Tritrichomonas foetus damages bovine oocytes in vitro

Tritrichomonas foetus is an extracellular parasite of the urogenital tract in cattle. It causes infertility and abortion, but there is no documented information on the susceptibility of bovine oocytes to the parasite, except by one article that claimed no effects of T. foetus on oocytes or embryos. The aim of the present study was to study the effects provoked by T. foetus when in interaction with bovine oocytes. Oocytes were obtained from cow ovaries and divided into two groups: (1) one group contained cumulus cells, whereas (2) a second group was denuded from these cells. Light microscopy, video microscopy and scanning electron microscopy (SEM) revealed that exposure of oocytes to T. foetus caused rapid adhesion of the trichomonads to cumulus cells and to the zona pellucida (ZP). Motile parasites were observed for 12 h. The ZP was completely damaged, and the parasites were able to infiltrate beneath the ZP and reached the oocytes directly when the oocytes were denuded of the cumulus cells. Both the oocytes and the cumulus cells exhibited morphological characteristics compatible with apoptosis after interaction with T. foetus, such as chromatin condensation, the presence of several cytoplasmic vacuoles, with intact cellular membranes and organelles. The results from this study demonstrate that when a large number of T. foetus interacts with oocytes in vitro damage and apoptosis are provoked in the cow's reproductive cells. The behavior of this parasite as one of the causes of cattle infertility is discussed.     

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1α

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1α) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1α was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.     

Experimental infection with Tritrichomonas suis in heifers.

Nine heifers were intravaginally challenged with 9.3x10(6) Tritrichomonas suis reference strains. Vaginal mucus and serum samples were collected weekly 4 weeks post-inoculation. Vaginal mucus was cultured for T. suis and sera was tested by ELISA against whole cell antigens for T. suis and Tritrichomonas foetus. All vaginal mucus cultures were T. suis-negative during the experiment. ELISA values for both antigens were similar and differences were not significant (P>0.05). Positive control serum samples from one heifer vaccinated against T. foetus showed anti-T. suis ELISA values. We concluded that T. suis intravaginal inoculation induced a low level of serum immune response in heifers measured by ELISA and both protozoa probably share a common antigen. However, under the experimental conditions of this trial, colonization of the heifers' genital tract was not possible in any of the nine animals.     

PCR detection of Tritrichomonas foetus in preputial bull fluid without prior DNA isolation

Tritrichomonosis is a widespread, economically important venereal disease caused by Tritrichomonas foetus. The traditional diagnosis of this disease, which causes infertility and abortion in cattle, is based on the culture of the parasite. This process is time consuming, has low sensitivity, and is prone to contamination with intestinal or coprophilic trichomonadid protozoa, resulting in false positive diagnostics of T. foetus. In order to avoid the shortcomings of the traditional method, we developed a simple PCR assay based on TFR3 and TFR4 primers, which does not require parasite culturing. The sensitivity of the PCR assay resulted comparable to that of the classical method, being able to detect as few as five T. foetus parasites. In addition the method is highly specific. The analysis of preputial fluid washing samples showed that 58 out of 203 samples were positive by both, the PCR and the culture method (+/+), 9 samples were positive by PCR and negative by the traditional method (+/-) and only one sample resulted negative by PCR and negative by culture (-/+). The samples for the PCR assay can be stored for a week at 4 degrees C or 72h at room temperature. In summary, our study demonstrated that the PCR assay is an effective method for the diagnosis of T. foetus from preputial samples, and that it compares advantageously to the classical method.     

Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity

The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. foetus, nor with killed organisms. Scanning electron microscopy (SEM) showed that human erythrocytes did not adhere to the trophozoites, in contrast horse erythrocytes adhered to the surface of the parasites and were phagocytosed for up to 90 min. The parasites are able to exert their cytopathic effects through: (a) physical contact established between the two cell surfaces, (b) toxins released from parasites into the interaction media, or (c) the association of both mechanisms. Further studies are necessary to clarify the importance of the hemolytic activity in the biology of T. foetus.     

