitic unicellular eukaryotes use EVs as vehicles for intercellular communication and host manipulation. Pathogenic protozoans are able to modulate the immune system of the host and establish infection by transferring a wide range of molecules contained in different types of EVs. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this review we cover the current knowledge on EVs from anaerobic or microaerophilic extracellular protozoan parasites, including Trichomonas vaginalis, Tritrichomonas foetus, Giardia intestinalis and Entamoeba histolytica, with a focus on their potential role in the process of infection. The role of EVs in host: parasite communication adds a new level of complexity to our understanding of parasite biology, and may be a key to understand the complexity behind their mechanism of pathogenesis.

Specific clones of Trichomonas tenax are associated with periodontitis.

Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases.

Studies on prevalence of infection with Trichomonas tenax identified by molecular techniques ­ in respect to oral health of patients with various systemic disease requiring immunosuppressive therapy

Trichomonas tenax, cosmopolitan flagellate inhabiting human oral cavity, is the etiological agent of oral trichomonosis associated with gingival and periodontium deterioration. Purpose of this studies was to investigate the prevalence of infection with Trichomonas tenax identified by molecular techniques amplifying the region of ITS1-5.8S rRNA-ITS2 specific for T. tenax. The study included 498 persons: diabetic, renal transplant, rheumatoid arthritis patients and the control group. Prevalence of T. tenax in oral cavity was 10.2% in control group, 14.1% in diabetics, 12.0% in renal transplant patients and 14.0% in rheumatoid arthritis patients. Comparative assessment of results showed symptoms of gingiva and periodontium deteriorations, at varying intensity in patients with various systemic diseases; higher prevalence of the trichomonad infection was revealed in adults in all groups. Simultaneously, renal transplantation, diabetes, rheumatoid arthritis and related therapy do not affect T. tenax incidences and no increased risk of the infection has been observed in the patients; the permanent medication used due to main disease should be taken into consideration as likely inhibitory factor.

Prevalence and genotyping of Trichomonas infections in wild birds in central Germany.

Avian trichomonosis is a widespread disease in columbids and other birds, caused by ingestion of the unicellular flagellate Trichomonas gallinae which proliferate primarily in the upper respiratory tracts. Studies using genetic analyses have determined some highly pathogenic lineages in birds, but the prevalence and distribution of potentially pathogenic and non-pathogenic T. gallinae lineages in wild birds is still not well known. We examined 440 oral swab samples of 35 bird species collected between 2015 and 2017 in Hesse, central Germany, for Trichomonas spp. infection and for determining the genetic lineages. Of these birds, 152 individuals were caught in the wild and 288 individuals were admitted from the wild to a veterinary clinic. The overall Trichomonas spp. prevalence was 35.6%. We observed significant differences between bird orders, with the highest prevalence in owls (58%) and columbids (50%), while other orders had slightly lower prevalences, with 36% in Accipitriformes, 28% in Falconiformes and 28% in Passeriformes. Among 71 successfully sequenced samples, we found 13 different haplotypes, including two previously described common lineages A/B (20 samples) and C/V/N (36 samples). The lineage A/B has been described as pathogenic, causing lesions and mortality in columbids, raptors and finches. This lineage was found in 11 of the 35 species, including columbids (feral pigeon, woodpigeon, stock dove), passerines (greenfinch, chaffinch, blackbird) and raptors (common kestrel, sparrowhawk, red kite, peregrine falcon and common buzzard). One new lineage (R) was found in a sample of a chaffinch. In conclusion, we found that the prevalence of Trichomonas spp. infection in wild birds was high overall, and the potentially pathogenic lineage A/B was widespread. Our findings are worrying, as epidemic outbreaks of trichomonosis have already been observed in Germany in several years and can have severe negative effects on bird populations. This disease may add to the multiple pressures that birds face in areas under high land-use intensity.

[CHARACTERISTICS OF PARASITIC PROTOZOA METABOLISM AND POSSIBILITIES OF ANTIPROTOZOA DRUG DEVELOPMENT (REVIEW)].

