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.

Ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no estado de Pernambuco, Brasil / Infection occurrence of Campylobacter fetus subsp. venerealis and Tritrichomonas foetus in buffaloes in the state of Pernambuco, Brazil

Objetivou-se com estudo determinar a ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no estado de Pernambuco, Brasil. Foram coletadas 133 amostras biológicas (muco cervicovaginal e raspado prepucial) de animais, procedentes de oito propriedades, de diferentes regiões do estado. O material biológico coletado foi transferido para solução salina tamponada (PBS) e, posteriormente, inoculado em meios de transporte específicos, Lander para diagnóstico de C. fetus subsp. venerealis e Diamond para T. foetus. Para o diagnóstico das infecções por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus, as amostras foram submetidas à reação em cadeia da polimerase (PCR) e cultivadas em meio ágar Columbia acrescido de antibiótico e Diamond, respectivamente. Para pesquisa de C. fetus subsp. venerealis, observou-se uma ocorrência de 1,8% (2/113) de animais positivos no exame microbiológico com confirmação pela PCR. Em relação à procedência, observou-se que 100% das amostras positivas pertenciam a dois machos do mesmo rebanho. Nenhum animal foi positivo na pesquisa de T. foetus. Este é o primeiro registro da infecção por C. fetus subsp. venerealis em búfalos no Brasil. Apesar da baixa ocorrência, recomenda-se adoção de medidas de controle, com o intuito de se evitar a disseminação do agente para outros rebanhos.(AU)

The objective this study was to determine the occurrence of infection with Campylobacter fetus subsp. venerealis and Tritrichomonas foetus in buffaloes in the State of Pernambuco, Brazil. Biological samples were collected (cervico vaginal mucus and shaved prepucial) of 113 animals, coming from 8 properties in different regions of the state. The biological material collected was transferred into phosphate buffered saline (PBS) and inoculated in the specific transport, Lander for diagnosis of C. fetus subsp. venerealis and Diamond for T. fetus subsequently. For the diagnosis of infection by Campylobacter fetus subsp. venrealis and Tritrichomonas foetus the samples were submitted to Polymerase Chain Reaction (PCR) grown in Columbia agar plus antibiotics and Diamond, respectively. There was an occurrence of 1.8% (2/113) of positive animals in the microbiological examination with confirmation by PCR, for C. fetus subsp. venerealis. We observed that 100% of positive samples were from two (2) males from the same herd. No animals were positive for T. foetus. This is the first report of infection with C. fetus subsp. venerealis in buffaloes in Brazil. Despite rare occurrence, control measures are recommended in order to prevent the spread of the agent to other herds.(AU)

Cysteine protease 30 (CP30) contributes to adhesion and cytopathogenicity in feline Tritrichomonas foetus.

Tritrichomonas foetus (T. foetus) is a flagellated protozoan parasite that is recognized as a significant cause of diarrhea in domestic cats with a prevalence rate as high as 30%. No drugs have been shown to consistently eliminate T. foetus infection in all cats. Cysteine proteases (CPs) have been identified as mediators of T. foetus-induced adhesion-dependent cytotoxicity to the intestinal epithelium. These CPs represent novel targets for the treatment of feline trichomonosis. However, cats also produce CPs that are part of life-critical systems. Thus, parasitic CPs need to be selectively targeted to reduce the potential for host toxicity. Previous studies have demonstrated the importance of a specific CP, CP30, in mediating bovine and human trichomonad cytopathogenicity. This CP has also recently been identified in feline T. foetus, although the function of this protease in the feline genotype remains unknown. Therefore, the study objectives were to characterize the presence of CP30 in feline T. foetus isolates and to evaluate the effect of targeted inhibition of CP30 on feline T. foetus-induced adhesion dependent cytotoxicity. The presence of CP30 in feline T. foetus isolates was identified by In gel zymography and proteomic analysis, indirect immunofluorescence (IF), and flow cytometry using a rabbit polyclonal antibody that targets bovine T. foetus CP30 (α-CP30). The effect of inhibition of CP30 activity on T. foetus adhesion and cytotoxicity was determined using CFSE-labeled feline T. foetus and crystal violet spectrophotometric assays in a previously validated co-culture model. CP30 expression was confirmed in all feline T. foetus isolates tested by all assays. Targeted inhibition of feline T. foetus CP30 resulted in decreased T. foetus adhesion to and cytotoxicity towards IPEC-J2 monolayers compared to rabbit IgG-treated T. foetus isolates. These studies establish that CP30 is expressed by feline T. foetus isolates and may be an important virulence factor in the cytopathogenicity of feline T. foetus. The results of these studies provide strong evidence-based justification for investigation of CP30 as a novel target for the treatment of feline trichomonosis.

