Multiyear Survey of Coccidia, Cryptosporidia, Microsporidia, Histomona, and Hematozoa in Wild Quail in the Rolling Plains Ecoregion of Texas and Oklahoma, USA.

We developed nested PCR protocols and performed a multiyear survey on the prevalence of several protozoan parasites in wild northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains ecoregion of Texas and Oklahoma (i.e. fecal pellets, bird intestines and blood smears collected between 2010 and 2013). Coccidia, cryptosporidia, and microsporidia were detected in 46.2%, 11.7%, and 44.0% of the samples (n = 687), whereas histomona and hematozoa were undetected. Coccidia consisted of one major and two minor Eimeria species. Cryptosporidia were represented by a major unknown Cryptosporidium species and Cryptosporidium baileyi. Detected microsporidia species were highly diverse, in which only 11% were native avian parasites including Encephalitozoon hellem and Encephalitozoon cuniculi, whereas 33% were closely related to species from insects (e.g. Antonospora, Liebermannia, and Sporanauta). This survey suggests that coccidia infections are a significant risk factor in the health of wild quail while cryptosporidia and microsporidia may be much less significant than coccidiosis. In addition, the presence of E. hellem and E. cuniculi (known to cause opportunistic infections in humans) suggests that wild quail could serve as a reservoir for human microsporidian pathogens, and individuals with compromised or weakened immunity should probably take precautions while directly handling wild quail.

A new species of Tritrichomonas (Sarcomastigophora: Trichomonida) from the domestic cat (Felis catus).

Feline trichomoniasis is an intestinal disease in cats resulting in chronic diarrhea, flatulence, tenesmus, and fecal incontinence. Bovine trichomoniasis is a sexually transmitted disease of cattle infecting the reproductive tract of cows causing pyometra and possible mid- to late-term abortions. The causative agent for both diseases has been reported to be the flagellated protozoan, Tritrichomonas foetus. However, several published reports support significant biological differences between T. foetus isolated from bovines and felines. In the present study, we describe Tritrichomonas blagburni n.sp. from the domestic cat (Felis catus) as the causative agent of feline intestinal trichomoniasis. We support our proposal based on results of experimental cross-infection studies between cats and cattle using both feline and bovine isolates of the parasite, differences in pathogenicity between the two parasites for the respective host species, and molecular gene sequencing differences between parasites obtained from domestic cats and parasites obtained from cattle.

Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization.

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral (Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents (Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

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

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.

Prevalence and morphology of tritrichomonas suis from the nasal cavity of domestic pigs, sus scrofa, without atrophic rhinitis in the southern Brazil

The Tritrichomonas suis (Gruby and Delafond) that occur in the nasal cavity of domestic pigs. Sus scrofa, was isolated, described and its prevalence studies in healthy swine of different areas of Rio Grande do Sul, Brazil. The parasite protozoa was found in 2 of 208 cultures of nasal washings, representing a prevalence of 0.96 porcent. The morphology study of the live specimens done by phase contrats microscopy, dark field microscopy and by examination of fresh and stained specimens, showed that the T. suis isolated has the same morphological characteristics as the nasal cavity of trichomonads described by othe authors

Prevalence of trichomoniasis among California beef herds.

Sixty cow-half herds of more than 50 cows each were randomly selected for a prevalence survey of bovine trichomoniasis in California. Herd size, as judged by the number of bulls, ranged from 1 to 210 bulls (median = 8; mean = 59 +/- 15.8). Preputial smegma was collected from 729 bulls (median = 6 bulls/herd) and cultured for Tritrichomonas foetus. Of 57 herds from which samples were collected, 9 (15.8%) had at least one infected bull. Of the 729 bulls from which samples were cultured, 30 (4.1%) were infected. Correcting for sensitivity of the diagnostic test yielded a prevalence of 5.0%. Infection rates for bulls greater than 3 years old and less than or equal to 3 years old were 6.7% and 2.0%, respectively (P less than 0.025). Median herd sizes were 14 bulls (range, 6 to 114) for infected herds and 7 (range, 1 to 210) for uninfected herds. These findings suggest that trichomoniasis is common in California beef herds. Because several bulls less than 4 years old were infected, we suggest that control measures stressing replacement of older bulls with younger ones should be combined with diagnostic procedures in those younger replacements, to ensure that they are not already infected.

Identification of Tritrichomonas foetus in sections of bovine placental tissue with monoclonal antibodies.

Sections of bovine placenta from cases of bovine trichomoniasis were examined for the presence of Tritrichomonas foetus by standard histological methods using phase-contrast microscopy and by indirect immunofluorescence assay (IFA) employing monoclonal antibodies (mAbs) specific for T. foetus. Parasites were identified readily in deparaffinized tissue up to 4 yr old by IFA with 2 mAbs previously shown to bind to the surface of living T. foetus. These results indicated that the IFA provided a rapid and specific method of identifying T. foetus in tissue sections as compared to standard histological methods.

