In vitro efficacy testing of ivermectin against Tritrichomonas foetus trophozoites.

There are no recommended drugs to treat cattle infected with the protozoan Tritrichomonas foetus (TF). Ivermectin, widely used in the treatment of intestinal parasites, was found effective against some protozoa growing in vitro. Here, its effectiveness against a TF line was investigated. Trophozoites were incubated in media with increasing concentrations of ivermectin and mortality was determined after 24 h. Ivermectin killed cells with a mean maximum effective concentration (EC50) of 2.47 µg mL-1. The effective concentration of ivermectin was rather high for a formulation suitable for systemic treatment. However, topical treatment of animals against TF could still be considered and tested.

Bovine neutrophils kill the sexually-transmitted parasite Tritrichomonas foetus using trogocytosis.

The protozoan parasite Tritrichomonas foetus (T. foetus) is the causative organism of bovine trichomonosis (also referred to as trichomoniasis), a sexually-transmitted infection that reduces fertility in cattle. Efforts to control trichomonosis on cattle farms are hindered by the discouragement of antibiotic use in agriculture, and the incomplete, short-lived protection conferred by the current vaccines. A more complete mechanistic understanding of what effective immunity to T. foetus entails could enable the development of more robust infection control strategies. While neutrophils, the primary responders to infection, are present in infected tissues and have been shown to kill the parasite in vitro, the mechanism they use for parasite killing has not been established. Here, we show that primary bovine neutrophils isolated from peripheral blood rapidly kill T. foetus in vitro in a dose-dependent manner, and that optimal parasite killing is reduced by inhibitors of trogocytosis. We also use imaging to show that bovine neutrophils surround T. foetus and trogocytose its membrane. These findings are consistent with killing via trogocytosis, a recently described novel neutrophil antimicrobial mechanism.

Identification of miRNA Associated with Trichomonas gallinae Resistance in Pigeon (Columba livia).

Trichomonas gallinae (T. gallinae) has a great influence on the pigeon industry. Pigeons display different resistance abilities to T. gallinae, so the study of the molecular mechanism of resistance is necessary in breeding disease resistant lines. MiRNA plays important roles in the immune response, but there are still no reports of miRNA regulating trichomonosis resistance. We used small RNA sequencing technology to characterize miRNA profiles in different groups. T. gallinae was nasally inoculated in one day old squabs, and according to the infection status, the groups were divided into control (C), susceptible (S) and tolerant (T) groups. We identified 2429 miRNAs in total, including 1162 known miRNAs and 1267 new miRNAs. In a comparison among the C, S and T groups, the target genes of differentially expressed miRNAs were analyzed via GO and KEGG annotation. The results showed that the target genes were enriched in immune-response-related pathways. This indicated that the differentially expressed miRNAs had a critical influence on T. gallinae infection. Novel_miR_741, which could inhibit the expression of PRKCQ, was down-regulated in the T group compared to the C group. It was proven that a decreased novel_miR_741 expression would increase the expression of PRKCQ and increase the immune response. This study brings new insights into understanding the mechanism of trichomonosis resistance.

[Prevalence and geographical distribution of bovine sexually transmitted diseases in the province of Formosa, Argentina]. / Prevalencia y distribución geográfica de las enfermedades de transmisión sexual de los bovinos en la provincia de Formosa, Argentina.

Bovine genital campylobacteriosis (BGC) and bovine trichomonosis (BT) are sexually transmitted diseases (STDs) that affect bovine breeding herds, decreasing their reproductive efficiency. The objective of this work was to estimate the prevalence of these diseases and their temporal-spatial distribution in the province of Formosa, Argentina. The cross-sectional study conducted between 2018 and 2021 included a total of 15,571 bulls, inter-herd prevalence being 29.62% and 17.23% for BGC and BT, respectively. The prevalence of positive animals was 2.05% for BGC and 0.43% for BT. The temporal-spatial analysis of BGC showed two distinct spatial groupings, one group had a low risk of contracting the disease (RR = 0.13; p < 0.001; 2018-2021) while the other group had a high risk (RR = 2.84; p < 0.001; 2020-2021). BT had a high-risk group for the disease (RR = 35.24; p < 0.001; 2019). This study shows that STDs are endemic in the region, providing updated and valuable information as a tool for the health management of these diseases.