A review of sexually transmitted bovine trichomoniasis and campylobacteriosis affecting cattle reproductive health.

The objective is to discuss sexually transmitted diseases caused by Tritrichomonas foetus (T foetus) and Campylobacter fetus (C fetus) subsp. venerealis, with a focus on prevalence, pathogenesis, and diagnosis in cows and bulls. Diagnosis and control are problematic because these diseases cause severe reproductive losses in cows, but in bulls are clinically asymptomatic, which allows the disease to flourish, especially in the absence of legislated control programs. We review research regarding prophylactic systemic immunization of bulls and cows with antigens of T foetus and C fetus venerealis and their efficacy in preventing or clearing preexisting infections in the genital tract. Current diagnostic methods of C fetus venerealis and T foetus (microbial culture and PCR) should be improved. Review of the latest advances in bovine trichomoniasis and campylobacteriosis should promote knowledge and provide an impetus to pursue further efforts to control bovine sexually transmitted diseases.

Apoptosis and cell proliferation in the mouse model of embryonic death induced by Tritrichomonas foetus infection.

Bovine tritrichomonosis is a sexually transmitted disease caused by the protozoon Tritrichomonas foetus and characterised by embryonic-death and abortion. During pregnancy, the processes of cell proliferation and death play a crucial role for blastocyst implantation and the subsequent maintenance of early pregnancy, and their misbalance may lead to the abortion. In this study, we aimed to investigate whether cell proliferation and death may be altered during tritrichomonosis. For this purpose, we used pregnant BALB/c mice as an alternative experimental animal model that has successfully reproduced the infection. We analysed the immunohistochemical expression of active caspase-3 and proliferating cell nuclear (PCNA) antigens in the endometrium of infected mice. We found an increase in the number of caspase-3 positive cells in infected mice that were not pregnant at the necropsy. Besides, the number of positive proliferating cells increased in the uterine luminal epithelium of infected animals killed at 5-7 days post coitum (dpc). Pregnant infected mice killed at 8-11 dpc showed higher proliferation than control animals. We suggest that the cytopathic effect induced by T. foetus in the uteri of infected mice may induce the apoptosis of the epithelial cells and, as a result, promote a compensatory proliferative response. The information described here will be helpful to further study the pathogenesis of the bovine tritrichomonosis.

Efficacy of ronidazole for treatment of cats experimentally infected with a Korean isolate of Tritrichomonas foetus.

To evaluate the efficacy of ronidazole for treatment of Tritrichomonas foetus infection, 6 Tritrichomonas-free kittens were experimentally infected with a Korean isolate of T. foetus. The experimental infection was confirmed by direct microscopy, culture, and single-tube nested PCR, and all cats demonstrated trophozoites of T. foetus by day 20 post-infection in the feces. From day 30 after the experimentally induced infection, 3 cats were treated with ronidazole (50 mg/kg twice a day for 14 days) and 3 other cats received placebo. Feces from each cat were tested for the presence of T. foetus by direct smear and culture of rectal swab samples using modified Diamond's medium once a week for 4 weeks. To confirm the culture results, the presence of T. foetus rRNA gene was determined by single-tube nested PCR assay. All 3 cats in the treatment group receiving ronidazole showed negative results for T. foetus infection during 2 weeks of treatment and 4 weeks follow-up by all detection methods used in this study. In contrast, rectal swab samples from cats in the control group were positive for T. foetus continuously throughout the study. The present study indicates that ronidazole is also effective to treat cats infected experimentally with a Korean isolate of T. foetus at a dose of 50 mg/kg twice a day for 14 days.