Characterization of Tritrichomonas foetus antigens, using bovine antiserum

Tritrichomonas foetus antigens were identified, using the serum of an Angus heifer that had been repeatedly immunized with suspensions of 1 X 10(8) organisms in Freund's complete adjuvant. Antibody activity against T foetus was determined by dot-blot analysis, using horse-radish peroxidase-conjugated anti-bovine immunoglobulin to detect bound antibody. The antiserum contained antibodies against surface and flagellar components of live or fixed T foetus, as determined by use of immunofluorescence. The antiserum reacted with approximately 38 proteins in a pool of 55 to 60 components resolvable by polyacrylamide-gel electrophoresis of T foetus extracts.     

Investigations on the prevalence and potential pathogenicity of intestinal trichomonads in pigs using in situ hybridization

In pigs, three different trichomonad species (Tritrichomonas foetus, Tetratrichomonas buttreyi and Tritrichomonas rotunda) have been described as commensals in the large intestine. The aim of this study was to gain further knowledge on the prevalence and pathogenicity of trichomonads in pigs by using a morphology-based approach. Chromogenic in situ hybridization (ISH) is a technique which allows direct localization of the protozoa in the intestinal tissue and correlation of the infection with pathologic changes. In the present study paraffin-wax embedded colon and ileum samples of 192 pigs were analyzed with this method. Using a probe specific for all known members of the order Trichomonadida (OT) 100 of the 192 pigs were tested positive. Thereof, about 10% showed moderate to high-grade parasitic load with trichomonads invading the lamina propria. Partial 18S ribosomal RNA gene sequencing of six of those animals showed a 100% sequence identity with T. foetus sequences. The majority of these animals were also tested positive for other enteropathogenic agents, such as Brachyspira sp., Lawsonia intracellularis, Escherichia coli, and porcine circovirus type 2. All OT-positive samples were further examined with another probe complementary to all known Tritrichomonas species sequences including T. foetus, T. augusta, T. mobilensis and T. nonconforma resulting in only 48 positives. These results suggest that T. foetus may not only be considered as an intestinal commensal but rather a facultative pathogen of pigs with a tendency for tissue invasion in the presence of other agents. Furthermore, the existence of other - yet to be identified - trichomonad species in the colon of pigs was shown.     

Experimental infection of cats with Tritrichomonas foetus.

OBJECTIVE: To determine whether infection with Tritrichomonas foetus causes diarrhea in specific-pathogen-free or Cryptosporidium coinfected cats. ANIMALS: 4 cats with subclinical cryptosporidiosis (group 1) and 4 specific-pathogen-free cats (group 2). PROCEDURE: Cats were infected orogastrically with an axenic culture of T. foetus isolated from a kitten with diarrhea. Direct microscopy and protozoal culture of feces, fecal character, serial colonic mucosal biopsy specimens, and response to treatment with nitazoxanide (NTZ; group 1) or prednisolone (groups 1 and 2) were assessed. RESULTS: Infection with T. foetus persisted in all cats for the entire 203-day study and resulted in diarrhea that resolved after 7 weeks. Group-1 cats had an earlier onset, more severe diarrhea, and increased number of trichomonads on direct fecal examination, compared with group-2 cats. Use of NTZ eliminated shedding of T. foetus and Cryptosporidium oocysts, but diarrhea consisting of trichomonad-containing feces recurred when treatment was discontinued. Prednisolone did not have an effect on infection with T. foetus but resulted in reappearance of Cryptosporidium oocysts in the feces of 2 of 4 cats. During necropsy, T. foetus was isolated from contents of the ileum, cecum, and colon. Tritrichomonas foetus organisms and antigen were detected on surface epithelia and within superficial detritus of the cecal and colonic mucosa. CONCLUSIONS AND CLINICAL RELEVANCE: After experimental inoculation in cats, T. foetus organisms colonize the ileum, cecum, and colon, reside in close contact with the epithelium, and are associated with transient diarrhea that is exacerbated by coexisting cryptosporidiosis but not treatment with prednisolone.     

