Immunity to infections in the lower genital tract of bulls.

The mucosa of the bovine prepuce has unique immunological characteristics critical to defense against sexually transmitted diseases. Tritrichomonas foetus and Campylobacter fetus subspecies venerealis persistently colonize the lower genital tract of bulls but usually do not cause either major clinical signs or inflammation. These microbes may be sexually transmitted to female cattle to cause reproductive failure. Although the male genital immune responses to T. foetus and C. fetus subspecies venerealis are inefficient in clearing infection, systemic immunization with T. foetus and C. fetus subspecies venerealis antigens does prevent or eliminate these infections with induction of IgG antibodies in genital secretions and serum.

Preputial cellular and antibody responses of bulls vaccinated and/or challenged with Tritrichomonas foetus.

Systemic and genital immune responses in bulls were determined after infection with Tritrichomonas foetus and systemic vaccination with whole cell antigens. Vaccinated bulls resisted infection, developed IgG1 and IgG2 antibodies against T. foetus in preputial secretions and serum, and had increased MHC II(+) and CD205(+) cells (probably dendritic cells), CD3(+) and CD8(+) T cells, and B cells including IgG1 and IgA plasma cells in the prepuce. Non-vaccinated bulls challenged with T. foetus were persistently infected and had no detectable antibodies to T. foetus in either preputial secretions or serum for 6 weeks post challenge. We conclude that genital and serum IgG antibodies to T. foetus accounts for resistance of vaccinated bulls to T. foetus infection and that the lack of an antibody response in infected bulls accounts for persistent infection.

Studies of trichomonad protozoa in free ranging songbirds: prevalence of Trichomonas gallinae in house finches (Carpodacus mexicanus) and corvids and a novel trichomonad in mockingbirds (Mimus polyglottos).

This study refutes the accepted dogma that significant pathogenic effects of Trichomonas gallinae are limited to columbiformes and raptors in free ranging bird populations in North America. Trichomonads were associated with morbidity and mortality amongst free ranging house finches (Carpodacus mexicanus), mockingbirds (Mimus polyglottos) and corvids (scrub jay: Aphelocoma californica; crow: Corvus brachyrhynchos; raven: Corvus corax) in northern California. Prevalence of trichomonad infection was 1.7% in house finches, 0-6.3% in corvids, and 0.9% in mockingbirds. Bird case fatality ratio was 95.5% in house finches, 0-100.0% in corvids, and 37.5% in mockingbirds. DNA sequences of parasites in house finches and corvids were identical to T. gallinae strain g7 (GeneBank AY349182.1) for the 5.8s ribosome. DNA sequences of parasites cultured from two mockingbirds were genetically distinct from that of available sequenced trichomonads. These isolates were clearly phylogenetically more closely related to the Trichomonadinae than the Tritrichomonadinae. While molecular techniques were required to differentiate between trichomonad species, wet mount preparations from the oral cavity/crop were a reliable and inexpensive method of screening for trichomonad infections in these species. Positive wet mount tests in house finches and corvids living in northern California were highly likely to indicate infection with T. gallinae, while in mockingbirds positive wet mounts most likely indicated a trichomonad other than T. gallinae.

Detection of trichomonad species in the reproductive tracts of breeding and virgin bulls.

