Direct detection of Tritrichomonas foetus in cattle genital fluid trough loop mediated isothermal amplification of elongation factor 1 alpha 1.

Testing for Tritrichomonas foetus and exclusion of infected animals is an effective way of improving the reproductive efficiency in a herd. Conventional PCR is inherently more specific than the culture method and quantitative PCR can significantly increase the detection limit. Loop Mediated Isothermal DNA Amplification (LAMP) is gaining interest because the method does not require expensive equipment, specificity and sensitivity can be as high as quantitative PCR. The object of this study was to develop a sensitive and friendly test for point-of-care detection of T. foetus. The LAMP test that targeted T. foetus elongation factor 1 alpha 1 sequences showed high specificity. Sensitivity was 100-1000 times higher than that reached through culture, polymerase chain reaction or with a previously developed LAMP for 5.8 ribosomal sequences. Moreover, T. foetus detection could be performed without DNA purification from infected cervical vaginal mucus (CVM) or smegma samples. The tf-ef1a1 LAMP method was tested for field detection with paper strips soaked in CVM from infected cows and the results were observed 90 min later. Direct detection of T. foetus in CVM with the tf-ef1a1 LAMP showed high sensitivity and specificity, and an overall diagnostic odds ratio of 56 (CI: 13.3-235.0). The tf-ef1a1 LAMP showed great potential for diagnosis and control of T. foetus in resource-challenged regions.

Improvements in Tritrichomonas foetus molecular testing.

Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.

Prevalence and risk factors associated with Tritrichomonas foetus infection in cattle in the state of Paraíba, Brazil.

The objective of this study was to determine the prevalence of Tritrichomonas foetus infection and to evaluate risk factors associated with this infection among cattle in the state of Paraíba in northeastern Brazil. Samples of cervicovaginal mucus from 290 females and smegma from 59 males [beef, 31; mixed aptitude (beef and dairy), 10; and dairy, 18] from 31 farms were collected. Modified Diamond's medium and polymerase chain reaction (PCR) were used for the laboratory diagnosis of T. foetus infection. Univariate analysis and logistic regression were performed to test for potential risk factors in addition to prevalence mapping. No sample was positive for T. foetus in culture, and the prevalence of T. foetus infection using PCR was 3.7% (13/349) [confidence interval (CI) 95%, 2.1%-6.4%]. In total, 19.3% (6/31) of the farms had at least one animal positive for T. foetus. The contact of females with males from other farms [Odds ratio, 5.9; 95% CI, 1.5-22.4; p = 0.009] was identified as a risk factor for T. foetus infection. This study demonstrates that T. foetus infection is prevalent among dairy cows in the state of Paraíba, Brazil. Sexual resting, removal of positive females, and avoiding contact of females with males from other farms are recommended to reduce the risk of infection.

Detection of Leishmania infantum in the smegma of infected dogs / Detecção de Leishmania infantum no esmegma de cães infectados

Considering the venereal transmission of visceral leishmaniasis from dogs to bitches, the aim of this study was to verify if the penile surface and smegma from infected dogs can be the source of parasites in bitches. Twelve Leishmania infantum infected dogs had semen and smegma samples collected for submission to PCR identification of the DNA of the parasite. Semen (41.7 percent) and smegma (50.0 percent) have similar positive incidence (P>0.05; Fisher's exact test), with 58.3 percent of the dogs positive for semen and/or smegma samples. The proportion of positivity for both semen and smegma was 33.3 percent, but 8.3 percent was positive only for semen, and 16.7 percent only for smegma, revealing a moderate agreement between tests (K=0.5; Kappa index). It was concluded that Leishmania infantum is present in the smegma of contaminated dogs and it can be a source of parasites for the semen and the bitch...