Assessment of in vitro efficacy for common surface disinfectants and antiseptics against Tritrichomonas foetus trophozoites.

The protozoan Tritrichomonas foetus causes early embryonic death in cattle, there are no legal options for treating this parasite in the United States, and there are few developed protocols for cleaning veterinary and obstetrical equipment that may have been contaminated with trophozoites. In this study, we evaluated bleach, ethanol, acetic acid, chlorhexidine gluconate, and hydrogen peroxide solutions for the ability to kill trophozoites in vitro. Our findings suggested that ethanol and bleach could adequately disinfect tools and equipment. Acetic acid, chlorhexidine, and hydrogen peroxide had applications as surface disinfectants in addition to potential as local topical treatments due to their past uses in veterinary theriogenology. Chlorhexidine gluconate demonstrated trophocidal effects by damaging parasite cell membranes and had the lowest effective concentration 50 (EC50) of any compound tested and was in the micromolar range. These findings, in conjunction with accepted clinical uses of chlorhexidine gluconate suggest that this is a convenient agent for disinfecting equipment. In addition, topical use of chlorhexidine is relatively common, setting the stage for further investigation of this compound as a topical therapeutic option for bovine trichomonosis.

True molecular phylogenetic position of the cockroach gut commensal Lophomonas blattarum (Lophomonadida, Parabasalia).

Lophomonas blattarum is a facultative commensal gut dweller of common pest cockroaches. Its cells are roughly spherical in shape with an apical tuft of ~50 flagella. Controversially, it has been implicated in human respiratory infections based on light microscopic observations of similarly shaped cells in sputum or bronchoalveolar lavage fluid. Here, we have sequenced the 18S rRNA gene of L. blattarum and its sole congener, Lophomonas striata, isolated from cockroaches. Both species branch in a fully supported clade with Trichonymphida, consistent with a previous study of L. striata, but not consistent with sequences from human samples attributed to L. blattarum.

Proteomic analysis of proteins released by Tritrichomonas foetus: Identification of potential targets for the development of new diagnostic methods.

Bovine trichomonosis (BT), a disease of the bovine urogenital tract, is caused by the protozoan Tritrichomonas foetus (Tf). Tf causes endometritis, infertility, and premature death of the embryo, which generates considerable economic losses. The proteins released can mediate fundamental interactions between the pathogen and the host, triggering factors associated with the symptomatology, immune evasion and pathogenesis characteristic of the species. However, little is known about the profile of the proteins released by Tf. In order to contribute to their knowledge, we performed an isolation protocol and a proteomic profiling of the supernatant (SN) content of six Tf isolates. A total of 662 proteins present in the SN of Tf were detected, out of which 121 were shared by the six isolates, while the remaining 541 were found in at least one of the isolates studied. The comparative analyses using the databases of Tf strain genome K revealed 32.9% of uncharacterized proteins. The bioinformatic analyses showed that the main molecular functions predicted were binding (47.9%) and catalytic activity (38.2%). Additionally, we performed immunodetection assays to evidence the antigenic potential of SN proteins. Interestingly, we observed great ability to detect SN proteins from all six isolates using serum from immunized mice and infected bulls. A complementary mass spectrometry assay allowed us to determine that the proteins that showed the strongest signal intensity in the immunoassays were Grp78 (A0A1J4IZS3) and Ap65 (A0A1J4JSR1). This work represents the first proteomic characterization of Tf SN proteins and their antigenic potential, which might be interesting for the future design of new diagnosis and treatment methods for BT.

Avian Oropharyngeal Trichomonosis: Treatment, Failures and Alternatives, a Systematic Review.

