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Unexpected Cross-Reaction with Honigbergiella-Like DNA in a PCR for Detection of Bovine Tritrichomonas foetus.

ABSTRACT: The prevalence of bovine Tritrichomonas foetus infection has decreased almost to zero in most European countries, such as Poland, which has been Tritrichomonas foetus-free since 1997. However, trichomonosis is a notifiable disease and there is a duty to examine samples from cattle. In this study, we present an unexpected cross-reaction with Honigbergiella-like DNA in a specimen from a bull. The bovine sample was submitted to the Department of Parasitology National Veterinary Research Institute in Pulawy (NVRI) for confirmatory testing after having been examined at the Regional Veterinary Laboratory, during a routine T. foetus diagnosis. Positive results from microscopic observation and cultures were confirmed. Noteworthily, parasites grew on Diamond's medium only after seven days of incubation, while optimal growth of trichomonads is generally observed after two to four days for this medium. Moreover, by using PCR we obtained positive results for the presence of T. foetus. However, sequencing of the amplification product revealed 99.62% identity with Honigbergiella sp. Our data suggest that false-positive results may occur in commonly used PCR tests. Thus, unexpected results should be carefully interpreted.

SUBMITTER: Dabrowska J

PROVIDER: S-EPMC8067958 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.

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Unexpected Cross-Reaction with Honigbergiella-Like DNA in a PCR for Detection of Bovine Tritrichomonas foetus.

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