The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis.

Trypanosoma equiperdum, which is the etiological agent of dourine, spreads through sexual intercourse in equines. Dourine (T. equiperdum) has been reported in Mongolia, where it is considered an economically important disease of horses. T. evansi has also been reported in Mongolian domestic animals. The objective of this study was to evaluate the potential application of recombinant T. evansi GM6 (rTeGM6-4r)-based diagnostic methods on a farm with an outbreak of non-tsetse transmitted horse trypanosomosis. Ninety-seven percent homology was found between the amino acid sequences of T. equiperdum GM6 and the GM6 of another Trypanozoon, which also shared the same cellular localization. This finding suggests the utility of rTeGM6-4r-based serodiagnostic methods for epidemiological studies and the diagnosis of both surra and dourine in Equidae. Fifty blood samples were examined from a herd of horses. The diagnostic value of an rTeGM6-4r-based ELISA and an rTeGM6-4r-based immunochromatographic test (ICT) were measured in comparison to a T. evansi crude antigen-based ELISA, which is a diagnostic method recommended by the OIE. However, this is not a perfect diagnostic method for trypanosomosis. Positive serum samples were detected in 46%, 42% and 28% of the tested horses using an rTeGM6-4r-based ELISA, crude antigen-based ELISA and rTeGM6-4r-based ICT, respectively. The sensitivity of rTeGM6-based ELISA was 81%, the specificity was 79%, and the agreement was moderate. We conclude that rTeGM6-4r-based ELISA and ICT represent alternative options for baseline epidemiological studies and the on-site diagnosis of horse trypanosomoses in the field, respectively.

Dourine: a neglected disease of equids.

Dourine is a venereal transmitted trypanosomosis causing a major health problem threatening equines worldwide. The origin and identification of Trypanosoma equiperdum within the subgenus Trypanozoon is still a subject of debate. Unlike other trypanosomal infections, dourine is transmitted almost exclusively by coitus. Diagnosis of dourine has continued to be a challenge, due to limited knowledge about the parasite and host-parasite interaction following infection. The pathological lesions caused by the diseases are poorly described and are observed mainly in the reproductive organs, in the nervous system, and on the skin. Dourine has been neglected by research and current knowledge on the disease, and the parasite is very deficient despite its considerably high burden. This paper looks in to the challenges in identification of T. equiperdum and diagnosis techniques with the aim to update our current knowledge of the disease.

Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia.

BACKGROUND: Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection. METHODS: Trypanosomes isolated from the urethral tract of a stallion with suspected dourine, were directly cultivated using soft agarose media at 37 °C in 5 % CO2. For molecular characterization, 18S ribosomal RNA (rRNA) gene, the internal transcribed spacer (ITS) and 8 maxicircle DNA regions were amplified by a PCR and their sequences were determined. To analyze the ratio of the kinetoplastic/akinetoplastic population, the kinetoplasts and the nuclei of trypanosomes were subjected to Hoechst staining and observed by fluorescence microscopy. RESULTS: In addition to the clinical symptoms and the molecular diagnosis, this stallion was definitively diagnosed with dourine by the detection of trypanosomes in the urethral mucosa. These results strongly suggested that the isolated trypanosome was true T. equiperdum. T. equiperdum isolated from the urethral tract was adapted in vitro using soft agarose media. Based on the results of a phylogenetic analysis of 18S rRNA and ITS, this T. equiperdum isolate was classified into the Trypanozoon clade. In a PCR of the maxicircle DNA region, only NADH-dehydrogenase subunits 4 and 5 was amplified. Clear kinetoplasts were observed in most of the T. equiperdum isolates. In contrast, most culture-adapted T. equiperdum were of the akinetoplastic form. CONCLUSION: We concluded that our isolated trypanosome was the first confirmed case of T. equiperdum in Mongolia and named it "T. equiperdum IVM-t1". T. equiperdum IVM-t1 was well adapted and propagated in soft agarose media, which indicates that this culture method is useful for isolation of T. equiperdum from horses with dourine.