Oropharyngeal avian trichomonosis is a potentially lethal parasitic disease that affects several avian orders. This review is focused on the disease treatments since prophylactic treatment is prohibited in most countries and resistant strains are circulating. A systematic review following the PRISMA procedure was conducted and included 60 articles. Successful and non-toxic treatments of avian oropharyngeal trichomonosis started with enheptin, a drug replaced by dimetridazole, metronidazole, ornidazole, carnidazole and ronidazole. Administration in drinking water was the most employed and recommended method, although hierarchy of the avian flocks and palatability of the medicated water can interfere with the treatments. Besides pigeons, treatments with nitroimidazoles were reported in budgerigars, canaries, finches, bald eagles, a cinereous vulture and several falcon species, but resistant strains were reported mainly in domestic pigeons and budgerigars. Novel treatments include new delivery systems proved with traditional drugs and some plant extracts and its main components. Ethanolic extracts from ginger, curry leaf tree and Dennettia tripetala, alkaloid extracts of Peganum harmala and essential oils of Pelargonium roseum and some Lamiaceae were highly active. Pure active compounds from the above extracts displayed good anti-trichomonal activity, although most studies lack a cytotoxicity or in vivo test.

A new inactivated Tritrichomonas foetus vaccine that improves genital clearance of the infection and calving intervals in cattle.

Bovine trichomonosis is a sexually transmitted disease that is a primary cause of early reproductive failure in cattle. The aim of the present study was to develop a vaccine formulation based on Tritrichomonas foetus trophozoites inactivated by lyophilization and Quil-A-adjuvanted. The safety, immunogenicity and efficacy of this new vaccine formulation (Trichobovis®) administered by two routes (subcutaneous: SC, and intravulvar: IVU) were compared with a commercial vaccine (TrichGuard®) in a well-established experimental bovine model of genital T. foetus infection. The new vaccine was considered safe in cattle because only mild local reactions were found in the vaccination area, which disappeared 3 weeks after administration. Cows immunized with Trichobovis cleared the infection faster than the non-immunized/challenged group (27-28 vs. 60 days; P < 0.05). Not significant differences were observed with the commercial vaccine respect to the positive control group, or between SC and IVU routes. The new vaccine stimulated high serum anti-T. foetus IgG and genital IgA levels and generated an IgG booster effect similar to TrichGuard. IgA levels were associated with significantly earlier genital clearance of T. foetus in cows immunized with Trichobovis by SC route (G1A) or TrichGuard (G2). The strongest association was found in the group G1A on day 14 post-infection (p.i.) (r = -0.74) and in G2 on day 35 p.i. (r = -0.71). The efficacy of vaccination using Trichobovis on the reproductive performance was also investigated under field conditions in a herd where T. foetus was present. The calving intervals were significantly reduced by 45.2 days (P < 0.05), calves were born 28 days earlier (P < 0.05) and an increase of 8.7% in the calving rate (P > 0.05) was observed in the vaccinated group. These results demonstrate that Trichobovis improved the reproductive performance under field conditions in herds where T. foetus infection is present.

Adaptation of the classical end-point ITS-PCR for the diagnosis of avian trichomonosis to a real-time PCR reveals Bonelli's eagle as a new host for Trichomonas gypaetinii.

Avian trichomonosis is a parasitic disease caused mainly by Trichomonas gallinae and other Trichomonas species. It can be asymptomatic, or it can produce a necrotic lesion in the upper digestive tract and spread to other organs, causing the death of the infected birds. In this study, we aimed to evaluate an adapted real-time PCR method for the diagnosis of different genotypes and species of avian oropharyngeal trichomonads. Fifty-six samples from the oropharynx of Bonelli's eagles (Aquila fasciata) obtained between 2018 and 2019 were analyzed using the real-time PCR and the end-point PCR, both targeting trichomonads ITS, and the results were compared by a coefficient of agreement. All positive samples were sequenced. The analysis showed a higher percentage of detection of real-time PCR ITS compared with end-point PCR ITS (64.3 vs 55.4%), and good agreement value (Kappa = 0.816). Melting temperature value for resulting amplicons of real-time PCR for avian trichomonads was 83.45 ± 0.72 °C. Genotypes A, D, and III were found among the sequences. Moreover, Trichomonas gypaetinii, a common species in scavenger birds, is reported for the first time in Bonelli's eagles.

Molecular prevalence of Tetratrichomonas gallinarum and Trichomonas gallinae in three domestic free-range poultry breeds in Anhui Province, China.