One of the most poorly studied areas of protozoology is metabolic processes of parasitic protozoa. Study of the biochemistry of parasites required for the development of effective chemotherapy of protozoal diseases. Some amitochondrial parasites of humans, such as Giardia intestinalis, Entamoeba histolytica, Trichomonas sp., living in an environment with low oxygen content, have specialized cellular organelles-hydrogenosomes (like mitochondria provide cells with simple energy). The study of the functioning of these organelles allows us to consider them as targets for the development of аntiprotozoal drugs. The target for chemotherapy in the treatment of trypanosomiasis can be processes related to the characteristics of the glycolytic pathway or a decrease in the level of energy substrate, such as glucose. This leads to a rapid decrease in ATP levels in the cell of the parasite, an overall loss of mobility and disappearance of trypanosomes from the bloodstream of the infected host. Also, glucose transporters located in the membrane of the parasite can be targets for drugs.

Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.

Trichomonas tenax and periodontal diseases: a concise review.

Periodontal diseases (gingivitis and periodontitis), result from a disruption of the host-oral microbiome homoeostasis. Whereas the pathological role of some specific bacterial strains during periodontal diseases is well documented, the impact of parasites in periodontium pathophysiology is still under debate. This review aims to collect data about the prevalence and the potential role of Trichomonas tenax during periodontal diseases. Data from 47 studies revealed that T. tenax prevalence in diseased periodontium ranged from 0 to 94·1%. The prevalence of oral protozoan infections was found to be largely greater in patients with periodontal diseases than with healthy periodontium. The parasite detection was mainly performed by direct microscopy. Trichomonas tenax presence was clearly correlated with periodontal disease. The high heterogeneity of its periodontal prevalence may be correlated with the diversity of the population screened (age, sex, systemic diseases), and the methods used for diagnosis. This protozoan seems to have the capacity to be involved in the inflammatory process of gum disease. Animal experimentation, using relevant physiopathological models of periodontitis, needs to be performed to investigate the ability of T. tenax to cause and/or worsen the disease. Further investigations using standardized experimental designs of epidemiologic studies are also needed.

High prevalence of Trichomonas gallinae in wild columbids across western and southern Europe.

BACKGROUND: Avian trichomonosis is known as a widespread disease in columbids and passerines, and recent findings have highlighted the pathogenic character of some lineages found in wild birds. Trichomonosis can affect wild bird populations including endangered species, as has been shown for Mauritian pink pigeons Nesoenas mayeri in Mauritius and suggested for European turtle doves Streptopelia turtur in the UK. However, the disease trichomonosis is caused only by pathogenic lineages of the parasite Trichomonas gallinae. Therefore, understanding the prevalence and distribution of both potentially pathogenic and non-pathogenic T. gallinae lineages in turtle doves and other columbids across Europe is relevant to estimate the potential impact of the disease on a continental scale. RESULTS: We examined 281 samples from four wild columbid species for Trichomonas infection and determined the genetic lineages. The overall prevalence was 74%. There were significant differences between the species (P = 0.007). The highest prevalence was found in stock doves Columba oenas (86%, n = 79) followed by wood pigeons Columba palumbus (70%, n = 61) and turtle doves (67%, n = 65), while three of five collared doves Streptopelia decaocto (60%) were infected. We found seven different lineages, including four lineages present in columbids in the UK, one lineage already described from Spain and three new lineages, one of those found in a single turtle dove migrating through Italy and another one found in a breeding stock dove. Stock doves from Germany and collared doves from Malta were infected with a potentially pathogenic lineage (lineage A/B), which is known to cause lesions and mortality in columbids, raptors and finches. CONCLUSIONS: Generally, turtle doves showed high prevalence of Trichomonas infection. Furthermore, the potentially pathogenic lineage A/B (or genotype B according to previous literature) was found in a recovering stock dove population. Both findings are worrying for these columbid species due to the occasional epidemic character of trichomonosis, which can have severe negative effects on populations.

The Prevalence of Canine Oral Protozoa and Their Association with Periodontal Disease.