Antigenic relationship among field isolates of Tritrichomonas foetus from cattle

Analysis of protein and antigen profiles of Tritrichomonas foetus isolates from cattle from 5 western states was accomplished by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, immunoblot, immunoprecipitation, and fluorography techniques. Total protein profiles of all isolates were compared by Coomassie brilliant blue staining of T foetus protein samples prepared by 4 protein-extraction methods. Antigenic tritrichomonas proteins were identified by immunoblot assay with polyclonal bovine or rabbit anti-T foetus serum. Additionally, [14C]glucosamine-labeled T foetus was used for total and antigenic glycoprotein analyses. Detectable differences in the composition of total proteins or antigenic tritrichomonal proteins were not observed among all isolates. However, intensity differences in some antigenic protein bands were apparent. Bovine and rabbit sera from immunized animals possessed antibodies to the same antigenic tritrichomonal proteins. Each T foetus isolate contained 4 to 7 molecular weight size classes of glycoprotein, which were labeled by [14C]glucosamine; however, only 3 to 4 glycoproteins were identified as antigens by bovine or rabbit antiserum.     

First record of natural Tritrichomonas foetus infection of the feline uterus

Tritrichomonas foetus was found in the uterus of a cat with pyometra and in the faeces of three other cats in the same household, one of which had chronic diarrhoea. This is the first report of a feline uterine infection with T. foetus and also the first time T. foetus has ever been diagnosed in animals in Norway. The diagnosis was made by microscopic examination and sequencing studies.     

Ultrastructural changes in Tritrichomonas foetus after treatments with AlPcS4 and photodynamic therapy

The Tritrichomonas foetus is an amitochondrial parasitic protist which causes bovine trichomoniasis, a major sexually transmitted disease in cattle. No effective drugs for this disease have been approved to this date. Photodynamic therapy (PDT) is an experimental treatment that shows great potential for treating bacteria, fungi, yeasts, and viruses. However, the cytotoxic effect of PDT on protozoan has been poorly studied. In this study, PDT with aluminum phthalocyanine tetrasulfonated (AlPcS4) photosensitizer was efficient in killing T. foetus. The mode of cell death in T. foetus after PDT was investigated by transmission electron microscopy. Morphological changes, such as membrane projections, nucleus fragmentation with peripheral masses of heterochromatin, endoplasmic reticulum proliferation, intense cytoplasmic vacuolization, fragmented axostyle-pelta complex, and internalized flagella could be observed. This is the first report to demonstrate cell death in T. foetus after PDT, and thus will open up new lines of investigation to develop new treatments for bovine trichomoniasis.     

In vitro cytopathic effects of a cysteine protease of Tritrichomonas foetus on cultured bovine uterine epithelial cells

OBJECTIVE: To evaluate the cytopathic effects of Tritrichomonas foetus and a purified cysteine protease (ie, CP30) of T foetus on cultured bovine uterine epithelial cells (BUECs) in vitro. SAMPLE POPULATION: 10 reproductive tracts were obtained from late-term bovine fetuses at a commercial abattoir. PROCEDURE: An in vitro culture system of BUECs was developed to study the cytopathic effects of T foetus and purified CP30 of T foetus on host cells. Cytotoxicity of T foetus or CP30 on exposed BUECs was determined. Fluorescence microscopy and flow cytometry analyses were used to detect apoptosis. A fluorometric assay was used to detect BUEC caspase 3 activation. The CP inhibitor E-64 and a caspase inhibitor were used to inhibit apoptosis. RESULTS: Cytopathic effects were observed in BUECs treated with parasites or CP30 and were concentration and time dependent. The BUECs underwent apoptosis in the presence of parasites or CP30. The specific CP inhibitor E-64 abolished the induction of apoptosis in BUECs by CP30. The caspase inhibitor reduced the amount of apoptosis in BUECs. CONCLUSIONS AND CLINICAL RELEVANCE: T foetus and its CP30 induce apoptosis in cultured BUECs in vitro. Induction of apoptosis by CP30 is correlated with protease activity. Endometrial cell death as a result of a T foetus infection is likely to be more important in mediating infertility than a direct effect on the conceptus. Provoking an apoptotic reaction in the host may mitigate an inflammatory reaction or immune response and therefore favor survival of the parasite in a chronic infection.     

