Cryopreserved bovine semen represents an accessible source of genetic material due to its widespread use in assisted reproductive technologies and the conservation of genetically valuable animals. However, DNA extraction from spermatozoa within this type of sample remains challenging due to the high protein content and the complex structure of the ejaculate, which can affect DNA yield and quality. The aim of this study was to identify and validate an efficient method for obtaining high-quality DNA from spermatozoa present in cryopreserved bovine semen for molecular diagnostic applications. Five DNA extraction protocols were evaluated: TRIzol™, MagMax™ Nucleic Acid Purification Kit, Rapid DNA™ Fecal/Soil Microbe Kit, a conventional phenol–chloroform protocol, and a modified phenol–chloroform–isoamyl alcohol protocol. All extracted genetic material was assessed by spectrophotometry (concentration and purity), and DNA integrity was evaluated by agarose gel electrophoresis. Statistical analysis revealed significant differences in DNA concentration among extraction methods (Friedman test, χ2 = 22.0, df = 4, p = 0.0002). Post hoc comparisons indicated that the modified phenol–chloroform–isoamyl alcohol protocol yielded significantly higher DNA concentrations compared to selected methods. This protocol showed the highest DNA concentration (1006.2 ± 829.4 ng/μL) and favorable purity values, and enabled consistent amplification in both conventional PCR and qPCR assays targeting the β-actin gene and Tritrichomonas foetus, respectively. These findings suggest that the modified protocol represents a suitable and promising approach for extracting genomic DNA from spermatozoa in cryopreserved bovine semen, with potential applications in molecular diagnostics and reproductive biotechnology.

Rational engineering of a novel cationic LL-37-temporin SHa hybrid peptide with anti-parasitic activity and effects on mitochondrial membrane potential in protozoan parasites.

Tritrichomonas foetus, a protozoan parasite responsible for bovine and feline trichomonosis, has traditionally been considered to form only pseudocysts. Here, we demonstrate that T. foetus produces true cysts characterized by a chitin-like, fibrillar wall, cytoskeletal reorganization, and resistance to detergent treatment. Using scanning and transmission electron microscopy, sarkosyl resistance assays, lectin and chitin-binding staining, and monosaccharide composition analysis, we identify a polysaccharide-rich cyst wall enriched in N-acetylated hexosamines. Cystogenesis is induced by environmental stresses, including nutrient deprivation, alkaline pH mimicking the bovine preputial environment, and metronidazole exposure. Flow cytometry analyses reveal increased DNA content and multinucleation in cysts, consistent with endoreplication. Encysted parasites remain viable and revert to proliferative trophozoites upon restoration of favorable conditions. Pharmacological inhibition of cyst wall biosynthesis enhances metronidazole susceptibility, supporting a functional role for cyst formation in drug tolerance. Transcriptomic profiling identifies pathways associated with carbohydrate metabolism, Myb-like transcription factors, and calcium/calmodulin-dependent kinases during encystation. Together, our findings establish true cyst formation in T. foetus and define a previously unrecognized survival state linked to environmental persistence and reduced drug susceptibility.

A recently developed Tritrichomonas foetus RT-rtPCR reports higher sensitivity and specificity, and lower limit of detection (LOD) than other diagnostic modalities at approximately 1 organism/100 μL. Clinical interpretation of cycle threshold (Ct) results could have major implications on beef cattle herd level decisions. The objective of this study was to develop likelihood ratios for T. foetus RT-rtPCR Ct results near the LOD. Six hundred samples inoculated with T. foetus at approximately LOD, and 600 samples left uninoculated, were submit­ted to a diagnostic laboratory for T. foetus RT-rtPCR. Category specific likelihood ratios (LRcat) were calculated to compare Ct results ≤ 35.00, 35.01-39.99 and 40.00+. The median Ct of inoculated samples was 32.93 (min = 22.39, max = 40.00), and of uninoculated samples was 40.00 (min = 32.81, max = 40.00). For Ct ≤ 35.00, LRcat = 432 (95% CI: 60.91-3063.85); Ct = 35.01-39.99, LRcat = 149 (95% CI: 20.92-1061.3); and Ct = 40.00+, LRcat = 0.03 (95% CI: 0.02-0.05). The high LR for categories Ct ≤ 35.00 and Ct = 35.01-39.99 show that those values were more likely to occur in samples that were inoculated than in sam­ples uninoculated. This suggests that any amplification could be indicative of a true positive, and results in the Ct = 35.01-39.99 category should be considered for retest­ing. The LR for category Ct = 40.00+ demonstrates that truly inoculated samples were less likely to have that result. Using LR to estimate post-test prob­ability could aid clinical interpretation of results and more accurate bull culling decisions.

