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Background Fecal polymerase chain reaction (PCR) testing for Tritrichomonas foetus is considered the most sensitive means for diagnosis of infection but results could be influenced by fecal collection technique and prior use of antimicrobial drugs. Objectives To establish any association between fecal collection technique or treatment history and results of fecal PCR testing for T. foetus. Animals Fecal samples from 1717 cats submitted by veterinarians between January 2012 and December 2017. Methods This study used a retrospective analysis. T. foetus PCR test results from 1808 fecal samples submitted for diagnostic testing were examined for their association with method of fecal collection and prior antimicrobial treatments. Data were collected from sample submission form. Results Positive T. foetus PCR test results were obtained for 274 (16%) cats. Fecal samples collected via fecal loop had increased probability of positive PCR test results (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.31-3.17, P = .002) compared to samples collected by colonic flush. There was no association between PCR test results and treatment history, treatment type, or prior treatment with ronidazole. After an initial positive PCR test, 4/19 (21%; 95% CI 2.7%-39.4%) cats treated with ronidazole had a second positive test result. Conclusions and clinical importance Results of this study support that fecal samples collected by loop might be better for PCR diagnosis of T. foetus infection. Lack of association of ronidazole with PCR test results and a 21% all-potential-causes failure rate of ronidazole in cats with preconfirmed infection are important limitations to use of this drug.

Almost 20 years has passed since trichomonosis was first recognized as a potential cause of diarrhea in domestic cats. Despite progress in confirming disease causation, developing means for diagnosis, and identifying approaches to treatment of the infection, we still know very little about how this parasite causes diarrhea. With increasing recognition of resistance of trichomonosis to treatment with 5-nitroimidazole drugs, new treatment strategies based on an understanding of disease pathogenesis are needed. In this review, lessons learned from the pathogenesis of venereal trichomonosis in people and cattle are applied to clinical observations of trichomonosis in cats in effort to generate insight into areas where further research may be beneficial.

Ronidazole (RDZ) is the only known effective treatment for feline diarrhea caused by Tritrichomonas foetus. This study aimed to develop guar gum-coated colon-targeted tablets of RDZ and to determine the pharmacokinetics of this delayed-release formulation in cats. Guar gum-coated tablets were administered orally once to five healthy cats (mean dose 32.3 mg/kg). The tablets were then administered once daily for 5 days to four cats (mean dose 34.5 mg/kg), and absorption studies repeated on day 5. Plasma was collected and analyzed for RDZ concentration, and pharmacokinetic noncompartmental and deconvolution analysis were performed on the data. There was negligible RDZ release until after 6 h, and a delayed peak plasma concentration (mean Cmax 28.9 μg/mL) at approximately 14.5 h, which coincides with colonic arrival in cats. Maximum input rate (mg/kg per hour) occurred between 6 and 16 h. This delayed release of ronidazole from guar gum-coated tablets indicates that release of RDZ may be delayed to deliver the medication to a targeted area of the intestine. Repeated dosing with guar gum tablets to steady-state did not inhibit drug bioavailability or alter the pharmacokinetics. Such targeted RDZ drug delivery may provide improved efficacy and reduce adverse effects in cats.

Background Bacterial and parasitic agents are commonly implicated as causes of diarrhea in cats, but there is a paucity of information evaluating epidemiological and prevalence factors associated with most of these organisms in cats. Objectives Determine the prevalence of selected enteropathogens in diarrheic and nondiarrheic cats. Animals A total of 219 diarrheic and 54 nondiarrheic cats. Methods Prospective study. Fresh fecal specimens were submitted for centrifugation flotation, culture, ELISA (Giardia, Cryptosporidium, Clostridium perfringens enterotoxin [CPE], and C. difficile toxin A [TcdA]) and polymerase chain reaction (PCR) testing (Tritrichomonas foetus and Campylobacter spp.). An epidemiologic questionnaire was completed for each cat. Results Campylobacter was isolated from significantly fewer diarrheic (21/219 or 9.6%) versus nondiarrheic cats (15/54 or 27.8%, P = .001), and was detected in 74 of 131 cats (56.5%) via PCR. Campylobacter jejuni, C. helveticus, and C. upsaliensis were detected in 6.8, 100, and 44.6% of the 74 cats. Multiple Campylobacter spp. were identified in 47.3% of these cats. All cats were negative on fecal culture for Salmonella and for C. difficile TcdA via ELISA. CPE was detected in 9/219 diarrheic (4.1%) and in 1/54 nondiarrheic cats (1.9%, P = .69). Cats < 2 years were significantly more likely to be infected with intestinal parasites (P