An improved polymerase chain reaction assay for the detection of Tritrichomonas foetus in cattle

An improved polymerase chain reaction test has been developed to detect Tritrichomonas foetus, the causative agent of trichomoniasis in cattle. The test amplifies a region of the 5.8S ribosomal RNA gene of T. foetus, and it is simple, sensitive, and specific when compared with traditional methods to examine field samples.     

An improved molecular assay for Tritrichomonas foetus

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment.     

Survival of a feline isolate of Tritrichomonas foetus in water, cat urine, cat food and cat litter

Feline intestinal trichomoniasis caused by Tritrichomonas foetus is associated with large bowel diarrhea in cats from many parts of the world. It has long been recognized as an economically important sexually transmitted disease that causes early abortion in cattle. Isolates of T. foetus from cattle are infectious for the large intestine of cats and isolates of T. foetus from cats are infectious for the reproductive system of cattle. The parasite is maintained by fecal-oral transmission in cats. The present study was conducted to examine the survival of a feline isolate of T. foetus, AUTf-12, under various conditions that are relevant to fecal-oral transmission in cats. Trophozoites were grown in TYM medium and then exposed to water, cat urine, dry cat food, canned cat food, clumping cat litter, or filter paper for various lengths of time and then re-cultured in TYM medium. Trophozoites survived exposure to distilled or tap water for 30 but not 60 min, while they survived for at least 180 min in urine. Trophozoites survived for 30 min on dry cat food but survived for 120-180 min in canned cat food. No survival of trophozoites was observed on cat litter but trophozoites survived for 15 min when placed on filter paper. Our results indicate that T. foetus can survive and be potentially infectious in water, urine, dry cat food and canned cat food.     

Interaction of Tritrichomonas foetus with the reproductive tract of experimentally infected female BALB/c mice: ultrastructural evaluation

The interaction of Tritrichomonas foetus with its host is a complex process that involves colonisation, attachment and persistence. The goal of the present study was to describe the interaction of T. foetus with the genital tract using a model of non-oestrogenised female BALB/c mice which had been intravaginally infected with a suspension of T. foetus during oestrus. Animals were sacrificed after 10 weeks and the uteri fixed and processed for light and electron microscopy. Ultrastructural analysis showed that the attached protozoa interacted with the mucosa through a somal projection. With an amorphous secretion at the protozoa-host cell interface. There was no direct contact between the protozoal plasma membrane and the epithelial cell membrane. Our results demonstrated the participation of an active phagocytosis and the destruction of T. foetus by eosinophils.     

A cross-sectional study of Tritrichomonas foetus infection among healthy cats at shows in Norway

Background

In recent years, the protozoan Tritrichomonas foetus has been recognised as an important cause of chronic large-bowel diarrhoea in purebred cats in many countries, including Norway. The aim of this cross-sectional study was to determine the proportion of animals with T. foetus infection among clinically healthy cats in Norway and to assess different risk factors for T. foetus infection, such as age, sex, former history of gastrointestinal symptoms and concurrent infections with Giardia duodenalis and Cryptosporidium sp.