Periodontal disease is one of the most important health concerns for companion animals. Research into canine forms of periodontitis has focused on the identification and characterization of the bacterial communities present. However, other microorganisms are known to inhabit the oral cavity and could also influence the disease process. A novel, broad spectrum 18S PCR was developed and used, in conjunction with next-generation sequencing analyses to target the identification of protists. Trichomonas sp. and Entamoeba sp. were identified from 92 samples of canine plaque. The overall prevalence of trichomonads was 56.52% (52/92) and entamoebae was 4.34% (4/92). Next-generation sequencing of pooled healthy, gingivitis, early-stage periodontitis, and severe periodontitis samples revealed the proportion of trichomonad sequences to be 3.51% (health), 2.84% (gingivitis), 6.07% (early periodontitis), and 35.04% (severe periodontitis), respectively, and entamoebae to be 0.01% (health), 0.01% (gingivitis), 0.80% (early-stage periodontitis), and 7.91% (severe periodontitis) respectively. Both genera of protists were statistically associated with plaque from dogs with periodontal disease. These findings provide the first conclusive evidence for the presence of oral protozoa in dog plaque and suggest a possible role for protozoa in the periodontal disease process.

RAPD analysis and sequencing of ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase as tools for genetic classification of potentially pathogenic isolates of Trichomonas gallinae.

Trichomonas gallinae is a worldwide parasite that causes oropharyngeal avian trichomonosis. During eight years, 60 axenic isolates were obtained from different bird species and characterized by three molecular methods: RAPD analysis and PCR-sequencing of ITS1/5.8S rRNA/ITS2 fragment and Fe-hydrogenase gene. We have found two genotypes of ITS1/5.8S rRNA/ITS2 widely distributed among bird populations, a new variant and also two sequences with mixed pattern. Genotype ITS-OBT-Tg-1 was associated with the presence of gross lesions in birds. We have found eight genotypes of the Fe-hydrogenase (A1, A2, C2, C2.1, C4, C5, C6 and C7), three of them are new reports (C5, C6 and C7), and also three sequences with mixed pattern. Subtype A1 of the Fe-hydrogenase was also related with the presence of lesions. RAPD analyses included most of the strains isolated from animals with lesions in one of the sub-clusters. Potentially pathogenic isolates of T. gallinae obtained in this study fulfill the following criteria with one exception: isolated from lesions+ITS-OBT-Tg-1 genotype+FeHyd A1+RAPD sub-cluster I2.

Detection of the European epidemic strain of Trichomonas gallinae in finches, but not other non-columbiformes, in the absence of macroscopic disease.

Finch trichomonosis is an emerging infectious disease affecting European passerines caused by a clonal strain of Trichomonas gallinae. Migrating chaffinches (Fringilla coelebs) were proposed as the likely vector of parasite spread from Great Britain to Fennoscandia. To test for such parasite carriage, we screened samples of oesophagus/crop from 275 Apodiform, Passeriform and Piciform birds (40 species) which had no macroscopic evidence of trichomonosis (i.e. necrotic ingluvitis). These birds were found dead following the emergence of trichomonosis in Great Britain, 2009-2012, and were examined post-mortem. Polymerase chain reactions were used to detect (ITS1/5·8S rRNA/ITS2 region and single subunit rRNA gene) and to subtype (Fe-hydrogenase gene) T. gallinae. Trichomonas gallinae was detected in six finches [three chaffinches, two greenfinches (Chloris chloris) and a bullfinch (Pyrrhula pyrrhula)]. Sequence data had 100% identity to the European finch epidemic A1 strain for each species. While these results are consistent with finches being vectors of T. gallinae, alternative explanations include the presence of incubating or resolved T. gallinae infections. The inclusion of histopathological examination would help elucidate the significance of T. gallinae infection in the absence of macroscopic lesions.

The protozoan parasite Trichomonas gallinae causes adult and nestling mortality in a declining population of European Turtle Doves, Streptopelia turtur.

