Fecal PCR Test for Tritrichomonas foetus/blagburni

Sample Requirements & Shipping Information

• 1 gram of fresh feces.
• To increase the probability of detecting infection, the cat should be sampled when it is having diarrhea.
• The fecal sample can be collected from a spontaneously voided stool, using a fecal loop, or using the colonic saline flush technique. A recent large, retrospective study suggests that the fecal loop collection technique might be superior to the colonic flush technique, potentially due to better targeting of mucosal trichomonads, but these results need to be confirmed. Spontaneously voided should be collected promptly after defecation and must be free from cat litter as some components of certain cat litters might inhibit PCR reactions.
• It is recommended to stop antiprotozoal agents, such as ronidazole or metronidazole, at least 2 weeks before sample collection.
• Feces can be stored in a refrigerator for up to one week following collection prior to shipping. Please ship cooled samples overnight with a gel ice pack (the samples do not have to be frozen, but these precautions will help prevent the samples getting overheated during transit, especially during summer months in Texas).

Turnaround

• 1-2 business days after receipt of samples.

General Information

Assay Performance

Tritrichomonas foetus/blagburni is an important cause of diarrhea in cats. This assay amplifies the DNA of T. foetus/blagburni in feces and has been shown to detect as few as 10 organisms per 1 gram of feces. All PCR reactions are performed with positive and negative controls, and a restriction enzyme digest is performed on positive samples to ensure the amplified DNA sequence is specific to T. foetus/blagburni.

Clinical Features

When evaluating feline patients with diarrhea, infectious etiologies are an important category to consider. One such infectious organism is Tritrichomonas foetus/blagburni, a flagellated protozoal parasite that was originally identified as a venereal pathogen of significance in cattle. T. foetus/blagburni has also been identified as an intestinal pathogen in cats, with large bowel diarrhea reported in cats after both experimental and natural infection.

Although the true prevalence of T. foetus/blagburni infection in cats is unknown, it is suspected to be relatively high. In one study, 31% of 117 cats examined at an international cat show were infected with T. foetus/blagburni. While cats of any age, breed, or sex can be infected, young cats that are densely housed (e.g., cats in catteries, animal shelters, or multi-cat households) seem to be at increased risk. Infection is most often seen in young cats (i.e., those that are less than 12 months of age), but older cats may also be infected. Abyssinians with appropriate clinical signs have been identified as having higher odds of a positive PCR test.

T. foetus/blagburniprimarily colonizes the surface of the colonic mucosa, leading to chronic large bowel diarrhea. Without appropriate treatment cats usually remain persistently infected. While diarrhea might spontaneously resolve, cats will often experience recurrent bouts of diarrhea after stressful events. Cats infected with T. foetus/blagburni generally appear healthy (e.g., maintain body weight, systemically healthy otherwise) but show an increased frequency of defecation with loose to liquid stools, which might contain blood and/or mucus. Fecal incontinence is also frequently observed. The anal region often appears edematous and may become painful with severe diarrhea. A rectal prolapse might ensue in some cases.

What Populations Should be Tested?

In general, kittens and young cats from multi-cat environments with chronic (particularly large or mixed bowel) diarrhea should be tested for T. foetus/blagburni infection. However, it should be noted that cats in single cat households and older cats can also get the disease. Thus, this parasite should also be ruled out in cats with clinical signs that do not have another identifiable cause for their diarrhea. Furthermore, cats that are unresponsive to treatment for suspected Giardia spp. infection should be tested, as the two parasites are sometimes confused on routine fecal smear evaluation.

Obtaining a Diagnosis

Diagnosis of a T. foetus/blagburni infection can be made by identification of tritrichomonads on a direct fecal smear examination (see T. foetus vs. Giardia for a video by Dr. Jody Gookin on light microscopic diagnosis), fecal culture, PCR analysis of fecal material, or by colonic mucosal biopsy. Disadvantages of direct fecal smear examination include a low sensitivity (14%), low specificity (T. foetus/blagburni can be misdiagnosed as Giardia spp. or the nonpathogenic Pentatrichomonas hominis), and the fact that only fresh fecal samples can be used.