Tritrichomonas foetus is a venereal protozoan parasite that causes significant reproductive losses in cattle. Bulls act as asymptomatic carriers, transmitting the pathogen to susceptible cows, leading to infertility, embryonic or fetal loss, and other reproductive disorders. With no FDA-approved treatments available, vaccination has become a key control strategy. This study evaluated the reproductive effects of a commercially available killed, whole-cell vaccine (TrichGuard®) administered to both heifers and T. foetus-positive bulls in a natural mating system. Heifers and bulls were randomly assigned to one of four vaccine treatment groups. Pregnancy status was monitored, and statistical models assessed pregnancy maintenance and loss. A significant treatment effect was observed on the probability of maintaining pregnancy (P=0.03), with control (uninfected) heifers showing the highest odds of retention. Vaccinated and infected groups all showed reduced odds of maintaining pregnancy, though post hoc comparisons showed no significant differences amongst any of the vaccination protocols. Pregnancy loss did not differ significantly between groups (P=0.22). Survival analysis revealed a treatment effect (P=0.04), driven by the absence of pregnancy loss in the control group. These findings suggest that vaccination with the commercially available vaccine Trichguard® may have variable efficacy in reducing reproductive losses in the face of natural infection.

Tritrichomonas foetus is a protozoan parasite implicated in intestinal infections in domestic cats, frequently associated with chronic diarrhea. Although reported on nearly all continents, its occurrence in Brazil is rare and has been restricted to the Southeast region so far. This study reports, for the first time, T. foetus infection in two Maine Coon cats originating from the Northeastern region of the country. Both animals exhibited persistent diarrhea unresponsive to initial therapeutic interventions and underwent clinical and laboratory evaluation. Fecal analysis performed on samples obtained by colonic lavage revealed motile trophozoites exhibiting morphological features consistent with T. foetus. Molecular confirmation was achieved through PCR amplification and subsequent sequencing, with sequences deposited under accession numbers PV857727 and PV857728. BLAST analysis demonstrated 100% nucleotide identity with reference sequences available in GenBank. Following diagnosis, both cats received appropriate treatment with Tinidazole (Drogavet, Brazil) at a dose of 30mg/kg, administered every 24h for 14 days, resulting in complete clinical remission and subsequent negative fecal examinations. These findings expand current knowledge on the geographical distribution of feline trichomonosis in Brazil, highlight the diagnostic challenges of T. foetus infection, and reinforce the importance of including this protozoan in the differential diagnosis of feline enteropathies.

Chronic diarrhea in cats is a frequent clinical problem, defined by gastrointestinal signs persisting for more than three weeks. Causes are diverse: intestinal dysbiosis, parasites, viral agents, or inflammation. Among protozoa, Tritrichomonas foetus and Cryptosporidium spp. are major contributors, while Feline Coronavirus (FCoV), though often subclinical, can worsen inflammation and dysbiosis, sometimes progressing to infectious peritonitis. This study reports a clinical case with multiple etiologies: T. foetus, Cryptosporidium spp., FCoV, and severe dysbiosis. Diagnostic workup included clinical examination, coproparasitological testing, PCR, and microbiome analysis. Treatment combined symptomatic measures with antiparasitic and antibiotic therapy. Co-infection caused persistent diarrhea and delayed therapeutic response, but PCR confirmation enabled a tailored protocol, gradually improving clinical signs. The case highlights the need for a comprehensive diagnostic approach, as concurrent infections and dysbiosis significantly affect prognosis and therapeutic strategies.

