Jody L. Gookin

Prevalence of and Risk Factors for Feline Tritrichomonas foetus and Giardia Infection

ABSTRACT Data were gathered for 117 cats from 89 catteries at an international cat show to examine prevalence and risk factors for feline Tritrichomonas foetus and Giardia infection. Prevalence of T. foetus was 31% among cats (36 out of 117) and catteries (28 out of 89) based on results of fecal smear examination (5 out of 36), fecal culture in modified Diamonds medium (9 out of 36), fecal culture in In Pouch TF medium (20 out of 36), or PCR amplification of the ribosomal RNA gene from feces with T. foetus-specific primers (34 out of 36). Catteries in which T. foetus was identified were more likely to have had a recent history of diarrhea, historical diagnosis of coccidia infection in adult cats, and a decreased number of square feet of facility per cat. Evidence did not exist for the ongoing transmission of T. foetus by water, food, or contact with other species.

TRITRICHOMONAS FOETUS AND NOT PENTATRICHOMONAS HOMINIS IS THE ETIOLOGIC AGENT OF FELINE TRICHOMONAL DIARRHEA

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, an organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

Single-Tube Nested PCR for Detection of Tritrichomonas foetus in Feline Feces

ABSTRACT Tritrichomonas foetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity with T. foetus in cats as well as misdiagnosis of the organisms as Pentatrichomonashominis or Giardia sp. when visualized by light microscopy. The diagnosis of T. foetus presently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. As T. foetus organisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of feline T. foetus infection. A single-tube nested PCR was designed and optimized for the detection of T. foetus in feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of the T. foetus internal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by using P. hominis, Giardialamblia, and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms.

Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism

Background:l-Arginine is a nutritional supplement that may be useful for promoting intestinal repair. Arginine is metabolised by the oxidative deiminase pathway to form nitric oxide (NO) and by the arginase pathway to yield ornithine and polyamines. Aims: To determine if arginine stimulates restitution via activation of NO synthesis and/or polyamine synthesis. Methods: We determined the effects of arginine on cultured intestinal cell migration, NO production, polyamine levels, and activation of focal adhesion kinase, a key mediator of cell migration. Results: Arginine increased the rate of cell migration in a dose dependent biphasic manner, and was additive with bovine serum concentrate (BSC). Arginine and an NO donor activated focal adhesion kinase (a tyrosine kinase which localises to cell matrix contacts and mediates β1 integrin signalling) after wounding. Arginine stimulated cell migration was dependent on focal adhesion kinase (FAK) signalling, as demonstrated using adenovirus mediated transfection with a kinase negative mutant of FAK. Arginine stimulated migration was dependent on NO production and was blocked by NO synthase inhibitors. Arginine dependent migration required synthesis of polyamines but elevating extracellular arginine concentration above 0.4 mM did not enhance cellular polyamine levels. Conclusions: These results showed that l-arginine stimulates cell migration through NO and FAK dependent pathways and that combination therapy with arginine and BSC may enhance intestinal restitution via separate and convergent pathways.

Efficacy of Ronidazole for Treatment of Feline Tritrichomonas foetus Infection

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 microg/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.

MOLECULAR CHARACTERIZATION OF TRICHOMONADS FROM FECES OF DOGS WITH DIARRHEA

Trichomonads are occasionally observed in the feces of dogs with diarrhea. On the basis of superficial morphological appearance, these infections have been attributed to opportunistic overgrowth of the commensal, Pentatrichomonas hominis. However, molecular characterization of canine trichomonads has never been reported. This study was performed to determine, by means of rRNA gene sequence analysis, the identity of trichomonads observed in feces from dogs with diarrhea. Total DNA was isolated from fecal samples obtained from a 3-mo-old mixed breed dog and litter of German Shepherd puppies having profuse liquid diarrhea containing numerous trichomonads. Total DNA was subject to PCR amplification of partial 18S rRNA gene or 5.8S, ITS1, ITS2, and partial 18S and 28S rRNA genes using species-specific and universal primers, respectively. Products of 642 and 1864 base-pair length were amplified and cloned. On the basis of rRNA gene sequence, the trichomonads observed in the single dog and the litter of puppies shared 100% identity with Tritrichomonas foetus and P. hominis, respectively. The present study is the first to establish the molecular identity of trichomonads infecting dogs with diarrhea. These studies validate the longstanding assumption that canine trichomoniasis may be attributed to P. hominis. Importantly, these studies additionally recognize that canine trichomoniasis may also be caused by infection with T. foetus.

Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria

Investigation of bacteria involved in pathogenesis of necrotizing enterocolitis (NEC) is limited by infant fragility, analysis restricted to feces, use of culture-based methods, and lack of clinically-relevant animal models. This study used a unique preterm piglet model to characterize spontaneous differences in microbiome composition of NEC-predisposed regions of gut. Preterm piglets (n=23) were cesarean-delivered and nurtured for 30 hours over which time 52% developed NEC. Bacterial DNA from ileal content, ileal mucosa, and colonic mucosa were PCR amplified, subjected to terminal restriction fragment length polymorphism (TRFLP) analysis and targeted 16S rDNA qPCR. Preterm ileal mucosa was specifically bereft in diversity of bacteria compared to ileal content and colonic mucosa. Preterm ileum was restricted to representation by only Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi. In piglets with NEC, ileal mucosa was uniquely characterized by increases in number of Firmicutes and diversity of phyla to include Actinobacteria and uncultured bacteria. Five specific TRFLP profiles, corresponding in closest identity to Clostridium butyricum, C. neonatale, C. proteolyticum, Streptomyces spp., and Leptolyngbya spp., were significantly more prevalent or observed only among samples from piglets with NEC. Total numbers of Clostridium spp. and C. butyricum were significantly greater in samples of NEC ileal mucosa but not ileal content or colonic mucosa. These results provide strong support for ileal mucosa as a focus for investigation of specific dysbiosis associated with NEC and suggest a significant role for Clostridium spp., and members of the Actinobacteria and Cyanobacteria in the pathogenesis of NEC in preterm piglets.

Feline diarrhoea associated with Tritrichomonas cf. foetus and Giardia co-infection in an Australian cattery.

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.

Observed occurrence of Tritrichomonas foetus and other enteric parasites in Australian cattery and shelter cats

Cattery-housed pedigree cats, located mostly within the USA, have the highest reported prevalence of Tritrichomonas foetus (T foetus) to date. This prospective, multi-institutional, cross sectional study examines the occurrence of T foetus and other enteric parasites in cattery-housed and shelter cats within Australia, where T foetus has only recently been identified. Faecal specimens were collected from 134 cats, including 82 cattery-housed pedigree cats and 52 shelter cats. Faecal examinations performed for most cats included concentration techniques, Snap Giardia test, culture in InPouch medium, and polymerase chain reaction (PCR) amplification of T foetus ribosomal ribonucleic acid (rRNA) genes using species-specific primers. Observed occurrence of T foetus, Giardia species, Isospora species and Toxascaris leonina for cattery-housed cats (and catteries) were 0%, 7.4 (13.8)%, 10.9 (22.6)% and 1.6 (3.2)%, respectively. Observed occurrence of T foetus, Giardia species, Isospora species and hookworms for shelter cats were 0%, 11.5%, 9.8% and 4.9%, respectively. These results suggest the prevalence of T foetus in cattery-housed cats is currently much lower in Australia than in the USA, while Isospora and Giardia species infections are common.

Evaluation of Four DNA Extraction Methods for the Detection of Tritrichomonas Foetus in Feline Stool Specimens by Polymerase Chain Reaction

Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect ≥10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.