Cytotoxic effects exerted by Tritrichomonas foetus pseudocysts.

The protozoan parasite Tritrichomonas foetus displays a pear-shaped form and a pseudocyst stage. However, little is known about the biology of the pseudocyst. The aim of this work was to assess whether pseudocysts exert cytotoxic effects during their interaction with MDCK cells (an epithelial kidney canine cell line) and compare their behavior to that of the pear-shaped parasites. Pseudocysts and pear-shaped parasites from both cultured and freshly isolated T. foetus were used. Electron microscopy revealed that the epithelial cells exhibited more signs of injury, such as depletion of microvilli, retraction from neighboring cells and swollen mitochondria with loss of electron density in the matrix, when the pseudocysts were used in interaction experiments. In addition, during the co-incubation with MDCK cells, pseudocysts exhibited a more intense amoeboid transformation than that found in pear-shaped parasites. The MTT viability assay demonstrated that the pseudocysts were more cytotoxic when in contact with host cells as compared to the flagellated pear-shaped parasites. The JC-1 viability assay revealed that pseudocysts induced a higher loss of mitochondrial membrane potential compared to pear-shaped parasites. Pseudocysts undergoing a budding process were observed after 2.5h of co-incubation with MDCK cells. Our results suggest that the T. foetus pseudocyst might be a more aggressive form.

Effect of vinyl sulfone inhibitors of cysteine proteinases on Tritrichomonas foetus infection.

Tritrichomonas foetus is a sexually transmitted protozoon that causes genital inflammation and adverse pregnancy outcomes in cattle. Cysteine proteinases (CPs) released by T. foetus degrade immunoglobulin G (IgG) antibodies, complement component 3 and matrix proteins as well as inducing apoptosis of bovine genital epithelial cells. In this study, the efficacies of the vinyl sulfone CP inhibitors K11777 and WRR-483 were tested against CPs of T. foetus. The activity of secreted T. foetus CPs in culture supernatants was decreased in the presence of vinyl sulfone inhibitors. Inhibitor K11777 reduced the in vitro cytopathogenic effects of T. foetus in bovine foetal trophoblast cells, which are relevant target cells since this pathogen interferes with pregnancy. Pre-treatment of T. foetus prior to intravaginal inoculation diminished genital infection in a murine model. Therefore, vinyl sulfone CP inhibitors reduce several effects of T. foetus-secreted CPs, including cytotoxicity on relevant target host cells and genital infection in a murine model. These inhibitors have potential as chemotherapeutic agents against bovine trichomoniasis. Generalisation to human trichomoniasis requires further study.

Immunity to infections in the lower genital tract of bulls.

The mucosa of the bovine prepuce has unique immunological characteristics critical to defense against sexually transmitted diseases. Tritrichomonas foetus and Campylobacter fetus subspecies venerealis persistently colonize the lower genital tract of bulls but usually do not cause either major clinical signs or inflammation. These microbes may be sexually transmitted to female cattle to cause reproductive failure. Although the male genital immune responses to T. foetus and C. fetus subspecies venerealis are inefficient in clearing infection, systemic immunization with T. foetus and C. fetus subspecies venerealis antigens does prevent or eliminate these infections with induction of IgG antibodies in genital secretions and serum.

The binding of Tritrichomonas foetus to immobilized laminin-1 and its role in the cytotoxicity exerted by the parasite.

