Application of a PCR Assay to Enhance the Detection and Identification of Tritrichomonas Foetus in Cultured Preputial Samples

Abstract

The traditional diagnostic test for Tritrichomonas foetus involves collection of preputial or vaginal samples followed by culture in a growth media and microscopic examination. Recently, polymerase chain reaction (PCR) techniques have been described for use as a diagnostic assay. The objective of this study was to evaluate a previously described PCR assay for detecting T. foetus in cultured preputial material. The detection limits of the assay for T. foetus organisms in a growth medium, in samples prepared from washing microscope slides, and in preputial material cultured in a growth medium were determined. Preputial samples were collected from 13 bulls uninfected with T. foetus. The PCR assay was able to detect 5 T. foetus organisms in the growth medium and the cultured preputial material. Amplification products were obtained from samples prepared from washes of microscope slides containing as few as 3 visualized organisms. The PCR assay was able to detect organisms in culture at a lower concentration than was possible by direct microscopic examination. This low detection limit may allow the PCR assay to be used to enhance the sensitivity of the current diagnostic test. In addition, the assay could be used to confirm the identification of T. foetus organisms observed by direct microscopic examination when other confirmation techniques, such as staining and phase microscopy, are not practical.

Comparison of the 5.8S rRNA Gene and Internal Transcribed Spacer Regions of Trichomonadid Protozoa Recovered from the Bovine Preputial Cavity

“…Because of the serious consequence of an incorrect diagnosis, distinguishing between T. foetus and these other trichomonads found in the bovine preputial cavity is important. Confirmatory diagnostic tests using a species-specific PCR assay have been described 5,11 and may be necessary for the accurate identification of trichomonads from the bovine preputial cavity, especially for isolates recovered from young bulls. …”

Identification of Trichomonadid Protozoa from the Bovine Preputial Cavity by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Typing

“…4,7 On the basis of 5.8S rRNA gene and internal transcribed spacer region (ITSR) sequence analysis, the non-T. foetus trichomonadid protozoa most frequently isolated from the bovine preputial cavity belong to the genera Pentatrichomonas or Tetratrichomonas. 8 However, accurate identification by sequencing requires specialized resources that may not be readily available to many laboratories to make this a routine identification method.…”

“…by sequencing, were identified as T. foetus by PCR using T. foetus-specific primers, TFR3 and TFR4, and morphologic features based on staining results 4,7 (Table 1). None of the 21 P. hominis samples nor the 16 Tetratrichomonas sp.…”

Construção de iniciadores e otimização de ensaios de PCR e de nested-PCR para a detecção específica de Tritrichomonas foetus

“…O teste de PCR oferece maior probabilidade de acerto, mesmo quando aplicado a amostras com parasitos inviáveis ou em cultivos com baixa concentração destes (PARKER et al, 2001). Esta propriedade propicia a redução do custo e do tempo de realização do diagnóstico, uma vez que pode reduzir o número de cultivos (PELLEGRIN: LEITE, 2003).…”