Background: The mainstays of treatment for clinically important trichomonad infections are the 5-nitroimidazoles. Metronidazole resistance of feline Tritrichomonas foetus is presumed because of common treatment failure, and tinidazole does not consistently eradicate infection. To date, ronidazole is the only drug demonstrated as effective for treatment of cats infected with T. foetus. Objective: To document in vivo treatment failure and identify underlying causes and in vitro conditions of resistance of feline T. foetus to ronidazole. Animals: Two intact male Abyssinians failing ≥5 courses of treatment with increasing doses of 5-nitroimidazole drugs. An intact male Abyssinian documented to clear infection after treatment with a single course of ronidazole. Methods: T. foetus isolates were cultured from feces and tested in vitro for susceptibility to ronidazole under aerobic and anaerobic culture conditions. A urogenital nidus of T. foetus infection was investigated by culture, polymerase chain reaction, or immunohistochemical testing of urogenital specimens. Results: Resistance to ronidazole under aerobic conditions was uniquely identified in T. foetus isolated from cats with well-documented treatment failure. Treatment failure could not be attributed to reinfection, inappropriate treatment protocol, or presence of a urogenital nidus of infection. Conclusions and Clinical Importance: Clinical resistance to metronidazole, low efficacy of tinidazole, and present documentation of in vivo and in vitro resistance to ronidazole in some cats are consistent with a high level of cross resistance of feline T. foetus to 5-nitroimidazole drugs. Current lack of alternative drugs with clinical efficacy against feline T. foetus suggests that active investigation of other treatment approaches is warranted.

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

A 10-week-old female Ocicat was presented at a primary care feline veterinary practice for failure to thrive and diarrhoea. Numerous trophozoites, atypical for Giardia sp., were detected on a direct faecal examination, in addition to Giardia cysts. Although the failure to thrive and diarrhoea resolved following treatment for giardiasis, further diagnostic tests performed on faecal specimens from the kitten and 15 other Ocicats from the same cattery, including culture of trophozoites in In Pouch™ medium, PCR testing and molecular sequencing of PCR amplicons, confirmed infection with Tritrichomonas cf. foetus. This is the first report in Australia of feline trichomoniasis, which appears to be an emerging infectious disease of cats. Pertinent information regarding the clinical features, diagnosis, therapy, and potential source of feline trichomoniasis within Australia are discussed.

Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate.

Tritrichomonas foetus, the agent of bovine trichomoniasis, is a flagellate protozoan responsible for substantial economic losses to the dairy and calf industries worldwide. As yet, there is no approved treatment nor is there a sensitive diagnostic method. All these problems suggest that immunization is the best control strategy. In view of this, we isolated and partially purified flagella of the parasite by vortex homogenization followed by low-speed differential centrifugation. The resulting enriched flagellar preparation termed "crude flagellar prep" was purified further by sucrose and percoll gradients. Microscopic analysis showed that the flagellar membrane was intact. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed three prominent protein bands of 42, 49, and >250 kDa, and several minor bands. Immunoblotting of flagellar and whole-cell extracts revealed many flagellar antigens.

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.

The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. It is found in several trichomonad species, protists living in oxygen-poor environments, as well as certain free-living ciliates, rumen ciliates, and some fungi. We performed a comparative microanalytical study (energy dispersive X-ray analysis and electron spectroscopic imaging) of different fixation methods for electron microscopy analyzing hydrogenosomes of the bovine parasite Tritrichomonas foetus. The study included the elemental composition and the mapping of calcium, phosphorus, and oxygen. A preparation of T. foetus cells, based on cryoimmobilization by high-pressure freezing and freeze-substitution, was compared to a second preparation based on chemical fixation followed by dehydration and routine processing. The ultrastructural preservation achieved by the cryotechnique was far superior to the chemical fixation, since it allowed the successful cryoimmobilization of intracellular ion contents. The detection of several cations (Al, Mg, Co, Ca, Fe) by X-ray microanalysis inside the peripheral vesicle of the hydrogenosome was only possible in cryofixed cells. The presence of aluminum and cobalt ion in the hydrogenosomal vesicle was established for the first time. Electron-spectroscopic images of calcium showed that this element, in addition to the vesicle compartment, is present in the hydrogenosome's membrane in varying concentrations, which might reflect changes in the physiology of this organelle. Microsc. Res. Tech. 53:87–92, 2001. © 2001 Wiley-Liss, Inc.

Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

The abilities of two isolates of Tritrichomonas foetus to survive and replicate in transport and Diamond's medium or in the InPouch TF system (Bio-Med Diagnostics) when exposed to different temperatures for different periods were determined in a series of experiments. Tubes containing thioglycollate transport medium or pouches were inoculated with 4000 to 5000 organisms and kept for up to seven days at 37 degrees C, 22 degrees C, 4 degrees C, or -20 degrees C. When the holding time had elapsed, the numbers of motile T foetus were counted. Samples in transport medium were transferred to Diamond's medium, and both the pouches and tubes containing Diamond's medium were incubated at 37 degrees C. The cultures were examined and counted four or five times during the 10 to 14 day culture period. The sensitivity of the test under the different conditions, expressed as the number of positive cultures/the total number of samples x 100, varied from zero to 100 per cent depending upon the combination of variables considered. In each medium, with both isolates of T foetus, all samples kept for up to four days at 22 degrees C or 37 degrees C were positive. All cultures of samples kept more than five days at 4 degrees C were negative. No positive cultures were detected when samples were kept more than three hours at -20 degrees C. The day on which the cultures reached mean peak concentration varied with the temperature at which the samples had been kept before they were cultured.

The arginine-specific reagents phenylglyoxal and butane-2,3-dione irreversibly inactivate the Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) and Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The inactivation of the tritrichomonal enzyme by phenylglyoxal follows time-dependent and concentration-dependent pseudo-first-order kinetics. Complete protection against inactivation is afforded by the addition of 25 microM GMP, whereas 5-phosphoribosyl-1-diphosphate (PRibPP) at 50-250 microM can only slow down the inactivation, without being protective. Digestion of [7-(14)C]phenylglyoxal-modified enzyme with trypsin and separation of the peptides by reverse-phase HPLC shows that only one radioactive peak is greatly diminished by incubation with 25 microM GMP or 1 mM PRibPP. Mass-spectral analysis identifies Arg155 as the target site of two molecules of phenylglyoxal that is protected by the substrates. This amino acid residue is positioned next to Tyr156, which is a highly conserved aromatic residue among all the purine phosphoribosyltransferases (PRT) and is always found stacked on top of the purine substrate. This may explain why phenylglyoxal labeling of Arg155 inactivates the enzyme and why GMP can protect Arg155 more effectively than PRibPP. Among the purine PRT in our possession, only schistosomal HGPRT, the only other enzyme that contains an arginine residue at the corresponding location (Arg187), was susceptible to phenylglyoxal and butane-2,3-dione. The presence of Lys185-Phe186 and Ser179-Trp180 at the corresponding locations in human HGPRT and Giardia lamblia GPRT, respectively, may explain their resistance to phenylglyoxal. Thus, Arg155 in T. foetus HGXPRT and Arg187 in S. mansoni HGPRT will be attractive targets for future studies.

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) of Tritrichomonas foetus was inactivated by the thiol reagents iodoacetate and 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). Iodoacetate inactivates the enzyme in a time-dependent and concentration-dependent manner that follows pseudo-first-order kinetics. However, the observation that total inactivation with iodoacetate was not achieved suggests that none of the reactive cysteine residues is directly involved in the catalytic activity of the enzyme. Nbs2 caused 50% inactivation rapidly, which was followed by gradual modifications of an additional three cysteine residues leading to complete enzyme inactivation. Analysis of the inactivation using the method developed by Tsou (1962) revealed that modification of two cysteine residues by Nbs2 is sufficient to impair the HGXPRTase activity. Tryptic digestion of HGXPRTase labeled with iodo[2-14C]acetic acid, followed by fractionation of the digest by HPLC and sequence analysis of the labeled peptides allowed the identification of Cys71, Cys129, Cys132, and Cys148 as the reactive cysteine residues. GMP and 5-phosphoribosyl-1-diphosphate provided complete protection against HGXPRTase inactivation by iodoacetate and against carboxymethylation of Cys129, Cys132, and Cys148, Cys71 was not protected by either substrate against iodoacetate, but its carboxymethylation caused no loss in enzyme activity either. There was also no substrate protection of Cys71 against Nbs2, which, however, caused 50% inactivation of the enzyme. Replacing the thionitrobenzoate (Nbs) moiety from Cys71 with cyanide resulted in a gradual recovery of the enzyme activity, which indicates that a steric hindrance at the active site was introduced by Nbs but removed by cyanide. Thus, our results demonstrate that although the reactive cysteine residues in HGXPRTase are not directly involved in the catalytic activity, modification of cysteine residues 129, 132, and 148 by iodoacetate or Nbs2 hinders substrate binding which can, in turn, protect the cysteine residues from modifications. The substrate protection of Cys129 and Cys148 is probably also indicative of a conformational change in the protein structure brought about by substrate binding.