The recognition and binding of pathogens to extracellular matrix glycoproteins may determine the outcome of infective processes. The interaction between the bovine urogenital parasite Tritrichomonas foetus and the major basal membrane glycoprotein laminin-1 (LMN-1) was investigated. The chemical nature of parasite molecules involved in the attachment of T. foetus to immobilized LMN-1 and the influence of LMN-1 in the toxicity exerted by the parasite to HeLa cells was studied. Attachment of T. foetus to LMN-1 resulted in notable morphological alterations of the parasite, which became amoeboid. T. foetus recognized LMN-1 through specific amino acid sequences (AG73, C16, A208 and A13) in the LMN-1 molecule, and the protein nature of the parasite molecules involved in the recognition was demonstrated by dot-blot analyses. Such molecular recognition was cation-dependent and five LMN-1-binding molecules (220, 200, 130, 125 and 80 kDa) were identified in T. foetus. Binding of T. foetus to LMN-1 rendered the parasite toxic to HeLa cell monolayers. Thus, LMN-1 appears to provide signalling cues that mediate important cell functions in T. foetus concerning its interaction with host cells.

Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence.

The effective acquisition of iron is a pre-requisite for survival of all organisms, especially parasites that have a high iron requirement. In mammals, iron homeostasis is meticulously regulated; extracellular free iron is essentially unavailable and host iron availability has a crucial role in the host-pathogen relationship. Therefore, pathogens use specialized and effective mechanisms to acquire iron. In this review, we summarize the iron-uptake systems in eukaryotic unicellular organisms with particular focus on the pathogenic species: Candida albicans, Tritrichomonas foetus, Trypanosoma brucei and Leishmania spp. We describe the diversity of their iron-uptake mechanisms and highlight the importance of the process for virulence.

Comparative histopathology and antibody responses of non-Tritrichomonas foetus trichomonad and Tritrichomonas foetus genital infections in virgin heifers.

The potential pathogenicity of non-Tritrichomonas foetus trichomonads (NTfTs) recently isolated from the prepuce of virgin bulls is not known. The purpose of this study was to determine the ability of these NTfTs to cause disease in the female reproductive tract relative to T. foetus. Forty-four virgin heifers were experimentally infected intravaginally with either one of two NTfTs (Pentatrichomonas hominis or Tetratrichomonas spp.), T. foetus, or sterile media and cultured weekly from 0 time until slaughter at 8 weeks. Serum and vaginal antibody responses during infection were assessed, and the reproductive tracts were histologically examined, scored, and compared based on numbers of neutrophils, eosinophils, lymphocytes, and plasma cells as well as the qualitative appearance of the reproductive tract. The NTfTs did not persist in the reproductive tract, while T. foetus persisted for at least 6-8 weeks. Further, no vaginal IgA response to infection was found in NTfT-infected and control heifers, but a vaginal IgA response was present in the T. foetus-infected group. Heifers infected with NTfT or controls showed little mucosal inflammatory response compared to T. foetus-infected heifers. Among the trichomonads studied, persistent infection by T. foetus alone seems responsible for uterine inflammatory lesions usually associated with pregnancy loss. The NTfTs studied in this work only transiently infected the vagina and were associated with strictly mild inflammatory changes, which probably do not cause significant disease, i.e., pregnancy loss.

Tritrichomonas foetus: pathogenesis of acute infection in normal, estradiol-treated, and stressed mice.

Environmental stress and endocrine control can affect pathogenesis of sexually transmitted diseases such as trichomoniasis. Acute Tritrichomonas foetus infection was compared in female BALB/c mice to infections in mice treated with high doses of estradiol or housed in constant bright illumination (stressed). In untreated mice, T. foetus readily colonized the reproductive tract, causing minimal epithelial damage and inflammation. Several fold increases of IFN-gamma, TNF-alpha, MCP-1, and IL-6 cytokines were detected after estradiol-treatment of mice, resulting in greatly enhanced inflammation and tissue damage throughout the reproductive tract. Interestingly, estradiol-treatment of mice resulted in reduced T. foetus colonization compared to untreated mice. Infection in stressed mice resulted in increased tissue damage, inflammation, and inflammatory cytokine expression, although parasite colonization within the reproductive tract was similar to that in untreated mice. These results indicate that either estradiol-treatment or stress result in pathogenesis often observed during severe disease. Alternatively, infection in non-treated mice results in chronic colonization, with little inflammation or pathology.

