Epididymitis in mice experimentally infected with Trypanosoma equiperdum: a histopathological and immunohistochemical study.

Trypanosoma equiperdum is a causative agent of dourine in equids and is transmitted from stallions to mares by coitus. Dourine-affected stallions develop orchitis and epididymitis, and these lesions are considered to be responsible for the sexual transmission of T. equiperdum during coitus. However, the parasitic site of trypanosomes in the reproductive organs and the mechanisms underlying transmission have not yet been elucidated histopathologically. We examined the reproductive organs of male mice infected with T. equiperdum histopathologically and identified severe epididymitis with sperm granulomas. Many trypanosomes were detected in the epididymal interstitium and a few were seen within tubular lumen. Interstitial inflammatory cells mainly consisted of Iba1-, iNOS- and CD204-positive cells with a few CD3-, FOXP3- or PAX5-positive cells. There was diffuse immunolabelling of tumour necrosis factor-α (TNF-α) within these inflammatory foci. While caspase-3-positive epithelial cells in the epididymis were not observed in control mice, they were detected multifocally in infected mice and were frequently associated with loss of immunolabelling of zonula occludens-1 (ZO-1), a major protein that forms tight junctions between epididymal epithelial cells. Anti-laminin immunofluorescence revealed an indistinct basement membrane of the epididymal duct. These results suggest that trypanosomes in the epididymal interstitium induce the infiltration of TNF-α-secreting macrophages. Secreted TNF-α may impair the tight junctions of the epididymal duct by inducing apoptosis and downregulating ZO-1 expression.

Molecular detection of 7SL-derived small RNA is a promising alternative for trypanosomosis diagnosis.

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), Trypanosoma brucei (nagana) and Trypanosoma evansi (surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected with Trypanosoma equiperdum by analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4°C, room temperature or 30°C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.

Histopathological lesions in reproductive organs, distal spinal cord and peripheral nerves of horses naturally infected with Trypanosoma equiperdum.

BACKGROUND: Dourine, a venereal transmitted trypanosomosis caused by Trypanosoma equiperdum, has different clinical signs related to the reproductive and nervous system. Pathologic tissue changes associated with the disease are poorly described. The present study describes the histopathological lesions in naturally T. equiperdum-infected horses in the chronical stage of dourine. RESULTS: Four chronically dourine diseased horses underwent a post-mortem examination. They were Woo test negative, but CATT/T. evansi positive, had a low packed cell volume (PCV) and exhibited obvious clinical signs of dourine. Post-mortem examination did not reveal gross lesions in the organs assumed to be responsible for the symptomatology. On histopathology, genital organs were affected, with mononuclear cell infiltration and erosions and degeneration of seminiferous tubules and perivascular lymphoplasmacytic cuffing in the uterus. In the nervous system, mononuclear cell infiltration was located in peripheral nerves, ganglia and in the spinal cord, leading to axonal degeneration. Real-time PCR using ITS primer revealed the presence of trypanosomes in these organs and conventional PCRs using maxicircle and RoTat1.2 primers further confirmed the involvement of T. equiperdum since the DNAs from the vagina, testicle, distal spinal cord, sciatic and obturator nerves found to be positive for maxicircle and negative for RoTat 1.2. CONCLUSIONS: The histopathological lesions in the spinal cord and peripheral nerves explain the incoordination of the hind legs in T. equiperdum-infected horses, whilst its presence in the genital tract exemplifies the venereal transmission.

Reduction of Trypanosoma equiperdum from equine semen by single layer centrifugation.

Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 102 to >5 × 106 trypanosomes, were added to semen samples. Subsequently, SLC was performed following standard procedures. The presence of the parasite in the purified semen was checked by wet smear examination, ITS1 PCR and in vivo inoculation in mice. Before SLC, all spiked semen samples, except the negative controls, were positive on PCR analysis. After SLC, all the pellets were found to be negative for T. equiperdum on microscopic examinations. Examination of the pellet by PCR could also not detect any parasite-DNA in the SLC-pellet of semen spiked with the lower number of parasites (102 to104 trypanosomes). However, in the SLC pellets spiked with 104 - 5 × 104 trypanosomes, only 1 out of the 4 replicates was negative for parasite DNA. All groups spiked with >5 × 104 trypanosomes were found to be positive on PCR. All mice in the positive controls exhibited parasitaemia (5/5). Mice inoculated with SLC-purified semen that was spiked with lower than 5 × 104 trypanosomes, remained free of parasitaemia, similar to the negative controls. However inoculation with SLC-pellets from samples with a higher number of trypanosomes (>5 × 104 - 5 × 106 and > 5 × 106), induced parasitaemia in 2 out of 5 and 3 out of 5 mice, respectively. This study indicates that single layer centrifugation can be used to clear T. equiperdum infected semen but that the success is dependent on the number of parasites.

Tissue (re)distribution of Trypanosoma equiperdum in venereal infected and blood transfused horses.