Tetratrichomonas gallinarum and Trichomonas gallinae can colonize the alimentary tract of domestic birds. However, little information is available on the epidemiology of the two trichomonad species in domestic free-range poultry in China. In this study, the occurrence and genetic characteristic of T. gallinarum and T. gallinae among free-range chickens, ducks, and geese in Anhui Province, China, were investigated. The 1910 fecal samples collected from 18 free-range poultry farms throughout Anhui Province were examined for the presence of T. gallinarum and T. gallinae by PCR and sequence analysis of the small subunit (SSU) rRNA gene of T. gallinarum and ITS1-5.8S-ITS2 sequence of T. gallinae. The overall occurrence of T. gallinarum in poultry was 1.2% (22/1910), with infection rates of 2.1% (17/829) in chickens, 0.2% (1/487) in ducks, and 0.7% (4/594) in geese. The constructed phylogeny tree using the concatenated ITS1-5.8S-ITS2 region and SSU rRNA indicated the T. gallinarum isolates detected in this study were closely related to previously defined genogroups A, D, and E, respectively. Nine (0.5%) fecal samples were positive for T. gallinae, with infection rates of 0.8% (7/829) in chickens, 0.4% (2/487) in ducks, and 0% (0/594) in geese. Sequence and phylogenetic analysis showed that four T. gallinae ITS1-5.8S-ITS2 sequences obtained from chicken feces and one duck fecal sample belonged to genotype ITS-OBT-Tg-1. This is the first report of the prevalence and genetic characterization of T. gallinarum and T. gallinae in free-range chickens, ducks, and geese in China.

Trichomonosis in Austrian Songbirds-Geographic Distribution, Pathological Lesions and Genetic Characterization over Nine Years.

In the early summer of 2012, sudden mass mortality among songbirds, particularly in greenfinches (Chloris chloris, syn: Carduelis chloris) was observed in Austria, which was caused by the protozoan parasite Trichomonas gallinae. This pathogen induced fibrinonecrotic ingluvitis and/or esophagitis, leading to impairment of food intake and ultimately death due to starvation. The pathogen was successfully detected within the lesions by polymerase chain reaction (PCR) and chromogenic in situ hybridization. The epizootic resulted in a significant decline in the Austrian greenfinch population. Continuing passive surveillance in the subsequent years (2013-2020) revealed that the condition occurred each year and was present in the entire country. Genetic characterization of the pathogen showed the presence of an identical strain irrespective of geographical location, bird species, and year.

Genetic characterization of Trichomonas gallinae (Rivolta, 1878) in companion birds in Japan and the genotypical relationship in the Asia region.

BACKGROUND/PURPOSE: Avian trichomonosis is a parasitic infection that affects a wide range of avian species, including free-ranging and pet birds worldwide, and Trichomonas gallinae has been considered as the only causative agent for decades. The sequence of the 5.8S ribosomal RNA with internal transcribed spacer (ITS) regions was widely used for identifying genotypes and determining inter-specific and intra-specific diversity. Moreover, the sequence of Fe-hydrogenase (FeHyd) was proposed as the second genetic marker for providing improved resolution of strain subtyping discrimination. Though the correlation between genetic variability and strain virulence is controversial, FeHyd analyses seemed to be useful to investigate the host or geographic origin of isolates. This study aimed to investigate the genetic characteristics of avian Trichomonas spp. METHODS: Forty-seven oral swabs and crop lavage fluids were collected from 9 avian genera, which were diagnosed as Trichomonas-positive by microscopy in animal hospitals in Japan, were analyzed. RESULTS: Genetic analysis of clonal isolates revealed the prevalence of the single genotype, ITS-OBT-Tg-1, by ITS region analysis, while two different subtypes, A2 and novel A3, were suggested by FeHyd gene analysis among Japanese companion birds. Phylogenetic analyses of available ITS sequences obtained from the Asia region (China, Iran, Iraq, and Saudi Arabia) were also preformed, revealing endemic ITS-OBT-Tg-1, ITS-OBT-Tg-2, ITS-OBT-Ttl-1, genotype III, and Saudi Arabia's unique lineages. Furthermore, ITS-OBT-Tg-2 predominance in these countries indicates different strains origination from Japan. CONCLUSION: This is the first report of the genetic characterization of T. gallinae in Japan with discovery of novel subtype A3.

Control and eradication of bovine trichomonosis in Wyoming, USA by testing and culling positive bulls.

