Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences

Abstract

Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples.

FIG. 1

Schematic representation of the primer sites used for PCR amplification and as a capture probe with respect to the rRNA gene unit of T. foetus. The positions of the boundaries between rRNA genes and internal transcribed spacer regions (numbers below the line) and the 3′ ends of primers TFR3, TFR4, and TFR8-Bio (numbers above line) are indicated in reference to T. foetus UT-1 (GenBank M81842) (4). The figure is drawn approximately to scale.

FIG. 2

Analysis of PCR amplification products with primers TFR3 and TFR4 by 2% agarose gel electrophoresis. Purified genomic DNAs from Tritrichomonas spp. (lanes 1 to 12) and other trichomonads (lanes 13 to 19) were used as templates. For documentation regarding numbering of lanes and respective species and strains, see Table 1. Lane 20, negative control; lane M, molecular size standard.

FIG. 3

Sensitivity of PCR amplification with primers TFR3-TFR4 and the UDG system. (A) Analysis of the products was by 2% agarose gel electrophoresis. (B) Detection of products by DEIA with biotinylated oligonucleotide TFR8-Bio. Tenfold serial dilutions of purified T. foetus genomic DNA were used as templates. Amounts of template were 0.3 fg (lane 2), 3 fg (lane 3), 30 fg (lane 4), 0.3 pg (lane 5), 3 pg (lane 6), 30 pg (lane 7), 0.3 ng (lane 8), 3 ng (lane 9), and 30 ng (lane 10). Lane 1, negative control; lane M, molecular size standard. The absorbance units (AU) presented in panel B are A₆₃₀ values subtracted from A₄₅₀ values.

FIG. 4

Sensitivity of PCR amplification with primers TFR3-TFR4 and the UDG system. Analysis of the products was by 10% acrylamide gel electrophoresis and silver staining. Genomic DNAs from serial dilutions of T. foetus organisms were used as templates. The numbers of T. foetus organisms were 1,000 (lane 1), 100 (lane 2), 50 (lane 3), 10 (lane 4), and 5 (lane 5). Lanes 6 and 7, 1 parasite; lane 8, negative control; lane M, molecular size standard.

FIG. 5

Application of PCR with TFR primers to diagnostic samples. (A) Analysis of products by 10% acrylamide gel electrophoresis. (B) Detection of products by DEIA with biotinylated oligonucleotide TFR8-Bio. Lanes 2 to 8, diagnostic samples from seven different bulls with suspected T. foetus infection; lane 1, T. foetus-positive control; lane 9, negative control. The absorbance units (AU) presented in panel B are A₆₃₀ values subtracted from A₄₅₀ values. Animals were retested at different times; results for only one sample per bull are shown.