Trichomoniasis is a sexually transmitted disease of cattle and a large bowel diarrheal disease of cats caused by Tritrichomonas foetus. Recently, other species of trichomonads have been identified from the prepuce of virgin bulls. It is not clear whether these non-T. foetus isolates are common (nor) or is it clear whether they are also present on the prepuce of breeding bulls. To answer these questions, we first developed an immunofluorescent assay (IFA) with T. foetus-specific monoclonal antibodies for comparison with a T. foetus-specific PCR assay. Results showed that all PCR positive isolates were also IFA positive, whether the isolates were from cats or cattle and PCR negative isolates were IFA negative. Bovine non-T. foetus (non-Tf) trichomonad isolates were detected by both assays in 14 virgin bulls, 10 breeding bulls, 21 bulls of undetermined breeding status (presumably breeding bulls) and 2 cows. These isolates from virgin bulls were mostly Tetratrichomonas spp. whereas the non-Tf isolates from most breeding bulls and the two cows were Pentatrichomonas hominis. All T. foetus isolates were from breeding bulls or bulls of undetermined breeding status. This IFA test which discriminates between T. foetus and non-Tf may be useful as a diagnostic assay, since no effective legal treatment is available, bulls positive for T. foetus are culled. With increasing reports of T. foetus large bowel infection in cats, these monoclonal antibodies may also be useful for diagnosis of feline infection. Since two isolates of non-Tf trichomonads were obtained vaginas of breeding cows, it may be that these parasites are sexually transmitted like pathogenic T. foetus.

Experimental infection of cats (Felis catus) with Tritrichomonas foetus isolated from cattle.

Tritrichomonas foetus is recognized as the causative agent of venereal trichomoniasis in cattle. It is characterized by embryonic and early fetal death and post-coital pyometra, and feline trichomoniasis, manifest as chronic, large bowel diarrhea. Many of the infected cats are less than 2 years old and specific routes of transmission remain unknown. We recently demonstrated that feline isolates of T. foetus can successfully infect heifers, resulting in pathologic changes similar, but not identical to those previously reported as representative of bovine trichomoniasis. In this study, we experimentally infected six cats less than 1 year of age with a bovine (D-1) isolate of T. foetus and one cat with a feline (AUTf-1) isolate of T. foetus. Within 2 weeks, the cat infected with the feline (AUTf-1) isolate was culture positive for trichomonads in weekly fecal samples. At the end of 5 weeks, only one cat infected with the bovine (D-1) isolate was fecal culture positive for trichomonads. At necropsy, the intestine of each cat was removed and divided into five sections (ileum, cecum, anterior, medial and posterior colon). Contents from each section were collected and cultured. The cat infected with the feline (AUTf-1) isolate was culture positive in the ileum, cecum, medial and posterior colon. Two cats infected with the bovine (D-1) isolate were culture positive in the cecum only. Additionally, each intestinal section was submitted to a pathologist for histopathological examination. The combined results indicate that there are demonstrable differences between the feline (AUTf-1) and bovine (D-1) isolates regarding their infectivity in cats.

Tetratrichomonas spp. and Pentatrichomonas hominis are not persistently detectable after intravaginal inoculation of estrous heifers.

Virgin heifers (44) were intravaginally inoculated at estrus with low (10(6)) or high (10(8)) doses of live Tetratrichomonas sp., Pentatrichomonas hominis (P. hominis), or Tritrichomonas foetus (T. foetus). Controls were inoculated with Diamond's trypticase yeast extract maltose media. Genital infection was determined by culture of cervico-vaginal mucus (CVM) in Schneider's media and InPouch TF as well as by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). The presence of trichomonads in fecal samples was determined by culture in Schneider's medium and PCR/RFLP. In CVM samples, tetratrichomonads were found by PCR/RFLP and Schneider's culture only sporadically at intermittent weeks. The presence of tetratrichomonads was not associated with the dose in the experimental vaginal inoculation since Tetratrichomonas sp. appeared more frequently in heifers inoculated with a low dose of tetratrichomonads than in heifers inoculated with a high dose of tetratrichomonads. Moreover, Tetratrichomonas spp. were isolated not only in heifers inoculated with tetratrichomonads but also in control heifers and in heifers inoculated with P. hominis. In feces, Tetratrichomonas spp. were frequently identified by culture in Schneider's and by PCR/RFLP in heifers of all groups. P. hominis was never found in CVM or feces by any method. Based on the common appearance of tetratrichomonads in feces and vaginal secretions, it appears that tetratrichomonads were detected periodically in the vagina of heifers as a consequence of repeated contamination from feces and not as a result of experimental infection. In summary, in this study, the strains of Tetratrichomonas sp. and P. hominis did not establish persistent infection in heifers.