Methods

The sample population consisted of 52 cats participating in three cat shows in Norway in 2009. Samples were examined for motile T. foetus by microscopy, after culturing and for T. foetus-DNA by species-specific nested PCR, as well as for Giardia cysts and Cryptosporidium oocysts by immunofluorescent antibody test (IFAT).

Results

By PCR, T. foetus-DNA was demonstrated in the faeces of 11 (21%) of the 52 cats tested. DNA-sequencing of five positive samples yielded 100% identity with previous isolates of T. foetus from cats. Only one sample was positive for T. foetus by microscopy. By IFAT, four samples were positive for Giardia cysts and one for Cryptosporidium oocysts, none of which was co-infected with T. foetus. No significant associations were found between the presence of T. foetus and the various risk factors examined.

Conclusions

T. foetus was found to be a common parasite in clinically healthy cats in Norway.     

Efficacy of tinidazole for treatment of cats experimentally infected with Tritrichomonas foetus

OBJECTIVE: To determine the efficacy of tinidazole for treatment of cats with experimentally induced Tritrichomonas foetus infection. ANIMALS: 8 specific-pathogen-free kittens. PROCEDURES: Tinidazole was tested for activity against a feline isolate of T foetus in vitro. Kittens were infected orogastrically with the same isolate and treated or not with tinidazole (30 mg/kg, PO, q 24 h for 14 days). Amoxicillin was administered 28 weeks after completion of tinidazole administration to induce diarrhea. Feces were repeatedly tested for T foetus by use of PCR assay and microbial culture for 33 weeks. RESULTS: Tinidazole killed T foetus at concentrations >or= 10 microg/mL in vitro. In experimentally induced infection, tinidazole administered at 30 mg/kg decreased T foetus below the limit of molecular detection in 2 of 4 cats. Recrudescent shedding of T foetus, as elicited by amoxicillin-induced diarrhea, was diminished in cats that received prior treatment with tinidazole. CONCLUSIONS AND CLINICAL RELEVANCE: Although tinidazole decreased the detection of T foetus and treated cats were resistant to later efforts to incite the infection, inability of tinidazole to eradicate infection in many cats poses a serious impediment to the drug's effectiveness in practice.     

The effects of Tritrichomonas foetus and nutritional status on the fertility of cows on a community pasture in Saskatchewan.

A prospective observational study of a breeding season in a Saskatchewan community pasture was carried out to determine the cause or causes of a chronic infertility problem. There were 774 cows, from 27 herds, divided into 4 breeding groups (A,B,C,D) on the pasture. Cows entering the pasture in May were weighed, had their body condition scored and height measured. All bulls received breeding soundness examinations and a preputial wash, which was cultured for Tritrichomonas foetus and Campylobacter foetus subsp. venerealis. In July, cows were also weighed and had their body condition scored and again when they left the pasture. In addition, cows were pregnancy checked when they left the pasture. Bulls were tested again for Tritrichomonas foetus at the end of the grazing season. Two breeding groups had T. foetus-positive bulls and an average pregnancy of 84%, which was significantly lower than that of the two T. foetus negative groups (93.5%) (P = 0.0001). A cow was 2.97 times less likely to be pregnant if she had been exposed to T. foetus-positive bulls. Cows with average daily gains above the mean for the pasture were 2.12 times more likely to be pregnant. Body condition score upon entering and leaving the pasture, height, age, and breeding group were significant predictors of average daily gain.     

Sequence differences in the diagnostic region of the cysteine protease 8 gene of Tritrichomonas foetus parasites of cats and cattle

In order to investigate the genetic variation between Tritrichomonas foetus from bovine and feline origins, cysteine protease 8 (CP8) coding sequence was selected as the polymorphic DNA marker. Direct sequencing of CP8 coding sequence of T. foetus from four feline isolates and two bovine isolates with polymerase chain reaction successfully revealed conserved nucleotide polymorphisms between feline and bovine isolates. These results provide useful information for CP8-based molecular differentiation of T. foetus genotypes.