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 Pentatrichomonas hominis 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, Giardia lamblia, 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.

FIG. 1.

Schematic representation of the primer sites used for PCR amplification with respect to the rRNA gene unit of T. foetus. The positions of the boundaries and the 3′ ends of the primers are indicated in reference to T. foetus UT-1 (GenBank accession no. M81842) (2).

FIG. 2.

Analysis of single-tube nested PCR amplification products with primers TFR3 and TFR4 and primers TFITS-F and TFITS-R by 1.5% agarose gel electrophoresis. From left to right, lanes show the following: molecular weight (MW) markers; T. foetus genomic DNA; sterile water (PCR contamination control); DNA extracted from feline fecal samples (200 mg) spiked with 20-μl aliquots of 10,000 to 0.01 cultivated T. foetus organisms; feline fecal DNA (no T. foetus) (NaCl); DNA extracted from sterile water (genomic DNA contamination control) (extraction control); and molecular weight (MW) markers.