Studies incorporating the ecology of clinical and sub-clinical disease in wild populations of conservation concern are rare. Here we examine sub-clinical infection by Trichomonas gallinae in a declining population of free-living European Turtle Doves and suggest caseous lesions cause mortality in adults and nestlings through subsequent starvation and/or suffocation. We found a 100% infection rate by T. gallinae in adult and nestling Turtle Doves (n = 25) and observed clinical signs in three adults and four nestlings (28%). Adults with clinical signs displayed no differences in any skeletal measures of size but had a mean 3.7% reduction in wing length, with no overlap compared to those without clinical signs. We also identified T. gallinae as the suggested cause of mortality in one Red-legged Partridge although disease presentation was different. A minimum of four strains of T. gallinae, characterized at the ITS/5.8S/ITS2 ribosomal region, were isolated from Turtle Doves. However, all birds with clinical signs (Turtle Doves and the Red-legged Partridge) carried a single strain of T. gallinae, suggesting that parasite spill over between Columbidae and Galliformes is a possibility that should be further investigated. Overall, we highlight the importance of monitoring populations for sub-clinical infection rather than just clinical disease.

Trichomonads in birds--a review.

Members of the family Trichomonadidae, mainly Trichomonas gallinae and Tetratrichomonas gallinarum, represent important parasites in birds with worldwide presence, since being reported in the 19th century. Especially Columbiformes, Falconiformes and Strigiformes can be severely affected by trichomonads, whereas the majority of infections in Galliformes and Anatiformes are subclinical although severe infections are occasionally reported. With the recent appearance of deadly infections in wild Passeriformes the protozoan parasite T. gallinae obtained greater attention which will be addressed in this review. Although light microscopy remains the method of choice to confirm the presence of trichomonads molecular studies were introduced in recent years, in order to characterize the parasites and to establish relationships between isolates. Isolation of trichomonads is a prerequisite for detailed in vitro and in vivo studies and different media are reported to obtain suitable material. The limited information about virulence factors will be reviewed in context with the pathogenicity of trichomonads which varies greatly, indicating certain strain heterogeneity of the parasites. Options for treatment characterized by the leading role of imidazoles whose activity is sometimes hampered by resistant parasites remains a challenge for the future. Introducing more standardized genetic studies and investigations concentrating on the host-pathogen interaction should be helpful to elucidate virulence factors which might lead to new concepts of treatment.

The role of extracellular vesicles in Plasmodium and other protozoan parasites.

Protozoan parasites and other microorganisms use various pathways to communicate within their own populations and to manipulate their outside environments, with the ultimate goal of balancing the rate of growth and transmission. In higher eukaryotes, including humans, circulating extracellular vesicles are increasingly recognized as key mediators of physiological and pathological processes. Recent evidence suggests that protozoan parasites, including those responsible for major human diseases such as malaria and Chagas disease, use similar machinery. Indeed, intracellular and extracellular protozoan parasites secrete extracellular vesicles to promote growth and induce transmission, to evade the host immune system, and to manipulate the microenvironment. In this review we will discuss the general pathways of extracellular vesicle biogenesis and their functions in protozoan infections.

The emergence and spread of finch trichomonosis in the British Isles.

Finch trichomonosis, caused by the protozoal parasite Trichomonas gallinae, was first recognized as an emerging infectious disease of British passerines in 2005. The first year of seasonal epidemic mortality occurred in 2006 with significant declines of greenfinch Carduelis chloris and chaffinch Fringilla coelebs populations. Here, we demonstrate that large-scale mortality, principally of greenfinch, continued in subsequent years, 2007-2009, with a shifting geographical distribution across the British Isles over time. Consequent to the emergence of finch trichomonosis, the breeding greenfinch population in Great Britain has declined from ca 4.3 million to ca 2.8 million birds and the maximum mean number of greenfinches (a proxy for flock size) visiting gardens has declined by 50 per cent. The annual rate of decline of the breeding greenfinch population within England has exceeded 7 per cent since the initial epidemic. Although initially chaffinch populations were regionally diminished by the disease, this has not continued. Retrospective analyses of disease surveillance data showed a rapid, widespread emergence of finch trichomonosis across Great Britain in 2005 and we hypothesize that the disease emerged by T. gallinae jumping from columbiforms to passeriforms. Further investigation is required to determine the continuing impact of finch trichomonosis and to develop our understanding of how protozoal diseases jump host species.