The morphogenesis of hydrogenosomes in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming enlongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.

The present paper describes in detail the complex movement of the protozoon Tritrichomonas foetus. By the use of analogue and digital video techniques, we were able to analyze frame by frame the beatings of the anterior flagella and discuss their role in the movement of the cell. We also measured the productive displacement of the cell during one flagellar beating cycle. The obtained data were digitally improved and compared to analogue quantifications. It is shown that during 1 s of recorded movement, T. foetus performs 4 complete anterior flagella beating cycles (with active-like and recovery-like beatings). In each cycle the cell swims ±6.5 μm forwards, after the recovery of ±1.5 μm of receded movement. These observations led us to conclude that the estimated average speed of T. foetus is 25 μm/s, and that all flagella participate in the cell movement. The recurrent flagellum continuously contribute to the forward movement of the protozoon. The cell also performs rotational movements. The obtained results led us to suggest a model for the movement of T. foetus. © 1996 Wiley-Liss, Inc.

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56-80%) in the non sedimentable fraction of both species. A large proportion (35-40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.

A neutrophil chemotactic factor (TfNCF) was isolated from the crude extract of Tritrichomonas foetus organisms by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S200. TfNCF showed homogenicity by both PAGE and SDS-PAGE. The molecular weight of TfNCF was estimated to be 22 and 24 kDa, by Sephacryl S200 gel chromatography and by SDS-PAGE under reducing conditions. Immunization of TfNCF caused almost complete protection against T. foetus infection in mice. Western blot analysis probed with anti-TfNCF antibody showed that the epitopes on TfNCF were not commonly shared on the other components of T. foetus organisms nor other helminthous parasite-derived components. Furthermore, pre-incubation of neutrophils with antigens of other helminthous parasites or N-formylmethionyl-leucyl-phenylalanine did not affect the neutrophil chemotactic activity for TfNCF. These results suggest that TfNCF is a novel NCF consisting of unique epitopes for both antigenicity and neutrophil chemotactic activity. The role of NCF in the initiation of the immune response is discussed.

The fine structure of normal and antibody-coated Tritrichomonas foetus cells and their interaction with rat peritoneal neutrophils was studied. Peritoneal neutrophils were obtained by glycogen stimulation. The neutrophils readily associated with and killed the parasites, which were subsequently ingested. The process involved activation of the respiratory burst, as demonstrated by the use of cytochemical methods. Images were obtained indicating that binding of parasites to the neutrophil surface triggers an exocytic response with release of oxygen-derived products. Cytochemical localization of acid phosphatase and peroxidase activities showed that leukocyte granules fused with the parasite-containing phagocytic vacuoles. We also showed the cytochemical localization of alkaline phosphatase in the parasite-neutrophil interaction.

Tritrichomonas foetus, a pathogenic protozoan, was used as a model to analyse microwave-stimulated fixation as a procedure of preparation of biological samples for electron microscopy of thin sections and freeze-fracture replicas. Good preservation of the protozoan structure was achieved by microwave-stimulated fixation and Epon polymerization. The membrane structure, as visualized in freeze-fracture replicas, was well preserved. © 1993 Wiley-Liss, Inc.