Dourine, caused by Trypanosoma equiperdum, is a life-threatening venereal disease in equidae. So far, there is no clear evidence on how and when stallions become infectious, nor which tissues are affected by the parasite in diseased animals. Post-infection, after a transient, temporary phase of parasitaemia, the parasite disperses to different tissues in an unknown distribution pattern. This study describes the distribution of the parasite after infection by artificial insemination (AI) or blood transfusion. Mares (N = 4) were artificially inseminated with T. equiperdum spiked semen whereas stallions (N = 4) were infected by blood transfusion. The course of the disease was monitored by parasitological (Woo) and molecular (PCR) tests and clinical signs and haematological parameters were recorded. At 120 days post infection, horses had a full necropsy, histopathology and PCR. A similar pattern of parasitaemia, disease progression and tissue distribution were seen in all horses. Ejaculated semen in the preclinical stage and epididymal semen in the chronic stage of the disease was positive on PCR and caused infection in mice. Cymelarsan® treatment in the chronic stage did not result in a clinico-haematological or histopathological improvement. At necropsy, lesions were observed in the nervous and reproductive system. Histopathological lesions were most severe in the peripheral nerves and associated ganglia, the testicles and genital mucosae with multifocal infiltration of lymphocytes, plasma cells and histocytes. The parasites disseminated to several tissues including the nervous system, testicles and semen. The results indicate that transmission of T. equiperdum is possible through semen even from symptomless stallions post-treatment.

Validation of a new experimental model for assessing drug efficacy against infection with Trypanosoma equiperdum in horses.

Trypanosoma equiperdum, the causative agent of dourine, may affect the central nervous system, leading to neurological signs in infected horses. This location protects the parasite from most (if not all) existing chemotherapies. In this context, the OIE terrestrial code considers dourine as a non-treatable disease and imposes a stamping-out policy for affected animals before a country may achieve its dourine-free status. The use of practices as drastic as euthanasia remains controversial, but the lack of a suitable tool for studying a treatment's efficacy against dourine hampers the development of an alternative strategy for dourine infection management. The present study reports on the development of an experimental infection model for assessing drug efficacy against the nervous form of dourine. The model combines the infection of horses by Trypanosoma equiperdum and the search for trypanosomes in the cerebrospinal fluid (CSF) through an ultrasound-guided cervical sampling protocol. After a development phase involving four horses, we established an infection model that consists of inoculating 5 × 104T. equiperdum OVI parasites intravenously into adult Welsh mares (Equus caballus). To evaluate its efficacy, eight horses were infected according to this model. In all these animals, parasites were observed in the blood at 2 days post-inoculation (p.i.) and in CSF (12.5 ± 1.6 days p.i.) and seroconversion was detected (8.25 ± 0.5 days p.i.). All eight animals also developed fever (rectal temperature > 39 °C), low hematocrit (< 27%), and ventral edema (7.9 ± 2.0 days p.i.), together with other inconstant clinical signs such as edema of the vulva (six out of eight horses) or cutaneous plaques (three out of eight horses). This model provides a robust infection protocol that induces an acute trypanosome infection and that allows parasites to be detected in the CSF of infected horses within a period of time compatible with animal experimentation constraints. We conclude that this model constitutes a suitable tool for analyzing the efficacy of anti-Trypanosoma drugs and vaccines.

Development of an indirect ELISA for the serological diagnosis of dourine.

Dourine is a parasitic venereal disease of equines caused by T. equiperdum. Humoral antibodies are found in infected animals, but diagnosis of dourine must include history, clinical, and pathological findings in addition to serology. Complement Fixation Test (CFT) is the Office International des Epizooties (OIE) recommended test for international trade; however, some uninfected equines may give inconsistent or nonspecific reactions in CFT due to the anticomplementary effects of their sera. In this study an Indirect Enzyme Linked Immunosorbent Assay (iELISA) was developed. This test could be used to confirm positive serological cases of dourine or to solve inconclusive results obtained by CFT, in addition to Indirect Fluorescent Antibody Test (IFAT) and a Chemiluminescent Immunoblotting Assay (cIB). Six-hundred-and-six CFT negative sera and 140 sera positive to CFT and IFAT were tested by iELISA using OVI T. equiperdum as antigen. Results were expressed as percentage of positivity and the optimum cut-off value determined sensitivity and specificity of 100%. All positive sera, tested by cIB, were confirmed as positive. Additionally, twenty seven sera, low-positive at CFT and negative by IFAT, were tested with iELISA and cIB. All samples resulted negative by cIB and one of them was positive in ELISA. Our results suggest that iELISA and cIB may be used as alternative or supplementary confirmatory tests whenever other recommended serological methods are inconclusive or doubtful.

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.

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules.

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay. cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healthy horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16-35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.

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.

Trypanosoma equiperdum: master of disguise or historical mistake?