Antigenic relationship among field isolates of Tritrichomonas foetus from cattle.

Analysis of protein and antigen profiles of Tritrichomonas foetus isolates from cattle from 5 western states was accomplished by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, immunoblot, immunoprecipitation, and fluorography techniques. Total protein profiles of all isolates were compared by Coomassie brilliant blue staining of T foetus protein samples prepared by 4 protein-extraction methods. Antigenic tritrichomonas proteins were identified by immunoblot assay with polyclonal bovine or rabbit anti-T foetus serum. Additionally, [14C]glucosamine-labeled T foetus was used for total and antigenic glycoprotein analyses. Detectable differences in the composition of total proteins or antigenic tritrichomonal proteins were not observed among all isolates. However, intensity differences in some antigenic protein bands were apparent. Bovine and rabbit sera from immunized animals possessed antibodies to the same antigenic tritrichomonal proteins. Each T foetus isolate contained 4 to 7 molecular weight size classes of glycoprotein, which were labeled by [14C]glucosamine; however, only 3 to 4 glycoproteins were identified as antigens by bovine or rabbit antiserum.

Intestinal trichomonads (Tritrichomonas mobilensis) in the natural host Saimiri sciureus and Saimiri boliviensis.

A retrospective study of cecal and colonic tissues from 28 squirrel monkeys (Saimiri sciureus and Saimiri boliviensis) demonstrated enteric trichomonads within luminal crypts. Twenty-one of 28 (75%) had trichomonads in the mucosal epithelium either in cup-like depressions or intraepithelial vacuoles. Organisms were also beneath the superficial luminal mucosal epithelium and between the basement membrane and crypt epithelial cells. Immunoperoxidase staining also identified organisms within the lamina propria and submucosa. Additional histologic changes included mucosal ulceration, multifocal cryptitis, and focal epithelial necrosis. Most areas containing trichomonads did not have an associated inflammatory response.

Adherence and surface properties of Tritrichomonas mobilensis, an intestinal parasite of the squirrel monkey.

Adherence properties of the potentially enteropathogenic Tritrichomonas mobilensis were studied in vitro. Axenically cultivated trichomonads readily attached to isolated intestinal epithelial cells and mucus of the squirrel monkey. The kinetics and nature of T. mobilensis cytadherence were microscopically evaluated in cell-suspension assay using Chinese hamster ovary (CHO) cells and in microplate hemagglutination assay with human erythrocytes. Adherence of the parasites to target cells was concentration- and time-dependent; it was inhibited by sialic acid (N-acetylneuraminic or N-glycolylneuraminic acid) and sialyllactose. Neither trypsinization of the flagellates nor their exposure to low temperature (4 degrees C) affected their cytadherence capacities. The data indicate the presence of adhesin(s) with lectin properties on T. mobilensis. Agglutination of live protozoa by animal and plant lectins with various carbohydrate-binding specificities as well as the occurrence of an electron-dense cell coat on plasma membrane suggest marked glycosylation of the parasite surface.

Tritrichomonas mobilensis n. sp. (Zoomastigophorea: Trichomonadida) from the Bolivian squirrel monkey Saimiri boliviensis boliviensis.

A trichomonad flagellate, Tritrichomonas mobilensis n. sp., is described from the large intestine of the squirrel monkey, Saimiri boliviensis boliviensis. The organism has a lanceolate body 7-10.5 micrometers in length; a well developed undulating membrane; a stout, tubular axostyle with periaxostylar rings that terminate in a cone-shaped segment projecting from the posterior end of the cell; and a moderately wide costa. The anterior flagella are about as long as the body, and the recurrent flagellum is of the acroneme type. All its characteristics suggest that the new species belongs in the Tritrichomonas augusta type of the subfamily Tritrichomonadinae.

Diagnosis of Tritrichomonas foetus infection in bulls using two sampling methods and a transport medium.

Preputial exudates were collected from 3 bulls infected with Tritrichomonas foetus by scraping the mucosa with a specially designed instrument and by aspiration. For diagnostic purposes the scraping method was superior direct microscopic examination but both methods were equally good when the samples were cultured within 2 hours of collection. The organism remained viable in a transport medium for 24, 48 and 72 hours showing a lineal decrease in viability with time which was more than 3 times greater in samples aspirated than in samples scraped.

[Follow-up of occurrence of protozoa in the nasal cavity of pigs in relation to atrophic rhinitis]. / Sledováni vÿskytu protozoi v nossni dutine prasat vzhledem k sipavkovému onemocnení

Ninety-five pigs, twelve suffering from atrophic rhinitis, were examined for the presence of protozoans in their nasal cavity. Tritrichomonas suis was isolated only in a single case in a diseased boar representing 1.09% of the set. Amoebae of the Vahlkampfia genus were detected in two pigs (2.1%). The axenic cultures of the two protozoans isolated from the pigs were not pathogenic to laboratory animals. No etiological relation of Tritochomonas suis to atrophic rhinitis was demonstrated.