Interaction of Tritrichomonas foetus and the bovine oviduct in an organ culture model.

Tritrichomonas foetus is an extracellular parasite of the reproductive tract in cattle. The mechanism by which T. foetus causes abortion in cattle is largely unknown. There are no studies of infection in the cow oviducts, almost all published papers are related to vagina infection and few articles focusing on the uterus. The aim of the present study was to establish a working model of bovine oviduct epithelial cells and submit these cells to Tritrichomonas foetus interaction. Twenty bovine oviducts were obtained from cows at a commercial abattoir and T. foetus was injected through the isthmus into the oviduct lumen. The whole oviduct was analyzed by scanning and transmission electron microscopy. The results reported here demonstrate that: (1) fresh whole oviducts can be used as a good model to study parasite-host cell interaction; (2) cow oviduct epithelium has been shown to consist of two cell types: ciliated and nonciliated secretory cells, and T. foetus displayed great specificity for the nonciliated cells localized in the deeper oviduct folds; (3) T. foetus adheres as single separate cells, and maintains the flagella externalized; (4) differently from T. vaginalis, T. foetus does not change its shape during the adhesion process; and (5) oviduct cells exhibited morphological characteristics of apoptosis after trichomonadal interaction.

Molecular identification of Tritrichomonas foetus-like organisms as coinfecting agents of human Pneumocystis pneumonia.

Trichomonads closely related to the bovid parasite Tritrichomonas foetus were identified in the bronchoalveolar lavage sample from a patient with AIDS in association with Pneumocystis pneumonia. This human case of T. foetus-like infection emphasizes the zoonotic potential of trichomonads, although the existence of a human-host-adapted T. foetus strain cannot be excluded.

Intra-strain clonal phenotypic variation of Tritrichomonas foetus is related to the cytotoxicity exerted by the parasite to cultured cells.

As observed in most of the investigated trichomonads, a strain of Tritrichomonas foetus includes different parasite subpopulations. Such population diversity might account for important properties such as the ability of the parasite to destroy host cells. The aim of this study was to characterize the cytotoxicity exerted by subpopulations (named as K1, K2, K3, K4 and K5) of an isolate of T. foetus on epithelial cultured cells. The five populations studied here destroyed epithelial monolayers at different rates (from 25% to 55%), even though the cytoadhesion level and whole-cell protease activity were closely related among them. We were also able to detect differences in contact-dependent and contact-independent cytotoxicity mechanisms among the five populations. An extracellular parasite protease had varying activity among the parasite populations. The intensity of contact-independent cytotoxicity was strictly related to the degree of enzyme activation, suggesting that such a protease might be involved in the cytotoxicity mediated by T. foetus.

Outcome of cats with diarrhea and Tritrichomonas foetus infection.

OBJECTIVE: To determine the long-term outcome of cats infected with Tritrichomonas foetus and identify treatment and management strategies influencing resolution of infection or associated diarrhea. DESIGN: Prospective study. SAMPLE POPULATION: 26 cats with T. foetus-associated diarrhea at least 22 months prior to the study. PROCEDURE: A standardized survey regarding clinical course and management was administered to owners of cats with T. foetus infection and associated diarrhea. Fecal samples were obtained from each cat; the presence of T. foetus was assessed via microscopic examination of smears, culture in commercial media, and polymerase chain reaction amplification of T. foetus rDNA involving species-specific primers. RESULTS: Survey responses were obtained from owners of all 26 cats. Twenty-three cats had complete resolution of diarrhea a median of 9 months after onset. Analysis of fecal samples obtained from 22 cats revealed persistent T. foetus infection in 12, with a median of 39 months after resolution of diarrhea. History of implementation of a dietary change, treatment with paromomycin, or higher numbers of cats in the household was associated with significantly longer duration of time to resolution of diarrhea. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested chronic T. foetus-associated diarrhea in most cats is likely to resolve spontaneously within 2 years of onset. Chronic infection with T. foetus (without clinical signs) after resolution of diarrhea appears to be common. Although often temporarily effective in decreasing severity of diarrhea, attempts to treat cats with T. foetus infection may result in prolongation of time to resolution of diarrhea.