Tendo em vista a transmissão venérea da leishmaniose visceral do cão para a cadela, o objetivo deste estudo foi verificar se a superfície peniana e o esmegma de cães infectados poderiam ser a fonte de parasitas para a fêmea. Amostras de sêmen e esmegma de 12 cães infectados com Leishmania infantum foram submetidas à identificação do DNA do parasita por PCR. As incidências de positividade no sêmen (41,7 por cento) e no esmegma (50,0 por cento) foram semelhantes (P>0,05; teste exato de Fisher), sendo 58,3 por cento dos cães positivos para sêmen e/ou esmegma. A positividade para sêmen e esmegma juntos ocorreu em 33,3 por cento, mas em 8,3 por cento dos casos apenas no sêmen, e em 16,7 por cento apenas no esmegma, o que revela uma concordância moderada entre os testes (K=0,5; índice Kappa). Conclui-se que a Leishmania infantum está presente no esmegma de cães contaminados, podendo ser a fonte de parasitas para o sêmen e a cadela...

Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus-colonized bulls.

The objectives of the current study were 1) to compare sample preparation workflows and quantitative real-time polymerase chain reaction assays (qPCR) as currently used in veterinary diagnostic laboratories with a study protocol utilizing commercially available reagents for individual Tritrichomonas foetus testing, 2) to assess the accuracy of pooling cultured smegma samples followed by extraction and qPCR testing as used in the study laboratory, and 3) to assess the specificity of the currently used primers and probes by sequencing all positive and presumptive positive samples identified in the study laboratory in an attempt to capture any nucleotide variability between T. foetus isolates and to rule out false-positive results possibly due to Simplicimonas moskowitzi. Eight hundred three cultured smegma samples were collected from different regions of the United States with the collaboration of 5 veterinary testing laboratories. The samples were processed individually by the respective laboratories, and then sent to the study laboratory and retested using the study protocol. Comparison testing showed an overall agreement of 95.89% between the veterinary testing laboratories and the study laboratory. One hundred seventy-six positive or presumptive positive samples plus 625 negative qPCR samples were combined and retested using a pooling protocol. Pools consisted of 1 positive sample and 4 negative samples (1/5). These pools were processed using the same study laboratory protocols, and 96% of the positive samples were detected in these pools. Nested PCR followed by sequencing confirmed 175 of the 178 samples classified as positive or presumptive positive in the study laboratory as containing T. foetus-specific DNA.

Spatial and temporal epidemiology of bovine trichomoniasis and bovine genital campylobacteriosis in La Pampa province (Argentina).

The venereal diseases bovine trichomoniasis (BT) and bovine genital campylobacteriosis (BCG) cause economic losses in endemic areas like La Pampa province in Argentina, where beef cattle are usually managed extensively. This study used data compiled under a Provincial Programme for the Control and Eradication of BT and BGC (PCE) to determine the spatio-temporal distribution of these diseases and identify spatial clusters. The study population comprised 29,178 non-virgin bulls drawn from 3766 herds, tested for BT and BGC in 2010. Preputial smegma samples were cultured for BT detection, while BGC was diagnosed by direct immunofluorescence testing of these samples. Campylobacter fetus infection was detected in 1.5% of bulls and 2.3% of herds, and Tritrichomonas foetus infection was found in 1.1% of bulls and 5.1% of herds. The proportion of positive tests was highest in February for BT, while in April it was highest for BCG, and was inversely related to the number of tests, which was greatest during the breeding season (spring). An elliptical spatial cluster of high risk for BGC and a circular cluster for BT were both identified in the south of La Pampa province, which could not be explained by cattle herd density. The spatial and temporal patterns identified in this study provide baseline data for monitoring the success of BT and BGC control activities in La Pampa.

Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus.

Tritrichomonas foetus is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to infertility and abortion. A test based on loop mediated isothermal amplification (LAMP) targeting the 5.8S rDNA subunit was designed for the specific identification of T. foetus. The LAMP assay was validated using 28 T. foetus and 35 non-T. foetus trichomonads strains. It did not exhibit cross-reaction with closely related parasites commonly found in smegma cultures like Tetratrichomonas spp. and Pentatrichomonas hominis. Bovine smegma did not show interferences for the detection of the parasite and, the sensitivity of the method (4×10(3) CFU/mL, approximately 10 cells/reaction) was slightly higher than that found for PCR amplification with TFR3 and TFR4 primers. The LAMP approach has potential applications for diagnosis and control of T. foetus and, practical use for low skill operators in rural areas.