IntroductionTritrichomonas foetus is a sexually transmitted flagellate that causes economic loss in the cattle industry throughout the world. In the United States, there are no approved treatments for the parasite. Owing to its transmission strategy, T. foetus typically infects cattle of breeding age. However, in vivo studies of treatment, diagnostic strategies, and vaccination are severely hampered by the maintenance and cost of maintaining adult cattle in research settings. In this study, we investigated the utility of infecting pre-pubescent heifer calves with T. foetus.MethodsFour independent cohorts of cross-bred prepubertal heifer calves were vaginally inoculated with T. foetus trophozoites previously derived from a naturally-infected bull. Infections were assessed by culture, PCR, DNA sequencing, histopathology, gross pathology, and lesion scoring. In addition, reproductive tract tissue was assessed for the presence of galectin-1, a putative receptor for T. foetus trophozoite adhesion.ResultsOur experiments revealed that despite being in anestrus, heifer calves were amenable to infection with trophozoites for as long as 42 days post-infection as determined by PCR and culture of the organism. Histopathology revealed inflammation throughout the reproductive tract of infected calves. Infection resulted in endometritis with lymphoplasmacytic infiltration and demonstrated that trophozoites could pass through the cervix even during anestrus in prepubescent heifers. In addition, immunohistochemistry of the vagina, cervix, and uterus demonstrated robust expression of galectin-1.ConclusionOur experiments demonstrated that prepubertal heifer calves are a suitable natural host model for bovine trichomonosis. This is a significant breakthrough in the field and also has potential for advancing the human trichomoniasis research agenda.

Ectophosphatase activities and phosphate transport mechanisms in Tritrichomonas foetus and their impact on parasite proliferation.

Transfection of bovine cells with synthetic mRNA to induce expression of functional antibodies against Tritrichomonas foetus surface antigen TF1.17.

The aim of the study was to evaluate the efficacy, pain response, and potential epithelial trauma of 3 devices (an equine infusion/insemination pipette, the Pizzle Stick, and the Trichit) used for sampling bulls naturally infected with Tritrichomonas foetus in the US. The study occurred between March 2023 and May 2024. Twelve bulls naturally infected with T foetus were sampled with the use of a Latin square design. Three devices were used to collect preputial samples, analyzed via reverse-transcription quantitative real-time PCR for diagnostic accuracy. Pain responses were scored by 2 veterinarians. The penis and prepuce of each bull were examined for epithelial trauma 24 hours after sampling. Histologic evaluation of biopsy samples from the penis and prepuce of 8 bulls was conducted after euthanasia to assess cellular damage. All devices provided comparable diagnostic accuracy. Pain scores differed significantly, with the Pizzle Stick causing the least pain and the Trichit causing the most. Epithelial trauma did not show statistical differences between devices, though some trauma was noted, particularly with the equine insemination pipette. Histologic analysis revealed epithelial hyperplasia of the penis and prepuce, unrelated to any device. While all devices effectively diagnosed T foetus, the Pizzle Stick demonstrated the most favorable pain scores, making it the recommended option for reducing discomfort and minimizing risk to both animals and veterinarians. Veterinarians have multiple diagnostic sampling methods for T foetus. The Pizzle Stick's lower pain score makes it the best choice, balancing animal welfare and diagnostic efficacy.