Bovine trichomonosis is caused by Tritrichomonas foetus. Thirty-three US states have state rules on this disease and render it reportable due to potential huge economic losses to cattle industry. The various rules of different states generally mandate testing and culling T. foetus-positive bulls as well as prohibiting import of T. foetus-positive animals. Wyoming has enforced these rules for over 20 year beginning in 2000. From 2017 to 2019, 3 years in a row, not even one T. foetus-positive bull has been detected throughout the entire state among over ten thousand bulls tested annually. Wyoming is the first US state to achieve total control and eradication of bovine trichomonosis by testing and culling T. foetus-positive bulls.

To get sick or not to get sick-Trichomonas infections in two Accipiter species from Germany.

Trichomonosis caused by the flagellate Trichomonas gallinae is one of the most important avian diseases worldwide. The parasite is localised in the oesophageal area of its host and mainly infects pigeon and dove species. During the last decade, a host expansion to passerine birds occurred, making the disease a potential threat for passerine predators as naïve host species. Here, we investigated the effect of the parasite on two Accipiter species in Germany which show a comparable lifestyle but differ in prey choice, the Northern goshawk (Accipiter gentilis) mainly hunting pigeons and the Eurasian sparrowhawk (Accipiter nisus) mainly feeding on passerines. We genetically identified the parasite strains using the Fe-Hydrogenase gene as marker locus and compared the incidence of parasite presence and clinical signs of trichomonosis between nestlings of the two Accipiter species. In total, we identified 14 strains, with nine strains unknown so far. There was a higher strain diversity and prevalence of Trichomonas spp. in goshawks than sparrowhawks (42.4% vs. 21.2%) whereas sparrowhawks when being infected more often displayed clinical signs of trichomonosis than goshawks (37.1% vs. 6.1%). Even though sparrowhawks were mainly infected with the finch epidemic strain and genetic data indicated some variation between isolates, no correlation with virulence could be detected. All in all, goshawks seem to be better adapted to Trichomonas infections, whereas to sparrowhawks, this is a novel disease with more severe manifestations, from individual morbidity to a higher risk of population decline caused by trichomonosis.

Bovine Trichomonosis Cases in the United States 2015-2019.

Tritrichomonas foetus is a sexually-transmitted protozoan parasite that causes early embryonic death in cattle. Tritrichomonas foetus is enzootic in the United States but is not a reportable disease at the national level. Thus, it is difficult to understand the prevalence and relative distribution of the disease for the purpose of developing appropriate control measures. In this study, a survey of state veterinarians was used to determine the number of reported cases in each state from 2015 to 2019. Our investigation revealed infections in 25 different states and a total of 3,817 reported cases nationwide. Infections occurred throughout different regions of the country, and numbers of cases were only weakly correlated with total number of cattle in each state. Tritrichomonas foetus is a significant pathogen in the United States and understanding the relative distribution of the parasite is useful for prioritizing surveillance and intervention strategies going forward.

Taguchi method for the optimization of three loop mediated isothermal amplification procedures for Tritrichomonas foetus detection.

Taguchi method was used to optimize loop mediated isothermal amplification tests aimed to amplify segments of the elongation factor 1a1 (tf-ef1a1), the 5,8 ribosomal gene (tf-5,8 r) or the beta tubulin 2 (tf-btub2) from the protozoan parasite Tritrichomonas foetus. L9 orthogonal array and quadratic loss functions that penalize deviations from prediction values revealed the effect of amplification reaction components. Analysis of variance (ANOVA) decomposed the contribution of individual factors to a small Ct. Confirmation experiments established that optimum conditions were predictable, verifiable and reproducible. Primers concentration conditioned the non-specific amplification of tf-ef1a1 while betaine and magnesium concentration contributed to accelerate the time to reach a positive threshold in tf-ef1a1, tf-5,8 r and tf-btub2. The general strategy of simple and robust experimental design holds potential as a general optimization protocol for LAMP tests in every diagnostic laboratory.

Not gone but forgotten: Tritrichomonas foetus in extensively-managed bulls from Australia's Northern Territory.