High prevalence of Tritrichomonas foetus infection in Asturiana de la Montaña beef cattle kept in extensive conditions in Northern Spain.

Bovine trichomonosis (BT) and bovine genital campylobacteriosis (BGC) are sexually transmitted diseases that can be important infectious causes of reproductive failure in extensively managed beef cattle where natural mating is a common practice. However, their prevalence in Europe was thought to be insignificant or very low. The purpose of this study was to investigate the prevalence and risk factors associated with BT and BCG in a representative beef cattle breed, Asturiana de la Montaña (AM), which is usually managed extensively in the mountain areas of Northern Spain and putative risk factors associated with the two diseases are present on most farms holding AM cattle. Preputial smegma samples were collected from 103 bulls belonging to 65 herds. Pathogen detection was undertaken using culture and PCR. Two scraping methods for sample collection (AI pipette and plastic scraper), as well as different culture media and DNA extraction methods were evaluated on field samples. Campylobacter fetus veneralis infection was not detected in any animal in any herd. However, Tritrichomonas foetus infection was demonstrated in 32% (33/103) and 41.5% (27/65) of bulls and herds tested, respectively. AM bulls older than 3 years (39.7%) were more likely to be infected than young bulls (16%) (OR=3.45, CI=1.07-11.19). An increase in repeat breeder cows was reported in herds from which T. foetus was detected (OR=5.2, CI=1.5-17.18). These findings highlight the re-emergence of this disease in extensively managed beef cattle in Spain. For routine diagnosis, the use of a culture technique and PCR in combination is advisable for testing smegma samples under field conditions.

Sensitivity and specificity of culture and PCR of smegma samples of bulls experimentally infected with Tritrichomonas foetus.

The sensitivity (Se) and specificity (Sp) of different testing schemes were estimated for detecting Tritrichomonas foetus (T. foetus) in smegma samples from experimentally infected bulls. Culture and polymerase chain reaction (PCR) on smegma samples were evaluated alone and in parallel testing. Mature dairy bulls (n=79) were intrapreputially inoculated with T. foetus (n=19); Campylobacter (C.) fetus venerealis (n=13); both T. foetus and C. fetus venerealis (n=11); Tetratrichomonas spp. (n=9); C. fetus fetus (n=8); or were not inoculated (n=19). For each bull, smegma samples were collected for 6 week post-inoculation and tested for T. foetus by In Pouch TF culture and PCR. Most T. foetus-inoculated bulls became infected, according to culture (86.7%), PCR (90.0%), and both tests together (93.3%). In T. foetus-inoculated bulls, both tests combined in parallel on a single sample had a Se (78.3%) and Sp (98.5%) similar to two cultures (Se 76.0%, Sp 98.5%) or two PCR (Se 78.0%, Sp 96.7%) sampled on consecutive weeks. The PCR on three consecutive weekly samples (Se 85.0%, Sp 95.4%) and both tests applied in parallel on three consecutive weekly samples (Se 87.5%, Sp 95.6%) were similar to the current gold-standard of six weekly cultures (Se 86.7% and Sp 97.5%). Both tests used in parallel six times had the highest Se (93.3%), with similar Sp (92.5%). Tetratrichomonas spp. were only sporadically detected by culture or PCR. In conclusion, we have proposed alternative strategies for T. foetus diagnostics (for the AI industry), including a combination of tests and repeat testing strategies that may reduce time and cost for bull surveillance.

An improved molecular assay for Tritrichomonas foetus.

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment.

Ultrastructural study of a tetratrichomonad species isolated from prepucial smegma of virgin bulls.

We present observations on an unusual tetratrichomonad species isolated from preputial smegma of virgin bulls. Ultrastructural studies were performed using scanning and electron microscopy techniques. This protozoan presents four anterior flagella of unequal length and a recurrent one forming the undulating membrane. It shows one anterior nucleus, a Golgi complex, an axostyle, and a costa. The hydrogenosomes are rather elongated, seen in groups, and presenting different electron densities. Vacuoles of different sizes containing bacteria and material in process of digestion were frequently found. PCR was also used in order to compare the species herein described with other trichomonad species. The amplification products were seen only with primers TFR1 and TFR2 (specific to trichomonads), but not with TFR3 and TFR4 (specific to Tritrichomonas foetus), suggesting that although collected from the genital tract of the bull, this protist was not T. foetus. We propose that the appearance of these tetratrichomonads were probably due to the sodomy practiced among bulls. Concomitant contamination of preputial cavity with feces could explain the presence of the opportunistic organism. The observations presented here show the importance of the correct diagnostic when investigating samples obtained from the urogenital tract of cattle. We also suggest that this flagellate belongs to the species Tetratrichomonas buttreyi.

Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls.

Portions of penis and prepuce were collected from 24 bulls with current or recent Tritrichomonas foetus infection. Epididymides were collected from seven of the bulls, and seminal vesicles and prostate were collected from four. Following immunohistochemical staining with two monoclonal antibodies (34.7C4.4 and TF1.15) prepared against T. foetus surface antigens, trichomonads were identified in sections from 15 of the bulls. Organisms were most often located in penile crypts in the midshaft and caudal regions and less often in preputial crypts. Trichomonads were not observed in sections from other genitalia or in subepithelial tissue. T. foetus antigen, however, was present in the cytoplasm of some epithelial cells and the cytoplasm of some mononuclear cells in subepithelial lymphoid aggregates and follicles. Preputial smegma was collected from 16 T. foetus-infected bulls and from 16 control bulls with negative T. foetus cultures. Preputial antibody levels to TF1.17, a surface antigen of T. foetus, were determined by an enzyme-linked immunosorbent assay. Preputial secretions from infected bulls contained specific antibody of each isotype and subisotype tested. IgG1 responses were the greatest, IgM and IgA responses were approximately equal, and IgG2 responses were low. Each isotype and subisotype response in infected bulls was significantly greater than that in the controls. These results confirm previous speculation concerning anatomical sites of infection and suggest that parasite antigen can be taken up and processed locally, resulting in deposition of specific IgG1, IgG2, IgA, and IgM antibodies in the preputial cavity.

Prevalence of Tritrichomonas foetus infection in beef bulls in northwestern Spain.

The purpose of this study was to investigate the prevalence of Tritrichomonas foetus infection in beef bulls in north-western Spain. The study area comprised of 350 herds (5909 cows and 102 bulls) managed under extensive or semi-extensive systems where natural mating alone or alternated with artificial insemination are used. The targets of this survey were bulls of 1 year of age or older. Preputial smegma samples were taken from 70 bulls (68.6% of bull population) servicing a total of 184 herds (52.6%) and 4136 cows (69.9%). Data collected at sampling included farm location, herd size, age, breed, private or communal bull and previous infertility problems in the herd. The percentage of infected bulls was 2.9% (2 of 70). Age of infected bulls was 5 and 7 years and both were of the local breed, Asturiana de los Valles. These results confirm the presence of T. foetus infection in Spain and the necessity to include this disease in the differential diagnosis of reproductive failure in rangeland grazing cattle.

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.

Efficacy of ipronidazole against trichomoniasis in beef bulls.

Preputial smegma samples from 195 beef bulls were collected repeatedly and cultured for Tritrichomonas foetus. Seventy-five (38.5%) of these bulls were positive for trichomonads on at least 1 culture. Sensitivity of the culture procedure (number of positive cultures/number of total cultures from known-positive bulls) was 81.6%. Storage of preputial smegma in lactated Ringer's solution at 5 C for 24 hours resulted in a 14% loss of sensitivity. Seventy-three of the 75 infected bulls were available for treatment and were alloted randomly to 2 groups. Bulls in both groups were treated with procaine penicillin (7,000 IU/kg, IM) for 2 days before ipronidazole treatment. Thirty grams of ipronidazole powder was dissolved in 60 ml of sterile water, and was given IM to group 1 bulls. Group 2 bulls were given a similar 30-g ipronidazole solution IM on day 1, and were given 15 g of ipronidazole dissolved in 30 ml of sterile water on days 2 and 3. Efficacy of treatment (ie, negative cultures of preputial smegma for trichomonads for 6 consecutive weeks after treatment) was 92.8% for the 42 bulls treated once and 100% for the 31 bulls treated 3 times.