Identification And Biological Characteristics Of A Trichomonad Isolate From Dog

There are two reported trichmonads isolated from canine intestines, whichinclude Tritrichomonas foetus and Pentatrichomonas hominis, and most of the caninetrichomonosis are infected by P. hominis commonly. As a species of zoonoticprotozoa parasitizing in the large intestine of human, dog, cat, nonhuman primate androdent, P. hominis is eliminated with excrement and the trophont can infect hostsdirectly. In recent years, some published cases reported that P. hominis was founded indiarrheic puppies’ feces, but at present there is still no systematic research onbiological characteristics of P. hominis from dog.An undetermined trichomonad was isolated from the feces of a naturally infectedpuppy with diarrhea in Changchun. Characteristics analysis of the trichomonad’cultivation in vitro, morphological identification, molecular biological identificationand animal regression experiment were accomplished in this study. Firstly, thecultivation and cryopreservation protocol in vitro were established. The resultsdemonstrated that the initial density of105/ml trichomonad’s subcultivation was better.As for the cryopreservation, DMSO demonstrated better than glycerol in protectioneffect. The long-term preservation of polypide could be placed in-80℃refrigerator orliquid nitrogen, and the preservation time was at least three months or one yearrespectively. Morphology data obtained by staining, scanning electron microscopyand transmission electron microscopy displayed that body size of the trichomonadwas6-12μm×5-10μm. The independent flagella which is unique for P. hominis wasobserved on the protozoa. Furthermore, other surface and internal structures were thesame as P. hominis. DNA sequence analysis of the partial18S rRNA, ITS1-5.8SrRNA-ITS2and EF-1α genes were accomplished after PCR amplified. Resultsdisplayed that the homology between undetermined trichomonad and P. hominis was99%,99%and99%by the three genes respectively. Phylogenetic tree of thetrichomonad sequence acquired in this study based on the nucleotide sequence of thepartial18S rRNA, ITS1-5.8S rRNA-ITS2and EF-1α genes were drawn. The results showed that the isolated trichomonad from dog was P. hominis or belonged toPentatrichomonas. As for the animal regression experiment,50days postnatal puppieswere successfully infected by taking orally5×10~7polypide. In conclusion, the isolatedTrichomonad from dog was identified as P. hominis.Biological characteristics of P. hominis including BALB/c mice’s infection, drugsensitive test in vitro and infection investigation in different animals wereaccomplished.Experimental infections were established through intragastric administration with106P. hominis polypide vaccinated into BALB/c mice which were5or6weeks old.Executing postmortem examination of the infected animals, movable polypides couldbe observed in large intestinal contents of mice. In addition, assay by nested PCR inwhich the extracted genomic DNA from intestinal contents was amplified displayed apositive result and the sequencing results were consistent with P. hominis as well.These data proved that the infection model was successfully established. Fecesexaminations of mice were carried out after infections and two peaks in excretion of P.hominis curve were detected during post infection6-7d and21-22d respectively,moreover, the amount of second excretion of P. hominis was obviously less than thefirst. The results of autopsy demonstrated that P. hominis was mainly parasitized inthe appendix and a small amount of parasites were detected in segmentum superius ofrectum of mice.Resistance of the trichomonad against four extracts with five variousconcentrations (10,5,2.5,1.25and0.625mg/ml) was tested in vitro. Growth curvesof the four extracts’ five different concentrations and inhibition ratio-time curves ofeach concentration of the four extracts were constructed to analyze their effects on P.hominis. The results showed that the four extracts with all of the five concentrationscould inhibit or kill P. hominis in various degrees, and the anti-trichomonaleffectivenesses enhanced with the increase of concentration. Aqueous extractsdemonstrated the strongest potent trichomonacidal activity among the four extracts,and its MLC against P. hominis at48h was5mg/ml.Nested PCR detection method which was based on partial18S rRNA gene of P.hominis was accomplished in order to investigate P. hominis infection in differentanimal species. In the northeast region of China,252dog feces,60monkey feces, and50diarrheal patient excrements were collected in total. Afterwards, existing samples were tested by the established detection method based on genomic DNA extractedfrom feces. The results displayed that dogs, monkeys, and diarrheal patients in thenortheast region of China were infected in varying degrees by P. hominis, and theinfection rates were27.38%,46.67%, and4%respectively. There are three differentGenotypes of P. hominis in all of the positive samples.