SIR, — We would like to draw colleagues' attention to an important new cause of diarrhoea in cats. Tritrichomonas foetus is a flagellated protozoan parasite that has recently been identified as a natural and experimental cause of large bowel diarrhoea in domestic cats in the usa ([Gookin and

Objectives: To determine the efficacy of ronidazole (RDZ), tinidazole (TDZ), and metronidazole (MDZ) against Tritrichomonas foetus in vitro and of RDZ for treatment of feline naturally occurring or experimentally induced T foetus infection. Animals: A cat naturally infected with T foetus infection and diarrhea. Ten specific-pathogen-free (SPF) kittens. Procedure: RDZ, TDZ, and MDZ were tested for activity against 3 different feline isolates of T foetus in vitro. RDZ then was administered to a naturally infected cat at 10 mg/kg PO q24h for 10 days. SPF kittens were infected orogastrically with feline T foetus and treated with either placebo or RDZ (10 mg/kg PO q12h for 14 days). Cats with relapsing infection or those receiving placebo were treated subsequently with RDZ (either 30 or 50 mg/kg PO q12h for 14 days). Feces were examined for T foetus by direct microscopy, culture, and polymerase chain reaction (PCR) testing weekly. Results: Both RDZ and TDZ killed T foetus at concentrations .0.1 mg/mL in vitro. In the naturally infected cat, RDZ abolished diarrhea and T foetus infection for 85 days after treatment, at which time infection and diarrhea relapsed. Retreatment with RDZ eradicated diarrhea and T foetus infection for over 407 days. In experimentally induced infection, RDZ at 10 mg/kg caused initial improvement, but infection relapsed in all 5 cats 2 to 20 weeks after treatment. At 30 or 50 mg/kg, 10/10 cats were negative for T foetus infection for follow-up durations of 21 to 30 weeks after treatment. Conclusions and Clinical Relevance: Oral administration of RDZ at 30 to 50 mg/kg q12h for 14 days resolved diarrhea and eradicated infection (on the basis of polymerase chain reaction [PCR] testing) in 1 naturally infected cat and 10 experimentally inoculated cats receiving a different isolate of T foetus.

Summary The accessory sex glands of 521 slaughtered bulls were examined for lesions; normal anatomical observations were also made. The average weights and measurements (length and width) of the seminal vesicles and bulbourethral glands of 110 bulls were 34.1 g and 9.1 × 2.5 cm, and 4.6 g and 2.9 × 1.9 cm, respectively. Weights of both glands were correlated to age, but bulbourethral gland weight was more closely correlated to bodyweight than age. The average length and diameter of the ampulla was 11.1 and 1.0 cm, respectively. Seminal vesiculitis and ampullitis were found in 9.0% and 3.8% of bulls respectively. The incidence of both conditions was highest in old bulls. Mesonephric duct anomalies (observed in 1.7% of bulls) and other congenital and inflammatory lesions of the accessory sex glands were also recorded. Macroscopically normal accessory sex glands of 86 bulls were studied histologically and a high percentage of all glands showed inflammatory changes. Attempts to isolate viral agents, Mycoplasma, and Chlamydia from accessory sex glands were unsuccessful. Corynebacterium pyogenes was isolated from two cases of seminal vesiculitis. IBR-IPV virus, Campylobacter fetus subsp. venerealis and Tritrichomonas foetus were frequently isolated from the prepuce of bulls, many of which also had positive serological titres to Leptospira pomona and Leptospira hardjo. No one factor capable of causing bull infertility was consistently identified, and the incidence figures for observed conditions of the accessory sex glands were generally comparable to those found in bull populations elsewhere.

Geographically diverse strains and clones of Tritrichomonas foetus have been examined with respect to their expression of a major surface antigen of approximately 150,000 relative molecular weight (Mr), designated the 150 Ag. Radioiodination and 13S-methionine labeling of T. foetus followed by immunoprecipitation with monoclonal antibodies (MAbs), separation of polypeptides by SDS-PAGE, and autoradiography or fluorography confirmed the parasite origin of the 150 Ag. The results of flow cytometry analysis employing a panel of MAbs against live T. foetus parasites revealed that from 5 to 84% of individuals in a given population of T. foetus expressed a particular epitope of the 150 Ag. All strains and clones were positive for surface expression of epitopes of this antigen. These results show that the 150 Ag is widely distributed in populations of T. foetus, confirm the surface location of this antigen, and suggest its importance as a target for protective immune responses.