Diagnosis of dourine in outbreaks in Italy.

Dourine is trypanosomosis that affects equids, it's mainly sexually transmitted. The disease was first eradicated in Italy in the 1940s, but there was then a serious epidemic in the mid-70s. After sporadic reports at the end of the 1990s, in May 2011 it was reported once more. Clinical diagnosis of dourine can be complex, as clinical signs and gross lesions are not always present. Direct laboratory diagnosis is also problematic, given the low number of parasites normally present in infected tissues and the mild, short-lasting parasitaemia. This article describes the epidemiological, clinical and laboratory data enabling confirmation of the suspicion of dourine in Italy in the 2011 epidemic.

A clinical case of dourine in an outbreak in Italy.

In May 2011, dourine was reported in Italy following the declaration of a positive result observed in a stallion undergoing routine testing for stud purposes. Clinical signs, anatomo-histopathological findings and laboratory results that resulted in the confirmation of diagnosis of dourine in a clinically affected mare, which was the likely source of infection in the stallion, are described.

Comparative diagnosis of parasitological, serological, and molecular tests in dourine-suspected horses.

Study on comparative sensitivity of parasitological, serological, and molecular tests on 237 horses originating from two dourine-suspected districts of Arsi-Bale highlands of Ethiopia was conducted to determine the prevalence of the disease and degree of agreement of the diagnostic tests. Accordingly, the prevalence of the disease was found to be 4.6%, 36.7%, and 47.6% by parasitological Woo test, RoTat 1.2 and 18S PCR tests, respectively. The seroprevalence of the disease was 27.6% in CATT/Trypanosoma evansi test. In Ethiopia, it was for the first time that trypanosomes from dourine suspected horses were demonstrated in 4.6% of the animals using Woo test. The findings of the present study disclosed that dourine is highly prevalent and one of the major diseases of horses in the area. There was no statistically significant difference (P>0.05) in prevalence of the disease between districts, sexes, and age groups of the animals. However, there was a statistically significant difference (P<0.05) in the prevalence of the disease between emaciated and animals with good body condition. Assessment of the degree of agreement of the diagnostic tests employed revealed low to fair (k = 0·1 - 0·4) with significantly higher sensitivity by PCR than other tests.

Serological and parasitological survey of dourine in the Arsi-Bale highlands of Ethiopia.

This study was conducted from August 2005 to January 2007 to determine prevalence and distribution of dourine in horses and to investigate the occurrence of clinical and carrier cases in donkeys and mules in the Arsi-Bale highlands. Study methodology was based on questionnaire, serological, clinical and parasitological survey. The questionnaire indicated that dourine is a major health problem of equines in the Arsi-Bale highlands. Though dourine is commonly observed throughout the year, it has a seasonal character and occurs mostly during the breeding season from June to late September. Serological screening of 646 horses showed a seroprevalence of 184 (28%), 161 (25%) and 125 (19%) for card agglutination test for trypanosomosis, LATEX and enzyme-linked immunosorbent assay, respectively. Risk factors were parity number, previous history of abortion and body condition score. No trypanosomes could be detected by Giemsa staining or by haematocrit centrifugation technique. Ten puppies inoculated with blood samples, genital washes and oedematous fluids remained parasitologically negative. Different characteristic signs of dourine were observed. During the genital stage, mares showed vaginal oedema, discharge and presence of depigmented scars over the external genitalia. In stallions, oedema of the scrotum and prepuce, prepucial and urethral discharge, and ulceration of the genital mucosae mainly of the penile were observed. In both sexes, lameness in one or both legs, partial dragging and stiffness of the hind legs and incoordination were the dominant signs observed as nervous form of the disease.

A field study to estimate the prevalence of Trypanosoma equiperdum in Mongolian horses.