After 100 years of research, only a small number of laboratory strains of Trypanosoma equiperdum exists, and the history of most of the strains is unknown. No definitive diagnosis of dourine can be made at the serological or molecular level. Only clinical signs are pathognomonic and international screening relies on an outdated cross-reactive serological test (the complement-fixation test) from 1915, resulting in serious consequences at the practical level. Despite many characterization attempts, no clear picture has emerged of the position of T. equiperdum within the Trypanozoon group. In this article, we highlight the controversies that exist regarding T. equiperdum, and the overlap that occurs with Trypanosoma evansi and Trypanosoma brucei brucei. By revisiting the published data, from the early decades of discovery to the recent serological- and molecular-characterization studies, a new hypothesis arises in which T. equiperdum no longer exists as a separate species and in which current strains can be divided into T. evansi (the historical mistake) and Trypanosoma brucei equiperdum (the master of disguise). Hence, dourine is a disease caused by specific host immune responses to a T. b. equiperdum or T. evansi infection.

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.

The current challenges of dourine: difficulties in differentiating Trypanosoma equiperdum within the subgenus Trypanozoon.

During its 20th annual meeting in Paris in May 1999, the OIE (World organisation for animal health) Ad Hoc Group on Non-Tsetse Transmitted Animal Trypanosomoses expressed the following concerns about dourine: the discrepancies in some of the results of the complement fixation test (CFT), which is the only international diagnostic test officially recognised by the International Organisation for the Transportation of Equidae; the persistence of suspected cases of dourine in some Asian, European and African countries; the impossibility of differentiating Trypanosoma equiperdum from Trypanosoma evansi and of isolating new strains of T. equiperdum from clinical cases that have appeared in various parts of the world since 1982. In the light of these concerns, it was decided, in agreement with the Directorate of the Federal Veterinary Services of Russia in Moscow, to perform comparative trials on the value of CFT/dourine at the OIE Reference Laboratory for dourine in Moscow (The All-Russian Research Institute of Experimental Veterinary Medicine) using reagents (antigens and sera) from seven countries with extensive experience in the field of dourine diagnosis, namely, South Africa, France, Italy, Germany, Russia, the United States of America and the People's Republic of China. It is thanks to the successful co-operation of these countries that the trials were made possible. Results showed an overall concordance and were submitted for consideration to the OIE Biological Standards Commission, the commission which is in charge of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. These trials serve as a starting point for further study, particularly in the following areas: the isolation of new strains of T. equiperdum from clinical dourine cases; the identification of specific markers for T. equiperdum which would make it possible to differentiate it from among the other species within the subgenus Trypanozoon; the experimental infection of horses with newly isolated T. equiperdum strains to compare their pathogenicity with those currently used in national diagnostic laboratories and with that of T. evansi; phylogenetic studies; the proposal and validation of new, internationally recognised diagnostic test(s) for dourine.

Detection of trypanosome DNA in serologically positive but aparasitaemic horses suspected of dourine in Ethiopia.

A field study of horses was conducted in the province of Bale, Ethiopian highlands. A rapid questionnaire analysis indicated that dourine, known as "Dirressa", is a major health problem of equines in this area. A total of 121 horses suspected of dourine were examined by use of clinical, parasitological, serological and DNA based techniques. Incoordination of hindlegs (76%), swelling of external genitalia (48.8%) and emaciation (39.7%) were the most common clinical signs observed. Using the haematocrit centrifugation technique (HCT), no trypanosomes were detected in blood, genital washes or tissue fluids. By contrast, trypanosome specific DNA products were amplified by PCR and subsequently detected by DNA probe hybridization in blood samples of 29 horses (29/104), all serologically positive by CFT and/or ELISA. Positive PCR results were significantly associated with swelling of external genitalia (P< 0.05). There is strong evidence, although there was no direct detection of T. equiperdum, that dourine is highly prevalent in the area, a finding which is in accordance with earlier reports. It is concluded, that this PCR assay provides a very sensitive tool in the diagnosis of active infections of dourine in endemic areas where trypanocidal drug use is common.

Observations on the transmission, immunology, clinical signs and chemotherapy of dourine (Trypanosoma equiperdum infection) in horses, with special reference to cerebro-spinal fluid.

This paper is a record of observations on the transmission and clinical signs of dourine in naturally infected cases of known duration, and of temporal and quantitative aspects of the immune response in blood and cerebro-spinal fluid. Included in the record are observations on the presence of Trypanosoma equiperdum parasites in these body fluids and methods for their detection. There is evidence that the occurrence of nervous symptoms and lesions in infected horses is associated with the presence of Trypanosoma equiperdum parasites in cerebro-spinal fluid. The suitability of cerebro-spinal fluid as an environment for the parasite and its relationship with nervous manifestations of the disease are discussed. Observations support the previously reported lesions of peripheral polyneuritis and suggest a possible correltation between the consitstent position of the nervous lesions and the drainage of cerebro-spinal fluid containing the parasite. Chemotherapy with an experimental drug MSbE was used with varying results in 4 horses at different stages of infection.