This paper describes a survey of 504 bull genitalia collected after slaughter at 3 Western Australian abattoirs over 18 months. Details of the gross pathology, histopathology, and microbiology of the genitalia, and physical dimensions of the testicles are presented. The major breeds represented were Hereford (30%), Shorthorn (16%), Friesian (14.3%), and Aberdeen Angus (13.9%). The mean age of 263 bulls (52.6%) was 4.4 years. The mean length and breadth of 469 paired testicles was 10.8 cm and 6.9 cm respectively. Pathological conditions of the testicles were present in 61/469 (13%) bulls and in the epididymides of 37/469 (7.9%) bulls. Campylobacter fetus was isolated from 20 (4%) bulls, Tritrichomonas foetus was isolated from 6 (1.2%) bulls, and infectious bovine rhinotracheitis virus was isolated from 14/158 (8.9%) bulls. Histories were supplied by stock owners for 307 (60.9%) bulls and these did not relate well to the findings of the survey. The findings of this survey were compared, in particular, to similar studies carried out in Australia.

Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.

SYNOPSIS A primitive trichomonad, Monocercomonas sp. (strain NS-1:PRR) from Natrix sipedon, was grown axenically in Diamond's medium. Activity of NADH oxidase, malate dehydrogenase, malate dehydrogenase (decarboxylating) and of the anaerobic enzymes, pyruvate synthase and hydrogenase as well as of several hydrolases was demonstrated in homogenates. The subcellular distribution of these activities was studied by means of analytical differential and isopycnic centrifugation of homogenates prepared in 0.25 M sucrose. NADH oxidase and malate dehydrogenase are in the nonsedimentable part of the cytoplasm. Malate dehydrogenase (decarboxylating), pyruvate synthase, and hydrogenase are associated with a large particle which equilibrates at density 1.22. In its properties, this particle corresponds to the microbody-like hydrogenosomes of Tritrichomonas foetus. The 5 hydrolases studied are associated with at least 2 different particle populations: a large particle population equilibrating at densities from 1.10 to 1.16 is the exclusive location of 3 enzymes (β-galactosidase, protease and β-N-acetylglucosaminidase), 2 of which have a pH optimum close to neutrality. These particles contain part of the acid phosphatase and β-glucuronidase. Another part of these 2 enzymes is associated with a separate population of smaller granules with equilibrium densities of 1.16 to 1.18. The 2 types of hydrolase-carrying particles are also biochemically very similar to their counterparts in T. foetus.

The cell surface of Tritrichomonas foetus was characterized by using 18 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose, and sialic acid. The specificity of the lectin-induced cell agglutination was verified by inhibition of the agglutination with the specific sugars. By using cytochemical techniques associated with electron microscopy, carbohydrates were detected on the cell surface of T. foetus. The following techniques were used: periodic acid--thiosemicarbazide--silver proteinate, concanavalin A--horseradish peroxidase, and ruthenium red. Anionic sites were detected on the cell surface of the protozoan at pH's 1.8 and 7.2 with the use of colloidal iron hydroxide and cationized ferritin particles, respectively. The binding of colloidal iron particles, as well as the agglutination induced by the lectin from Limulus polyphemus, indicated the presence of sialic acid on the cell surface of T. foetus.

The 115,000-molecular-weight antigen of Trichomonas vaginalis was characterized using monoclonal antibodies developed to three different strains of T. vaginalis and one strain of Tritrichomonas foetus. The antigen was found to be present on all strains or isolates of T. vaginalis examined and was demonstrated to be located on the external surface plasma membrane by agglutination assays and complement-mediated lysis assays. Characteristics of the antigen were assessed with a proteolytic enzyme and periodate oxidation. Periodate treatment of whole T. vaginalis abrogated binding for eight antibodies while use of pronase-treated antigen resulted in loss of antibody binding for two different antibodies. Screening of 19 axenized clinical isolates of T. vaginalis and one strain each of T. foetus and Giardia lamblia with type-specific antibodies delineated three major groups of T. vaginalis based on antigenic specificities (epitope distributions) within the 115,000-molecular-weight antigen. In addition, one epitope of the 115,000-molecular-weight antigen was found only on the immunizing strain. Two epitopes were present on all T. vaginalis isolates as well as T. foetus and G. lamblia. One epitope was common to all T. vaginalis except one. A minimum of six different epitopes of the 115,000-molecular-weight antigen were identified. Antigens purified with type-specific or "common" monoclonal antibodies shared the same partial peptide maps demonstrating relatedness.

Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca++-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl2 and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.

A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné, Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal; emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.

Two strains of Trichomonas vaginalis, JH162A , with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces ( PFs ) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad . In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of approximately 9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae , as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta -type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.

Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9-12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/micron2 and 468/micron2 respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per micron2 in the nuclear membrane.