From May to July 2000, a cross-sectional study was conducted to estimate the prevalence of Trypanosoma equiperdum in the horse population of the central province (Tuv aimag) of Mongolia. On average, four herds were selected from each of the 29 aimag subdivisions (119 herds). From each herd, 10 horses were sampled in proportion to sex and age categories in the respective herds (1190 horses). Sera from 1122 horses were analysed for T. equiperdum antibodies using two serological assays, the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA). The crude estimate of the CFT and the ELISA seroprevalence was 7.6 and 6.7%, respectively. Concordance between the CFT and ELISA results was high (96%). The highest number of CFT positive animals was detected in one herd in Möngönmorit (6/10), followed by herds in Bayandelger (5/10) and in Bayantsagaan (5/10). Poor body condition was significantly correlated with positive serological status in both CFT and ELISA. A history of abortion appeared to be a risk factor for both CFT and ELISA seropositivity. Blood samples of all horses belonging to herds with at least three (3/10) seropositive animals (CFT and/or ELISA) were analysed by light microscopy and by PCR using a Trypanosoma (Trypanozoon) brucei specific primer pair. No trypanosomes or any other haemoparasites could be detected in Giemsa stained thin blood smears. Eight out of the 130 samples (6.2%) analysed by PCR gave positive signals. Seven out of the eight PCR positive horses were also serologically positive. One PCR (and ELISA) positive stallion from Möngönmorit showed emaciation, scrotal and preputial oedema and an oedematous skin plaque. From the serological and DNA-based results it is concluded, that trypanosome infections occur in horses in the Tuv aimag of Mongolia. Since at present neither serological nor DNA-based tests allow a subspecies specific identification within the subgenus Trypanozoon, no definitive diagnosis can be given for T. equiperdum. Whether the examined herds are infected with T. equiperdum or with T. evansi, the causative agent of surra, remains an open question. However, based on the clinical findings, the negative parasitological results and the concentration of conspicuous seroprevalences in single herds, circumstantial evidence supports the existence of infections with the causative agent of dourine.

Apparent prevalence of dourine in the Khomas region of Namibia.

A 15-year record of the results of horse sera from the Khomas region of Namibia tested by the complement fixation test for dourine at the Central Veterinary Laboratory in Windhoek before clearing the respective animals for export and competitive sport were subjected to statistical analysis. The range of percentage positive, taken as the apparent prevalence of dourine for the region, during the period of study, was 0-29.09%; the average regional level of apparent prevalence was 8.33%. These figures were thought to be lower than the real situation due to some bias in the sampling criteria. For more accurate results, the more reliable enzyme-linked immunosorbent assay techniques are recommended for use in sero-surveys for dourine in Khomas and other regions of Namibia to provide a basis for development of effective control strategies against the disease.

Dourine in Southern Africa 1981-1984: serological findings from the Veterinary Research Institute, Onderstepoort.

The distribution of positive dourine cases found on the complement fixation test at the Veterinary Research Institute, Onderstepoort from 1981 to 1984, is recorded. Within the Republic of South Africa, foci of infection occurred in the Johannesburg, Pretoria, Potchefstroom, Rustenburg, Upington, Lichtenburg, Kroonstad, Louis Trichardt, Middelburg (Cape) and Mossel Bay state veterinary districts. In Bophuthatswana, Transkei, Lesotho, South West Africa and Swaziland, positive cases were also recorded. Anti-complementary activity of horse sera does not present a problem. In donkey and mule sera, however, sera show anticomplementary activity in approximately 50% of cases. This can be reduced by diluting the sera 1/2 before inactivating at 63 degrees C for 1 h.

The prevalence of dourine in southern Africa.

The geographical distribution of positive dourine cases found on serological testing at Onderstepoort during the period 1954 to 1975 is recorded, showing major foci of infection occurring in the northern Cape Province, northern Orange Free State, western and north-western Transvaal. Positive cases are also recorded from South West Africa, Botswana, Lesotho, Swaziland and Rhodesia.