Evaluation of factors that affect embryonic loss in dairy cattle.

OBJECTIVE: To identify potential risk factors for embryonic loss before 35 to 42 days of gestation in dairy cattle. DESIGN: Prospective observational study. ANIMALS: 381 cows. PROCEDURE: Body condition score was determined at the time of artificial insemination (AI; day 0) and on days 20, 23, and 27 and between days 35 and 41; serum progesterone concentration was measured on days 0; 20 or 21; and 23, 24, or 25. Cows were excluded from analyses if day 0 serum progesterone concentration was > or = 1.0 ng/mL and classified as pregnant on day 23 if serum progesterone was > 1.5 ng/mL on day 20 or 21 and day 23, 24, or 25. Cows were examined via transrectal ultrasonography on day 27 or 28 and rectally palpated for pregnancy on days 35 to 41. RESULTS: 39% of cows that were pregnant on day 23 lost their embryo by day 27, and 18% of cows that were pregnant on day 27 or 28 were not pregnant on days 35 to 41. Breeding a pregnant cow posed the greatest risk for embryonic loss at both time periods. Mean serum progesterone concentrations on day 21 or 22 and day 23, 24, or 25 were lowest for cows that lost an embryo between days 24 and 28. Cows with a linear somatic cell count score > 4.5 before AI were twice as likely to lose the embryo by 35 to 41 days, compared with cows with a score < 4.5. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that embryonic loss could be reduced by more accurate estrus detection, reducing mastitis, and strategies to improve progesterone concentration after breeding.

Development of a simulation model to evaluate the effect of vaccination against Tritrichomonas foetus on reproductive efficiency in beef herds.

OBJECTIVE: To develop a model to evaluate the effect of vaccination against Tritrichomonas foetus on reproductive efficiency in beef herds. SAMPLE POPULATION: A beef herd of 300 cows and 12 bulls (8 bulls < or = 3 years old and 4 bulls > 3 years old). PROCEDURE: The model was developed by use of data for various risk factors and vaccine efficacy. The reference herd was considered to be one in which T. foetus had been diagnosed and bulls were tested for T. foetus before the breeding season. Five thousand iterations were run for each of 13 simulations, with each simulation representing a separate combination of risk factors. RESULTS: In all simulations, vaccination resulted in significantly higher calving incidence than nonvaccination. Shared grazing was found to be the most significant risk factor for a decrease in calving incidence attributable to T. foetus infection, followed in importance by lack of testing before the breeding season and a higher proportion of old bulls. Combinations of risk factors contributed to a loss of income of up to 22%, some of which could be blunted by vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Highest calving incidence is achieved when all bulls are tested for T. foetus before the breeding season and all bulls with positive culture results are culled. Avoiding all risk factors is better than vaccinating, but when this is not feasible for a given herd, the results of this simulation indicate that proper vaccination can decrease economic losses attributable to abortions caused by T. foetus.

Attempts to enhance production of porcine chimeras from embryonic germ cells and preimplantation embryos.

Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.

Effect of post-insemination supplementation with PRID on pregnancy in repeat-breeder Holstein cows.

Embryonic mortality contributes to repeat-breeding in dairy cows; luteal insufficiency is a known cause of embryonic mortality. The objective of this study was to assess the efficacy of supplementation with exogenous progesterone for 14 days on pregnancy maintenance in inseminated repeat-breeder dairy cows. On Day 5 after insemination, treated cows ( n=143 ) received a modified PRID (i.e. without estradiol capsule), which was removed on Day 19. Control cows ( n=148 ) did not receive any treatment. Overall there was no effect of PRID supplementation on pregnancy rates. However, when the study population was stratified by parity and stage of lactation, PRID supplementation significantly improved pregnancy rate in first and second parity late lactation cows (risk ratio = 3.26; 95% CI 1.22, 8.69). Pregnancy rates did not differ between PRID-treated cows with ( n=81 ) and without vaginitis. Control cows tended ( P=0.077 ) to have a higher proportion of abortions than PRID-treated cows (7/50 versus 2/51, respectively). In conclusion, young late lactation repeat-breeder cows benefited from progesterone supplementation, in terms of maintaining pregnancy until traditional time of pregnancy diagnosis.