SUMMARY The effect of Tritrichomonas foetus var. brisbane infection on calf production by Hereford cows was determined. The mean number of calves produced by cows that were kept continuously with bulls infected with T. foetus for 3 years was 17.6% less than the mean number produced by cows kept with a non-infected bull. Losses in production due to trichomoniasis occurred each year, but were greatest in the first 2 years in cows experiencing infection for the first time.

SYNOPSIS. When grown in CPLM medium or in a similar medium containing glucose instead of maltose as its sugar, Tritrichomonas foetus, Strain O, was much more sensitive to injury when frozen to -21d C. in the presence. of 1 m glycerol during the initial and logarithmic phases of its population growth curve than at its peak and for some time thereafter. In 7 experiments in which the population peak occurred an average of 28.1 hours after inoculation, the average culture age at which the protozoa first survived freezing was 20.3 hours, at which time the population had reached 52.6% of its peak. The optimum culture age for survival after 1 day of freezing averaged 37.7 hours at which time the population averaged 75.6% of its peak. The optimum culture age for survival after 7 to 15 days of freezing averaged 32.3 hours, at which time the population averaged 82.7% of its peak. Better survival upon freezing was obtained in those experiments in which the population peak was reached relatively rapidly.

Tritrichomonas foetus, Pentatrichomonas gallinarum, and nasal and fecal forms of Trichomonas suis grew well in NIH Thioglycollate Broth with 1% beef serum, and produced acid end-products. Succinic acid was the major acid produced by T. foetus P. gallinarum, and the nasal T. suis, accounting for over 50% of the total acid for each. The fecal T. suis produced more lactic than succinic acid; about 45% of the total acid was not accounted for. Pyruvic acid was found in small amounts in all cultures. The lactic/pyruvic ratio was about 2.5 for the two swine forms, and about 1.0 for the other two. The nasal form of T. suis produced much more total acid than the others. O2 uptake, CO2 production, H2 evolution, and anaerobic acid formation varied with age of organism. Respiratory activity was highest at 12 hours but varied considerably thereafter. The nature of the buffer used in manometric experiments also affected the results. Glucose, mannose, fructose and galactose most stimulated respiration. Disaccharides were slowly utilized (lactose and sucrose were not used by P. gallinarum). In a few cases lactate, pyruvate, formate, and malate stimulated O2 uptake. Evidence was obtained for hexokinase, phosphohexoisomer-ase, phosphofructokinase, aldolase, and glucose-6-phcsphate dehydrogenase in all 4 organisms. Phosphoglucomutase was demonstrated in T. foetus and the fecal T. suis. T. foetus and P. gallinarum showed evidence of phosphoglyceromutase and enolase. T. suis (fecal) and P. gallinarum had malic dehydrogenase, and P. gallinarum gave evidence of “malic enzyme” activity. Formic dehydrogenase activity was marked in all and presumptive evidence for a formic hydrogenlyase system was obtained for T. foetus and P. gallinarum. Study of reactions involving pyruvate and malate (with a possible linkage to a hexose monophosphate shunt) might perhaps provide a key to a better understanding of trichmonad metabolism.

Tritrichomonas foetus (Riedmuller) which occurs in the genital tract of domestic cows and which appears to be associated with pathological conditions, including sterility and abortion, has been found in American cows and its morphology studied. The spindle-shaped body is 10 to 25 μ in length and bears three anterior flagella and a posterior flagellum which constitutes the marginal filament of the nearly full-length undulating membrane and continues posteriorly as a trailing flagellum. There is an accessory filament in the undulating membrane. The axostyle is of the thick, hyaline type and contains endoaxostylar granules in the capitulum and bears a chromatic ring at the point of emergence from the body. There is a well-developed costa under the undulating membrane and a simple type of parabasal body. In its morphology this species more nearly resembles such intestinal forms as T. augusta and T. muris than it does Trichomonas vaginalis of man, since the latter has four anterior flagella, a short undulating membrane without an accessory filament, no trailing flagellum, a different type of axostyle, and a parabasal filament along with its larger parabasal body.