Tritrichomonas foetus/blagburni can be cultured in-house using the commercially available culture system In Pouch™ TF (Biomed Diagnostics, San Jose, CA). Pouches should be inoculated with less than 0.1 g of freshly voided feces and then incubated at 25°C. Pouches need to be evaluated under a microscope every couple of days. Results are usually obtained between 1 and 11 days of setting up a pouch. While fecal culture is more sensitive than direct fecal smear examination, difficulties in interpreting results, the necessity of using freshly voided feces, and the fact that results might not be available for up to 11 days are noteworthy disadvantages of this method.

Tritrichomonas foetus/blagburni DNA can be amplified from fecal samples by PCR. This diagnostic modality has been shown to be the most sensitive method for detecting T. foetus/blagburni in fecal samples and is ideally suited for direct diagnosis of this organism. Advantages of PCR testing compared to culture include a higher sensitivity, faster turnaround time, and greater sample handling and storage ease as DNA is relatively stable under various temperature conditions. Using a standard fecal loop or the colonic saline flush technique (see video) to obtain a fecal sample directly might help increase the probability of detecting T. foetus/blagburni. The sediment of the sample of fluid obtained from a colonic saline flush should be separated by centrifugation or allowing it to settle. This material is then submitted for PCR. A recent large, retrospective study suggests that the fecal loop collection technique might be superior to colonic flush technique, potentially due to better targeting of mucosal trichomonads. No matter which technique is used, fecal samples should be collected when the cat is having diarrhea.

Treatment

Ronidazole is the only therapeutic proven to successfully resolve diarrhea and eradicate T. foetus/blagburni in some cats. Based on a study of the pharmacokinetics of ronidazole in cats the current treatment recommendation is a dose of 30 mg/kg by mouth once daily for 14 days. Retesting may be performed 2 weeks after the last dose of ronidazole. Apparent treatment failure may be due to lack of patient or owner compliance, administration of an inappropriate dose or duration of ronidazole, reinfection from the environment or from other cats, or infection with a ronidazole resistant strain of T. foetus/blagburni. Ronidazole can cause neurotoxicity in cats so patients should be monitored carefully during treatment, and treatment should be discontinued if any neurological or other clinical signs develop. Some infected cats do not have diarrhea so testing non-diarrheic cats in the same household is warranted. Where possible, in multi-cat households separation of infected and non-infected cats is advised. Infection is via the fecal-oral route and environmental contamination might be important in the epidemiology of T. foetus/blagburni. Studies have documented trophozoite survival in water, urine, and cat food but not cat litter. Further information on T. foetus/blagburni for cat owners is available from Dr. Jody Gookin’s research laboratory at North Carolina State University (Tritrichomonas Diagnostic Laboratory | College of Veterinary Medicine).

Unfortunately, currently there is no pharmaceutical grade ronidazole available on the international marketplace. While non-pharmaceutical grade ronidazole is available, use of such is not recommended as pharmaceutical purity cannot be assured. One other nitroimidazole drug, tinidazole, has anecdotally been used off label for T. foetus/blagburni treatment. It is important to disclose to owners that the pharmacokinetics and efficacy of this drug in cats are unknown, and while it might be appropriate to use, owner consent should be obtained

Several compounding pharmacies that have historically compounded ronidazole are Diamondback Drugs (tel: +1.866.646.2223; diamondbackdrugs.com), Pet Health Pharmacy (tel: +1.800.742.0516), Abrams Royal Pharmacy (tel: +1.800.458.0804), Westlab Pharmacy (phone: +1.800.493.7852), and Creative Compounding (tel: +1.800.672.2177) and likely would do so again once pharmaceutical grade ronidazole becomes available again.