Detection of Tritrichomonas foetus by polymerase chain reaction in cultured isolates, cervicovaginal mucus, and formalin-fixed tissues from infected heifers and fetuses.

A rapid, reliable polymerase chain reaction (PCR) assay, originally developed for definitive laboratory identification of the bovine venereal pathogen Tritrichomonas foetus from cultures of male reproductive tract fluids, was used for testing the following: 1) cultured, geographically disparate trichomonad isolates, 2) formalin-fixed tissues from infected heifers and naturally infected fetuses, and 3) cervicovaginal mucus (CVM) from experimentally infected females. In 12 of 12 Western Hemisphere isolates of pathogenic T. foetus (isolated from outbreaks of clinical trichomoniasis or from screening surveys) and in 1 of 1 American Type Culture Collection strain of Tritrichomonas suis, PCR yielded a positive result, i.e., a 347-base pair amplicon in the 5.8S ribosomal RNA and internal transcribed spacer (5.8S-ITS) region of the genome, whereas cultures of Trichomonas vaginalis and Trichomonas gallinae did not produce a PCR product. The PCR assay was also positive in formalin-fixed, paraffin-embedded endometrial samples from 4 of 4 experimentally infected heifers, as well as in archived tissues from 2 of 2 T. foetus-infected aborted bovine fetuses that were submitted to the diagnostic laboratory from a natural outbreak. It was negative in fixed, embedded uterine tissues of 2 of 2 uninfected virgin heifers used as negative controls and in archived fixed gut tissue of a T. gallinae-infected pigeon. In another experiment, CVM aspirated from 4 of 4 experimentally infected heifers in the fifth or sixth postinfection week yielded a positive PCR product of the expected size, whereas CVM from 2 of 2 controls were PCR negative. Pending validation in larger clinical studies, the PCR assay for the 5.8S-ITS coding region of the T. foetus genome offers the prospect of definitive identification of this agent directly from CVM or from formalin-fixed tissues or when false-positive culture results are suspected.

Increased efficiency of transgenic livestock production.

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.

Experimental transmission of epizootic bovine abortion (foothill abortion).

Advances in defining the biology of epizootic bovine abortion (EBA), including identification of the etiologic agent, have been hampered by the inability to reproduce the disease with confidence. Experimental reproduction of EBA, by feeding the tick vector Ornithodoros coriaceus on susceptible pregnant heifers, is not reliable. The primary objectives of this study were to identify specific tissue(s) obtained from EBA-infected fetuses that could transmit the disease, and then utilize such an infectious challenge system to better define the pathogen, host immunity and geographic distribution of the agent. Described here is the ability to routinely reproduce EBA following inoculation of cryopreserved suspensions of homogenized thymus into susceptible pregnant heifers. This challenge system permitted experiments demonstrating the agent was non-filterable, inactivated upon sonication and susceptible to antibiotics. These findings suggest a prokaryotic microbe and represent a major advance in EBA research. Additional experiments demonstrated that inoculation of the cryopreserved EBA-infectious tissue into heifers, prior to breeding, conferred immunity. Furthermore, such immunized heifers were resistant to challenge with heterologous sources of infectious tissue, suggesting monovalent vaccine development might be feasible. Lastly, challenge studies employing animals from Central Nevada, an area considered free of EBA, demonstrated partial immunity, suggesting the pathogen, and possibly the disease, enjoy a broader distribution than previously thought.