Bovine trichomonosis, caused by infection with the protozoan parasite Tritrichomonas foetus, is globally recognised as a cause of reproductive failure in cattle. Maintained in clinically normal bulls, T. foetus infection results in infertility and abortion in infected cows. In Australia's Northern Territory (NT), logistical limitations associated with extensive livestock production inhibit wide-scale testing and diagnosis, allowing the parasite to persist undetected. In the present study, T. foetus was detected in 18/109 preputial cultures collected from bulls on a property in the NT with a history of low birth rates and reproductive failure using real-time PCR testing. Of the T. foetus-positive samples, 13/18 were genotyped using the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S rDNA unit. Selected samples were further characterised using the protein-coding genes of cysteine proteases (CP-1, 2, 4-9) and cytosolic malate dehydrogenase 1 (MDH-1) to determine if the isolates were 'bovine', 'feline' or 'Southern Africa' genotypes. All samples were 100% identical to the T. foetus 'bovine' genotype across all markers. This is the first reported case of trichomonosis in Australian cattle since 1988 and is a reminder that T. foetus should be considered whenever reproductive failure occurs in extensive cattle systems.

Molecular characterization of the Fe­hydrogenase gene marker in Trichomonas gallinae isolated from birds in Riyadh, Saudi Arabia.

Trichomonas gallinae causes avian oropharyngeal trichomonosis. This pathogen affects a large number of bird species and may cause substantial economic losses to the poultry industry. Al-Azizia poultry market in Riyadh, Saudi Arabia is among the largest poultry markets in the Arabian Gulf. Birds traded in this market may be exposed to a variety of T. gallinae strains. Genetic diversity of T. gallinae among birds in the market was examined using Fe­hydrogenase gene sequences. These sequences were amplified by PCR for twenty-nine isolates of T. gallinae from four different avian species, including 21 feral pigeons, one common mynah, three chickens, and four turkeys. Sequence analysis showed ten variant gene sequences. Nine sequences comprise a new subtype, including A(KSAF1), C(KSAF1) and C(KSAF3) with 34.48% (n = 10), 6.90% (n = 2), 6.90% (n = 2) of the isolates, respectively. Analyses also showed an additional five new sequences (KSAF1.1., KSAF2, KSAF13, KSAF14, KSAF15), representing 17.24% of the isolates. Subtype II (KSAF) was found in four feral pigeons (13.80%). To our knowledge, this report is the first to describe genotypes of T. gallinae from pigeons in Saudi Arabia using Fe­hydrogenase gene sequences for subtyping. Subtype analysis infers the presence of multiple genotypes of T. gallinae in Saudi avian populations.

Anti-trichomonad activities of different compounds from foods, marine products, and medicinal plants: a review.

Human trichomoniasis, caused by the pathogenic parasitic protozoan Trichomonas vaginalis, is the most common non-viral sexually transmitted disease that contributes to reproductive morbidity in affected women and possibly to prostate cancer in men. Tritrichomonas foetus strains cause the disease trichomoniasis in farm animals (cattle, bulls, pigs) and diarrhea in domestic animals (cats and dogs). Because some T. vaginalis strains have become resistant to the widely used drug metronidazole, there is a need to develop alternative treatments, based on safe natural products that have the potential to replace and/or enhance the activity of lower doses of metronidazole. To help meet this need, this overview collates and interprets worldwide reported studies on the efficacy of structurally different classes of food, marine, and medicinal plant extracts and some of their bioactive pure compounds against T. vaginalis and T. foetus in vitro and in infected mice and women. Active food extracts include potato peels and their glycoalkaloids α-chaconine and α-solanine, caffeic and chlorogenic acids, and quercetin; the tomato glycoalkaloid α-tomatine; theaflavin-rich black tea extracts and bioactive theaflavins; plant essential oils and their compounds (+)-α-bisabolol and eugenol; the grape skin compound resveratrol; the kidney bean lectin, marine extracts from algae, seaweeds, and fungi and compounds that are derived from fungi; medicinal extracts and about 30 isolated pure compounds. Also covered are the inactivation of drug-resistant T. vaginalis and T. foetus strains by sensitized light; anti-trichomonad effects in mice and women; beneficial effects of probiotics in women; and mechanisms that govern cell death. The summarized findings will hopefully stimulate additional research, including molecular-mechanism-guided inactivations and human clinical studies, that will help ameliorate adverse effects of pathogenic protozoa.