BackgroundInflammatory bowel disease (IBD) is a chronic autoimmune disorder driven by gut microbiota dysbiosis. As an essential immune checkpoint, Programmed death-ligand 1 (PD-L1) has been implicated in modulating gut microbiota composition. However, the precise role of PD-L1 in shaping metagenomic profiles during IBD-associated colitis remains unexplored.MethodsDSS-induced colitis was established in both PD-L1 knockout (Pdcd1lg1-/-) mice and wild-type (wt) control mice. Clinical parameters, including disease activity index (DAI), body weight changes, colon length, and histopathological alterations, were systematically evaluated using non-parametric Kruskal-Wallis tests and ANOVA to compare colitis severity between genotypes.ResultsPD-L1 knockout mice exhibited exacerbated colitis, manifesting significantly greater weight loss (p<0.05 vs. wt_DSS), colonic shortening (p<0.05), and DAI scores (p<0.05) and inflammatory changes. PD-L1 knockout mice showed distinct dysbiosis, with enriched pathobionts (Escherichia coli, p=0.006; Bacteroides thetaiotaomicron, p=0.015) and depletion of commensals (Tritrichomonas foetus, p<0.001; Ligilactobacillus murinus). Alpha diversity analysis using Chao1 index revealed statistically significant differences between experimental groups (p=0.05). The transporters downregulate anti-inflammatory SCFA metabolism. KEGG enrichment analysis of differentially expressed genes (DEGs) revealed significant associations with immune and inflammatory pathways in PD-L1 knockout mice.ConclusionPD-L1 deficiency aggravates colitis by driving pathogenic microbiota alterations and impairing microbial metabolic homeostasis, highlighting its dual regulatory roles in immune homeostasis and microbiome dynamics.

Tritrichomonas foetus is a parasitic protozoan that causes large-bowel disorders in domestic cats worldwide. Between 2020 and 2024, 1606 feline stool samples were tested for T. foetus. The aim of this study was to determine the prevalence of T. foetus infection among cats with diarrhea reported within the last 6 months, and to assess age and sex as risk factors for T. foetus infection. The diagnostic method of choice was nested PCR with modifications to reduce potential reaction inhibition. Overall, T. foetus DNA was detected in 10.6% (170/1606) of cats, with the highest prevalence observed in animals younger than one year, 14.9% (106/711). This study confirms that T. foetus infection is common in domestic cats in Poland. Therefore, this parasite should be included in the differential diagnosis protocol for feline patients with large-bowel diarrhea.

Tritrichomonas foetus is a sexually transmitted protozoan that causes early embryonic death in cattle. A challenge in trichomonosis research is that in vivo studies of treatments, diagnostic strategies, and vaccines are severely hampered by the logistical challenge and cost of maintaining adult bulls. Since natural infections are diagnosed in postpubescent animals, the paradigm is that only mature breeding bulls can be infected. In this study, we hypothesized that prepubescent bull calves could be artificially infected with T. foetus trophozoites for the purpose of conducting research trials. Initial attempts to directly infect bull calves with two different parasite isolates resulted in the sporadic and transient detection of parasite DNA but not culturable trophozoites. In vitro and in vivo studies suggested that urine directly inhibited trophozoites, likely by osmotic damage and mechanical flushing action. Studies utilizing a perineal urethrostomy to remove urine flow from the prepuce resulted in the ability to colonize the prepuce, with live organisms being cultured for as long as 15 days post-inoculation. Future studies optimizing this technique have the potential to accelerate the pace of bovine trichomonosis research and may have applications in the study of human trichomoniasis.

Tritrichomonas foetus is a notable reproductive pathogen in cattle, and the processes of sample collection, handling, transportation, and testing present considerable challenges for surveillance initiatives. Advancements include the development of a direct detection method for T. foetus using reverse-transcription quantitative real-time PCR. We compared the stability of samples in 3 different media: PBS, sterile saline, and lactated Ringer solution over periods of 24-120 h, with storage temperatures of 0°C, 20°C, 37°C, and 54.5°C to mimic shipping of samples in various environmental temperatures.