SUMMARY. The addition of 1.0–1.1 M (9–10%) glycerol to CPLM medium of Johnson and Trussell permits Tritrichomonas foetus to survive when frozen slowly to -20d or -76dC. and stored at either temperature. After freezing to -76dC. at the rate of 1dC. per minute, 38% of the protozoa were alive after 1 day, and some survived more than 128 days. After freezing to -20dC. in 1–2 hours, 63% of the protozoa were alive after 1 day, 46% after 8 days, and some survived 32 to more than 128 days in different experiments. After freezing to -20dC. in about 7 hours, 68% were alive after 1 day, 36% after 8 days, and some survived 16 to more than 64 days in different experiments. A concentration of 0.55 M (5%) glycerol is about as effective as 1.1 M, but 0.14 and 0.28 M are much less effective. If the protozoa are snap-frozen to -76dC., glycerol does not protect them, nor do they survive slow freezing without glycerol. In CPLM medium containing 1.0 M ethylene glycol, 72% of the protozoa were alive 1 day after having been frozen to -20dC. in about 7 hours, 29% were alive after 8 days, and some survived from 16 to more than 32 days in different experiments. The above figures are averages. In parallel tests at a concentration of 1.0 M in CPLM medium following slow freezing to -20dC. and storage at that temperature for 1 day, ethylene glycol was 80–90% as effective as glycerol; 1,2,3,4-butanetetrol was 67% as effective; 1,2-propanediol was 57% as effective; 2,3-butanediol, 14%; mannose, 6%; and mannitol, 1%. Glycerol monoacetate and polyvinyl alcohol were inactive; and 2,3-dimercaptopropanol was toxic.

SUMMARY. The metabolism of Tritrichomonas foetus (strain, BP-1) and trichomonads from the nasal cavity and cecum of swine was studied manometrically under similar experimental conditions. At pH 6.4, quantitative and qualitative differences were observed. The cecal (probably T. suis) and nasal trichomonad used glucose, galactose, fructose, mannose, lactose, sucrose, raffinose, and trehalose. T. foetus used all except lactose and raffinose. All three were inhibited by iodoacetate and arsenite. T. foetus and the nasal form were significantly inhibited by fluoride and 8-hydroxyquinoline, whereas the cecal trichomonad was not. At varied pH, all failed to oxidize Krebs' cycle intermediates. The amounts of oxygen consumed by T. foetus and the nasal trichomonad in the presence of lactate and pyruvate were at levels similar to those with disaccharides; the cecal trichomonad was indifferent toward both substances. Anaerobically, lactate and pyruvate increased the evolution of gas by all three trichomonads. Aerobic acid formation was demonstrated for all three forms. Anaerobically, metabolic CO2 and gas(es) that were not absorbed by KOH were evolved by all three. Pure oxygen was inhibitory to glucose utilization and stimulatory to the endogenous respiration of all trichomonads; the nasal form was affected the least. The writer believes that the cecal trichomonad is different from T. foetus and the nasal trichomonad of swine. The relationship between the nasal trichomonad and T. foetus remains in doubt.

Tritrichomonas foetus survives when frozen in the presence of 1 M glycerol after growth in cysteine-peptone-liver-maltose (CPLM) medium. In the present study, survival upon storage was better in a chest-type freezer at a constant −21°C than in an upright freezer from which were recorded cyclic temperature fluctuations between −23 and −25° or in a dry-ice chest with a nominal temperature of −72°. By the use of special thermocouples inside the freezing tubes, it was found that the temperature fluctuated between −22 and −24° in the chest-type freezer, between −19 and −30° in the upright freezer, but rising as high as -2° when the door was opened and samples were removed, and markedly in the dry-ice chest as the dry ice melted and was replaced, sometimes rising to as high as −27°. The poorer survival in the latter 2 freezers was considered due to temperature fluctuation. When equilibration with glycerol was carried out at room temperature, survival upon subsequent freezing was better following rapid equilibration (glycerol added all at once; equilibration time, 1 hour) than following slow equilibration (1/6 of the final amount of glycerol added each hour for 6 hr.; equilibration time, 7 hr.). Survival was extremely poor following either rapid or slow equilibration in the refrigerator (4°). Since T. foetus can grow indefinitely at 37° in the presence of 10% glycerol and since it is protected by glycerol when frozen, there is a critical zone near 4° in which glycerol appears to be toxic. Buffering the storage medium to pH's 6.3 to 7.1 with glycyl-glycine increased survival upon freezing, but buffering to the same pH's with triethanolamine had no significant effect upon survival, and buffering to pH's 7.1 to 7.5 with phosphates decreased survival upon freezing.