Evidence of spread of the emerging infectious disease, finch trichomonosis, by migrating birds.

Finch trichomonosis emerged in Great Britain in 2005 and led to epidemic mortality and a significant population decline of greenfinches, Carduelis chloris and chaffinches, Fringilla coelebs, in the central and western counties of England and Wales in the autumn of 2006. In this article, we show continued epidemic spread of the disease with a pronounced shift in geographical distribution towards eastern England in 2007. This was followed by international spread to southern Fennoscandia where cases were confirmed at multiple sites in the summer of 2008. Sequence data of the ITS1/5.8S/ITS2 ribosomal region and part of the small subunit (SSU) rRNA gene showed no variation between the British and Fennoscandian parasite strains of Trichomonas gallinae. Epidemiological and historical ring return data support bird migration as a plausible mechanism for the observed pattern of disease spread, and suggest the chaffinch as the most likely primary vector. This finding is novel since, although intuitive, confirmed disease spread by migratory birds is very rare and, when it has been recognised, this has generally been for diseases caused by viral pathogens. We believe this to be the first documented case of the spread of a protozoal emerging infectious disease by migrating birds.

Trichomonad infection in endemic and introduced columbids in the Seychelles.

Island endemic avifaunas face many threats, including the now well-documented impacts of pathogens. The impacts of pathogens on the endemic Seychelles avifauna, however, have been little studied. The protozoan parasite Trichomonas gallinae has been shown to reduce survival and reproductive success of the endemic Pink Pigeon Columba mayeri on the nearby island of Mauritius. I investigated trichomonad infection prevalence and pathogenicity in endemic Seychelles Blue Pigeons, Alectroenas pulcherrima, and two introduced species of columbid, the Madagascar Turtle-dove, Streptopelia picturata, and the Barred Ground Dove, Geopelia striata, on the Seychelles island of Mahé during September-October 2007. I asked whether: 1) trichomonad infections occur in these species; 2) prevalence varies among species; and 3) birds show any signs of pathogenicity consistent with tricho-monosis. I use the results to assess the potential threat of this pathogen to A. pulcherrima. All three species were infected with trichomonads, and the overall prevalence was 27.5%. Alectroenas pulcherrima had higher prevalence (47.1%) than the two introduced species combined (24.3%). No infected individuals showed any signs of disease. These findings suggest that trichomonad parasites should be considered as a potential disease threat to the A. pulcherrima population.

A clonal strain of Trichomonas gallinae is the aetiologic agent of an emerging avian epidemic disease.

Trichomonas gallinae is a protozoan parasite that is well characterised as a cause of trichomonosis in columbid and raptor species world-wide. The parasite emerged as a novel infection of British passerines in 2005, leading to epidemic mortality associated with significant declines of breeding populations of greenfinches (Carduelis chloris) and chaffinches (Fringilla coelebs). We characterised the extent of T. gallinae genotypic heterogeneity within the affected wild British avifauna by analysing individual isolates from 17 of the species affected. To do so, we employed improved platform-based multilocus typing tools as well as the hydrogenosomal Fe-hydrogenase gene as a single marker locus for fine-typing. We found no evidence of heterogeneity amongst the parasites infecting British passerines, indicating that a clonal strain of T. gallinae is the causative agent of this emerging infectious disease.

Effects of Tritrichomonas muris on the mouse intestine: a proteomic analysis.

Although Tritrichomonas muris is a common parasite often detected in experimental animals including mice, its pathogenesis in host animals remains unclear. Proteomics can be used to specifically analyze biochemical host-parasite interaction and immune responses of the host to parasites. However, proteomics have not yet been applied to T. muris studies. Here, the effects of T. muris on the host were analyzed by proteomics. We found that 10 different proteins were expressed in T. muris-infected mice intestines compared with non-infected intestines. The identified proteins represented several functions mainly related to stress, immune response, metabolism and signal transduction. The results suggest that T. muris infection may affect processes that are acclimatizing to the environmental changes caused by the infection in the mouse intestine.