Cats both naturally and experimentally infected with T. foetus/blagburni might also have concurrent dysbiosis based on recent literature. It is unknown if dysbiosis precedes infection and creates a more welcoming environment for the parasite, or it is secondary to trichomonad colonization. While one study identified a reduction in successful trichomonad adherence to intestinal epithelial cells in vitro following treatment with an enterococci probiotic, these results cannot be extrapolated to in vivo infection. Many questions remain, including the benefits of dietary therapy and microbiome modulation (e.g., addition of pre- and probiotics, fecal microbiota transplantation), which will surely comprise future areas of research for improvement in the quality of life for cats infected with T. foetus/blagburni. We encourage owners and veterinarians to visit the resources provided below for further information.

Further Reading

• General NC State Research Laboratory Website: Tritrichomonas Diagnostic Laboratory | College of Veterinary Medicine
• CAPC Website: Companion Animal Parasite Council | Tritrichomonas blagburni
• Video of wet mount light microscopy: T. foetus vs. Giardia
• Video of colonic saline flush: Colon Flush Technique

1. Gookin JL, Hanrahan K, Levy MG. The conundrum of feline trichomonosis – the more we learn the “trickier” it gets. Journal of Feline Medicine & Surgery 2017;19:261-274.
2. Hedgespeth BA, Stauffer SH, Gookin JL. Association of fecal sample collection technique and treatment history with Tritrichomonas foetusPCR test results in 1,717 cats. Journal of Veterinary Internal Medicine 2020;34:734-741.
3. Foster DM, Gookin JL, Poore MF, et al. Outcome of cats with diarrhea and Tritrichomonas foetus Journal of the American Veterinary Medical Association 2004; 225:888-892.
4. Gookin JL, Stebbins ME, et al. Prevalence of and risk factors for feline Tritrichomonas foetusand giardia infection. Journal of Clinical Microbiology 2004; 42:2707-2710.
5. Gookin JL, Birkenheuer AJ, et al. Single-tube nested PCR for detection of Tritrichomonas foetusin feline feces. Journal of Clinical Microbiology 2002; 40:4126-4130.
6. Gookin JL, Copple CN, Papich MG, et al. Efficacy of ronidazole for treatment of feline Tritrichomonas foetus Journal of Veterinary Internal Medicine 2006; 20:536-543.
7. LeVine DN, Papich MG, Gookin JL, et al. Ronidazole pharmacokinetics after intravenous and oral intermediate-release capsule administration in healthy cats. Journal of Feline Medicine & Surgery 2011;12:244-250.
8. Gookin JL, Stauffer SH, Dybas D, Cannon DH. Documentation of in vivo and in vitro aerobic resistance of feline Tritrichomonas foetusisolates to ronidazole. Journal of Veterinary Internal Medicine 2010; 24:1003-1007.
9. Rosado TW, Specht A, Marks SL. Neurotoxicosis in 4 cats receiving ronidazole. Journal of Veterinary Internal Medicine 2007; 21:328-321.
10. Rosypal AC, Ripley A, Stockdale Walden HD, et al. Survival of Tritrichomonas foetusin water, cat urine, cat food, and cat litter. Veterinary Parasitology 2012; 185:279-281.
11. Bierlein M, Hedgespeth B, Azcarate-Peril A, Stauffer SH, Gookin JL. Dysbiosis of fecal microbiota in cats with naturally-occurring and experimentally-induced Tritrichomonas foetusPLoS One 2021 Feb 19;16(2):e0246957. BioProject ID PRJNA692463.
12. Dickson R, Vose J, Bemis D, Daves M, Cecere T, Gookin JL, Steiner J, Tolbert MK. Effect of enterococci on feline Tritrichomonas foetusinfection in vitro. Veterinary Parasitology 2019;273:90-96.

Notice

• This service is performed pursuant to an agreement with Roche Molecular Systems, Inc.