Tritrichomonas foetus pseudocysts adhere to vaginal epithelial cells in a contact-dependent manner.

Tritrichomonas foetus is a parasitic protist of the urogenital tract of cattle. It presents the trophozoite stage, a motile elongated form that constitutes most of the cells in a normal population, and a pseudocyst stage, an immotile rounded form that appears under unfavourable environmental conditions. In the present report pseudocysts were studied in natural conditions and after induction by chemicals or cycles of cooling and warming of cultures. The capacity of T. foetus to adhere to vaginal epithelial cells (VECs) was compared for both trophozoite and pseudocyst forms. By the use of video-enhanced-contrast microscopy, scanning and transmission electron microscopy, and immunofluorescence microscopy techniques, we present evidence that: (1) T. foetus easily internalizes the flagella and forms pseudocysts under several unfavourable conditions; (2) T. foetus in both pseudocyst and trophozoite forms is able to adhere to VECs; (3) the adhesion rate is higher for pseudocysts than for trophozoites; (4) the adhesin Tf190 is expressed in both forms during interaction; (5) the adhesion process of pseudocysts seems to occur in a contact-dependent manner. Thus, we propose that the pseudocyst stage is not a degenerative form, but a functional life form that is able to interact with and firmly adhere to VECs.

Detection of Tritrichomonas foetus by polymerase chain reaction in cultured isolates, cervicovaginal mucus, and formalin-fixed tissues from infected heifers and fetuses.

A rapid, reliable polymerase chain reaction (PCR) assay, originally developed for definitive laboratory identification of the bovine venereal pathogen Tritrichomonas foetus from cultures of male reproductive tract fluids, was used for testing the following: 1) cultured, geographically disparate trichomonad isolates, 2) formalin-fixed tissues from infected heifers and naturally infected fetuses, and 3) cervicovaginal mucus (CVM) from experimentally infected females. In 12 of 12 Western Hemisphere isolates of pathogenic T. foetus (isolated from outbreaks of clinical trichomoniasis or from screening surveys) and in 1 of 1 American Type Culture Collection strain of Tritrichomonas suis, PCR yielded a positive result, i.e., a 347-base pair amplicon in the 5.8S ribosomal RNA and internal transcribed spacer (5.8S-ITS) region of the genome, whereas cultures of Trichomonas vaginalis and Trichomonas gallinae did not produce a PCR product. The PCR assay was also positive in formalin-fixed, paraffin-embedded endometrial samples from 4 of 4 experimentally infected heifers, as well as in archived tissues from 2 of 2 T. foetus-infected aborted bovine fetuses that were submitted to the diagnostic laboratory from a natural outbreak. It was negative in fixed, embedded uterine tissues of 2 of 2 uninfected virgin heifers used as negative controls and in archived fixed gut tissue of a T. gallinae-infected pigeon. In another experiment, CVM aspirated from 4 of 4 experimentally infected heifers in the fifth or sixth postinfection week yielded a positive PCR product of the expected size, whereas CVM from 2 of 2 controls were PCR negative. Pending validation in larger clinical studies, the PCR assay for the 5.8S-ITS coding region of the T. foetus genome offers the prospect of definitive identification of this agent directly from CVM or from formalin-fixed tissues or when false-positive culture results are suspected.