In this pilot study, the genetic diversity of protozoan parasites was analysed according to their different transmission routes (life cycle strategies), focusing on those species which were recently discovered or molecularly analysed for the first time in Hungary or its geographical region. The results showed that among four apicomplexan parasites (Babesia gibsoni, Cytauxzoon europaeus, Sarcocystis morae and Hepatozoon felis) the latter had the highest genetic diversity as reflected by its 18S rRNA gene sequences showing high (1.75%) maximum intraspecific pairwise distance, and also, based on its phylogenetic clustering. This is probably related to the long evolutionary history of H. felis, the absence of its intravascular division and other life cycle characteristics precluding direct transmission between hosts. On the other hand, among non-apicomplexan protozoa (Trichomonas gallinae, Pentatrichomonas hominis, Tritrichomonas foetus and Acanthamoeba castellanii), the latter proved to have the highest genetic diversity (7.73%), most likely due to its long evolutionary history, lateral gene transfer, homologous recombination and the absence of direct host-to-host dispersal. Transmission mode had a significant impact on the genetic diversity among protozoan parasites, depending on life cycle strategies and consequent frequency/chance of sexual reproduction vs binary fission. In particular, the absence of direct transmission between hosts is a common trait of H. felis and A. castellanii, contributing to their high genetic diversity.

The limit of detection for T. foetus RT-qPCR is 1 organism/ex­traction in phosphate buffered saline (PBS) without prior cul­ture. The objective of this study was to determine if 0.9% ster­ile saline (SAL), a readily available media, was not inferior to PBS as a transport media for T. foetus RT-qPCR testing at that concentration.

"Correction: Tritrichomonas foetus is not located in the accessory glands, epididymis, or testicles of infected bulls, and post mortem changes influence recovery of organism" published on 03 Mar 2025 by American Veterinary Medical Association.

Nowadays, even in light of all the massive advances in cell biology, we still find some cellular structures that are not entirely understood. Among those, we highlight the costa, a structure from the mastigont system existent only in some members of the orders Trichomonadida and Tritrichomonadida, including the pathogens of venereal diseases in humans and cattle, Trichomonas vaginalis (T. vaginalis) and Tritrichomonas foetus (T. foetus), respectively. The costa is a prominent striated fiber and, although part of the cytoskeleton, differs from its classical components, and its molecular composition is still not fully characterized. Using proteomics of T. foetus’s costa fraction, we previously identified hypothetic proteins, and among these, the protein ARM19800.1 positively localized in the costa and named costain-1. In this study, two other protein candidates were analyzed. To achieve the specific localization of 11810 and 32137 proteins in T. foetus’s cells, it was used expansion microscopy and immunocytochemistry. The immunofluorescence revealed the presence of both proteins throughout the whole costa but with different intensities. Immunocytochemistry using negative staining, LR-White, and Epon embedding revealed further analyses of the protein’s localization. All techniques confirmed the distinct and distributed localization of both proteins: costain-2 (11810) and costain-3 (32137). Also, AlfaFold3 was used to generate 3D models of the three identified proteins, showing a major prevalence of α-helical spans. Nonetheless, the identification and further characterization of these unique proteins can help understand their functional role in the assembled costa and, therefore, better understand the organization and function of this structure in these organisms.

To determine the presence of Tritrichomonas foetus in the accessory sex glands, epididymis, and testicular tissue of T foetus-positive bulls and to assess the impact of post mortem tissue acidity on quantitative reverse-transcription PCR results. 8 bulls were euthanized from June 12 through June 15, 2023, and samples from their accessory sex glands, testicles, and epididymis were collected for T foetus testing. Preputial smegma was obtained before and after euthanasia and tested for T foetus by quantitative reverse-transcription PCR. The pH of the penile and preputial epithelial surfaces was measured. Tritrichomonas foetus was not detected in samples from the testicle, epididymis, or epididymal semen. A post mortem decrease in the pH of the penis and prepuce correlated with a rise in cycle threshold values. Tritrichomonas foetus was not present in samples taken, suggesting its absence in these specific reproductive tissues. A decrease in the pH of the penis and prepuce over time post mortem correlated with an increase in cycle threshold values in the samples. This relationship indicates that changes in pH may impact the detectability or stability of genetic material in these tissues, possibly affecting diagnostic outcomes as time from death progresses. This research enhances the understanding of T foetus pathology in bulls and suggests that epididymal semen recovery could be a viable method for preserving genetic material from valuable bulls that test positive for T foetus. It also emphasizes the need for timely post mortem sampling to ensure accurate detection and management of T foetus in the cattle industry.