Tritrichomonas foetus is a motile flagellated protozoan measuring 10–15 × 5–10 μm. The trophozoite form has three anterior and one posterior flagellae and one undulating membrane along the long axis of the organism.^1,2 The pseudocyst form usually occurs when trophozoites are subjected to stressful conditions. During the transformation from trophozoite to pseudocyst, the organism loses its teardrop shape, becoming rounder, and internalizes its flagella. While existing as a pseudocyst, internal cellular structures continue to replicate resulting in a multinucleated organism.^1,2 The organism is microaerophilic, surviving best in areas of low oxygen tension. When observed in wet mount preparations, T. foetus moves in a rolling, jerky pattern.¹ Although the appearance and movement of the organism in culture media is quite characteristic, diagnosticians should be aware that trichomonads other than T. foetus may be present in cultures of preputial scrapings or cervical mucus. These organisms can be a cause of false-positive test results^1–7 (Figure 56.1).

Clinical signs

Clinical signs of trichomoniasis in the male are rare to nonexistent, although mild balanoposthitis has been associated with T. foetus. In the female, infections are characterized by early embryonic loss, post-coital pyometra, and occasionally abortion.^1,3,6,8 Trichomoniasis should be suspected in herds with low overall pregnancy rates and widely distributed gestational ages. History of exposure to nonvirgin bulls of unknown trichomoniasis status, along with signs of early embryonic loss and post-coital pyometra, should raise suspicion of T. foetus in herds with poor reproductive performance.^1,4,8

Following infection, bulls may become inapparent carriers and fail to eliminate infection. It has been long-standing dogma that older bulls have different histological architecture of the penile and preputial epithelium compared with younger bulls, with crypts and folds deepening and enlarging as bulls age.¹ Although this has been related to older bulls more commonly becoming chronic carriers of T. foetus, Strickland et al.⁹ showed no statistical difference in histological samples from younger bulls compared with those of older bulls with respect to preputial and penile epithelium and number of and area contained within epithelial folds. Infected cows and heifers mount an immune response and are often able to eliminate the organism 6–12 weeks following infection. Females less frequently develop a carrier state that persists throughout pregnancy and into the next breeding season or longer.^1,3,4,6,8

Diagnosis

Diagnostic tests commonly utilized to detect T. foetus include culture with microscopic identification, and polymerase chain reaction (PCR).^1,3 The organism may occasionally be detected with microscopic examination of direct samples, but this method lacks sensitivity. For both culture and PCR, the sample is incubated in Diamond’s media or, more commonly, in a commercially available, two-chambered, media-filled pouch (InPouch™).⁴ For culture, the sample is incubated for up to 6 days at approximately 36°C. The sample is inspected under a light microscope daily to check for the presence of T. foetus.⁵ Identification of T. foetus in culture is based on the morphological and movement characteristics of the organism.¹

Test sensitivity can be defined as the proportion of actual positive animals which are correctly identified. Test specificity can be defined as the proportion of actual negative animals which are correctly identified. Sensitivity and specificity of culture and PCR for T. foetus can be influenced by factors occurring at every level of the testing process, including sample acquisition, handling and shipping, as well as laboratory procedures and the type of PCR test utilized by the laboratory.¹⁰ Reported sensitivity for a single culture ranges from 84 to 96% under experimental conditions, but may be as low as 65% in suboptimal conditions in the field.⁷ Specificity of culture can be impaired by the presence of morphologically similar trichomonads.^1,3,5–7,10

PCR allows the detection of small quantities of the genetic material of the target organism by amplification of specific DNA fragments unique to the organism.¹⁰ PCR may also be used to confirm the identity of organisms recovered by culture to avoid false-positive results from morphologically similar trichomonads.^{1,3,4,7,10,11} With PCR being able to detect smaller numbers of T. foetus than culture, samples may be handled in a different manner than culture.^10,11 These options include, but are not limited to, immediate media sample testing with PCR, incubation for 1–2 days in culture media followed by PCR, incubation for 1–2 days in culture media and then freezing the sample prior to shipping, and direct sample PCR testing without any type of culture media utilized.^5,7,10,12 Because the sensitivity and specificity of these different methods vary, the receiving laboratory should be consulted prior to shipment to attain the specific requirements of that laboratory.

Sensitivity and specificity of a single PCR test have been shown to be greater than those for a single culture, although both parameters can be improved by using serial testing, as well as combining PCR and culture tests on the same sample.⁵ Comparison of different testing regimens utilizing different combinations of PCR and culture, as well as different field conditions and sample handling, reveals a wide range of sensitivities and specificities achieved.^3,5,7,12

As with most testing regimens, increasing sensitivity can have a negative impact on specificity. Although all types of PCR tests offer specificities of greater than 90%, veterinarians and producers alike should be aware that false positives can occur, though this has been shown to be diminished by serial and/or combination testing.^5,7 Because of the possibility of false positives, veterinary, laboratory, and regulatory personnel should use proper discretion regarding animals that test positive, particularly until the test results have been confirmed.

Trichomoniasis is reportable to state boards of animal health and several states require nonvirgin bulls to have preputial samples tested either by one PCR or three weekly cultures. These regulations often change and they differ from state to state. Data from field studies utilizing common methods of testing, as well as from experimental epidemiological models, do not support the practice of equating one PCR test with culture of three weekly preputial samples.^5,7 Cobo et al.⁷ showed sensitivity of a single culture to be 67.8%, while sensitivity of a single PCR was 65.9%. Combining both tests on a single sample increased the sensitivity to 78.3%, which was equivalent to two PCR tests taken 1 week apart. Ondrak et al.⁵ reported real-time PCR to be less sensitive and specific than gel PCR when results were compared with samples taken from bulls which were either positive or negative on three cultures taken 1 week apart. Montilla et al.¹² showed excellent sensitivity utilizing a single gPCR when the samples were incubated for 24 hours and then frozen for shipment. Unpublished experimental epidemiological data formulated with models devised by Sanderson show a significant decrease in sensitivity and specificity when a single PCR is compared to three weekly cultures (M. Sanderson, personal communication). Because of the complexities involved in the studies cited, testing regimens should be tailored to the prevalence of trichomoniasis in the herd and surrounding area, the age and history of the bull being tested, individual state regulations, and the laboratory utilized.

Producers should be made aware of the limitations of these protocols and how the sensitivity and specificity can be improved by using more intensive protocols than the regulations may require.^5,7 Since one test protocol does not fit all situations, these protocols should be based on the risk involved, with increased levels of testing in areas of higher trichomoniasis incidence, as well as in individual bulls of higher risk such as older bulls known not to be virgin.^8,11 The practice of 6 weekly cultures has historically been considered the gold standard in the AI industry.⁷ Any number of weekly cultures, PCR tests, or combinations of both should be employed to satisfy the risk level of the bull being tested, regardless of the minimum testing requirements to satisfy state regulations.

Proper sampling of a bull by a veterinarian requires training, attention to detail, and basic knowledge of the organism and how it behaves in the transport/culture media.¹³ Materials required for sampling include an infusion pipette, 12-mL syringe, marking pen, receptacle to maintain trich pouches in an upright position after inoculation, scissors, disinfectant, paper towels, examination gloves, 60-mL syringe, saline, InPouch™, secondary shipping containers, and an insulated shipping box^5,7,13 (Figure 56.2).