An improved molecular assay for Tritrichomonas foetus.

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment.

Comparison of the 5.8S rRNA gene and internal transcribed spacer regions of trichomonadid protozoa recovered from the bovine preputial cavity.

Sequence analysis of the 5.8S rRNA gene and the internal transcribed spacer regions (ITSRs) was used to compare trichomonadid protozoa (n = 39) of varying morphologies isolated from the bovine preputial cavity. A multiple sequence alignment was performed with bovine isolate sequences and other trichomonadid protozoa sequences available in GenBank. As a group, Tritrichomonasfoetus isolates (n = 7) had nearly complete homology. A similarity matrix showed low homology between the T. foetus isolates and other trichomonads recovered from cattle (<70%). Two clusters of trichomonads other than T. foetus were identified. Eighteen isolates comprised 1 group. These isolates shared >99% homology among themselves and with Pentatrichomonas hominis. The other non-T. foetus cluster (n = 14) did not exhibit a high degree of homology (<87%) with other bovine isolates or any of the trichomonad sequences available in GenBank. The sequence homology among isolates in that cluster was >99%, except for 1 isolate that varied from the others in both ITSRs (approximately 2% dissimilarity). Sequence analysis of the 5.8S rRNA gene and ITSRs was useful for comparing trichomonadid protozoa isolated from the bovine preputial cavity and demonstrated that 2 distinct types of trichomonads constituted the non-T. foetus isolates recovered from the bovine preputial cavity.

Comparison of diagnostic methods for detection of active infection with Tritrichomonas foetus in beef heifers.

OBJECTIVE: To compare sensitivity of a generic trypticase-yeast extract-maltose (TYM) medium versus a commercial nutrient medium in the diagnosis of Tritrichomonas foetus infection in heifers and to assess sensitivity when incubation of samples inoculated into commercial medium pouches is delayed overnight. DESIGN: Prospective study. ANIMALS: 30 virgin beef heifers. PROCEDURES: 20 heifers vaccinated with a trichomonad antigen and 10 unvaccinated control heifers were exposed at synchronized estrus by intravaginal instillation of 10(6) T foetus organisms. Cervicovaginal mucus samples were collected every other week for 10 weeks from controls and once (10 weeks after exposure) from vaccinated heifers. Samples were inoculated into both media and immediately incubated at 37 C (98.6 F). A duplicate inoculation from controls was made into commercial medium, and the pouch was shipped overnight to a diagnostic laboratory without prior incubation. RESULTS: For 40 of 50 samples from control heifers, there was agreement on diagnoses between media. There was agreement on a positive diagnosis for 3 of 20 samples from vaccinated heifers and on a negative diagnosis for 15 of these 20 samples. For samples shipped overnight before incubation, there were 10% fewer positive diagnoses, compared with samples incubated immediately in commercial medium and 10% more positive diagnoses, compared with samples immediately incubated in TYM. CLINICAL IMPLICATIONS: Use of the commercial medium is a more sensitive indicator of current infection in heifers than use of generic TYM medium. In herds where infection prevalence is high, this method is likely to identify more infected females, an important consideration when control programs include culling of infected cows.

Serological response to in vitro-shed antigen(s) of Tritrichomonas foetus in cattle.

We developed a serological assay for detection of (l) an erythrocyte-adhering molecule(s) shed by the bovine venereal pathogen Tritrichomonas foetus and (II) serum antibodies to this antigen(s) in exposed cattle. Sera from exposed and unexposed cattle were tested for their ability to induce complement-mediated lysis of bovine erythrocytes that had been previously incubated overnight at room temperature in pH-adjusted supernatants of T. foetus culture media. Eight of 180 serum specimens from six groups of presumably unexposed cows or heifers showed a positive (> or = 1:2) hemolytic titer (specificity = 95.6%). Thirteen of 14 females in two experimentally infected groups showed a positive hemolytic titer following infection (sensitivity = 94%). In experimentally infected heifers, there was little correlation (r2 = 0.33) between serum hemolytic titers with respect to shed antigen and titers obtained in serum enzyme-linked immunosorbent assays in which whole T. foetus served as the antigen. Serum hemolytic titers rose 3 to 4 weeks sooner than did previously described vaginal mucus immunoglobulin G1 or immunoglobulin A titers with respect to whole-cell antigen or TF1.17 subunit antigen, respectively. Among 14 chronically infected bulls, only 6 (43%) showed a positive hemolytic titer. This study is the first, to our knowledge, to show a specific serological response in the host to an in vitro-shed antigen(s) of T. foetus and suggests a useful diagnostic test for potentially exposed herds.