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The hemoparasite Trypanosomaequiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)8], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His)8 was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His)8 did not bind either [3H] cAMP or [3H] cGMP up to concentrations of 0.40 and 0.65 μM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA RIα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein.

Dourine, caused by infection with Trypanosoma equiperdum, is one of the trypanosomiasis in equids. The clinical course of dourine is long-term, ranging from 1-2 months to several years. Since the pathogenesis of dourine has not yet been elucidated, experimental studies using mouse infection models are needed. Although mice are not susceptible to most T. equiperdum strains, some strains can infect mice. Even in such strains, infected mice develop rapidly transient parasitemia and die within 2-8 days. Therefore, mice experimentally infected with these T. equiperdum strains are not suitable for mouse infection models to analysis the pathogenesis of dourine. A sequential method of isolating parasites from dourine-affected horses and adapting them to in vitro cultures using soft agarose media was recently developed. Various T. equiperdum strains adapted to in vitro conditions have been established using this technique. We used one of these strains, the T. equiperdum IVM-t2 strain. In the present study, T. equiperdum IVM-t2 strain inoculated mice developed periodic parasitemia during the experimental period of 60 days. Histopathologically, vaginitis and dermatitis were observed. These findings were comparable to those of dourine-affected horses. Therefore, mice infected with T. equiperdum IVM-t2 strain may be a valuable tool for pathological, immunological, and parasitological in vivo research, and will contribute to investigations on the mechanisms underlying the disease process and the host-parasite relationship. [ABSTRACT FROM AUTHOR]

Homologous proteins of the cAMP-dependent protein kinase (PKA) regulatory and catalytic subunits have been identified in Trypanosoma equiperdum (TeqR-like and TeqC-like, respectively). Partially purified TeqR-like from parasites isolated in the presence of glucose migrated as an apparent 55 kDa/57 kDa polypeptide doublet when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a single polypeptide of 57 kDa was obtained when parasites were deprived of glucose, a condition that has been shown to activate a TeqC-like enzyme. As revealed by immunoblots using anti-phospothreonine antibodies, the 57 kDa band corresponded to a form of TeqR-like that was phosphorylated in threonine residues. TeqR-like phosphorylation was reversible since the level of phospho-TeqR-like decreased once glucose was readded to glucose starved-parasites. Dephospho- and phospho-TeqR-like proteins are monomers with native molecular masses of 54.93–57.41 kDa, Stokes radii of 3.42–3.37 nm, and slightly asymmetric shapes (frictional ratio f/fo = 1.36–1.32). A protein kinase of ∼40 kDa was also partially purified from glucose deprived-trypanosomes, which corresponded to the TeqC-like enzyme by its ability to phosphorylate kemptide, its inhibition by PKA-specific inhibitors, and its immunorecognition by anti-PKA catalytic subunit antibodies. TeqR-like and TeqC-like did not coelute following anion-exchange chromatography, revealing that these proteins are not associated forming a complex in T. equiperdum. Yet, when TeqR-like was incubated in vitro with TeqC-like in the presence of Mg2+ and ATP, the 55 kDa dephospho form of the 55kDa/57 kDa polypeptide doublet of TeqR-like was converted into the 57 kDa phospho form, demonstrating that TeqR-like is a substrate for TeqC-like. • Native TeqR-like exists as monomeric dephosphorylated and phosphorylated variants. • The phosphorylation of TeqR-like is reversible and prompted by parasite starvation. • Phospho-TeqR-like is phosphorylated in threonine residues. • TeqR-like is a substrate of TeqC-like even though both proteins are not associated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dourine is a deadly protozoan disease in equids caused by infection with Trypanosoma equiperdum. Neurological signs in the later stage of infection may be caused by peripheral polyneuritis and related axonal degeneration. This neuritis involves T lymphocytes, B lymphocytes, and macrophages, and is observed in cases without obvious neurological signs. However, the pathogenesis of neuritis remains unclear. We identified M2 macrophages and CD8 T cells as the predominant phenotypes in neuritis of dourine-affected horses with or without neurological signs. In contrast, the populations of M1 macrophages and CD4 T cells were small. This result indicates that inflammation was chronic and suggests that dourine-associated neuritis occurs at the early stage of infection. [ABSTRACT FROM AUTHOR]

Two horses were infected with distinct non-tsetse transmitted Trypanozoon Venezuelan stocks, namely TeAp-N/D1 Trypanosoma equiperdum and TeAp-El Frio01 Trypanosoma evansi. Preceding reports have revealed that a 64-kDa antigenic glycopolypeptide (p64), which is the soluble form of the predominant variant surface glycoprotein from TeAp-N/D1 T. equiperdum, can be used as a good antigen for immunodiagnosis of animal trypanosomosis. Here, the course of the experimental acute infection in both horses was monitored by evaluating total anti-p64 IgG and particular anti-p64 γ-specific IgG and μ-specific IgM isotypes in sera using indirect enzyme-linked immunosorbent assays. Both equines showed a maximum of whole anti-p64 antibody generation, which dropped to readings below the maximum but always above the positive cutoff point. Levels of specific IgG and IgM isotypes oscillated throughout the course of the experiments. Essentially, the γ-specific IgG response remained very close to the cutoff point, whereas the μ-specific IgM response displayed values that were mostly above the positive cutoff point, showing a major peak that coincided with the maximum of complete anti-p64 IgG production. These results showed that horses infected with non-tsetse transmitted Trypanozoon parasites developed an immune reaction characterized by a dominant IgM generation against the p64 antigen. [ABSTRACT FROM AUTHOR]

An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5–20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T. equiperdum PKA regulatory subunit-like protein (TeqR-like) on the basis of Western blot and mass spectrometry analyses, and behaved as an uncompetitive or anti-competitive inhibitor of the parasite TeqC-like protein, with respect to a fluorescently labeled substrate (kemptide, sequence: LRRASLG), showing a K i of 1.17 μM. The isolated protein possesses a molecular mass of 57.54 kDa, a Stokes radius of 3.64 nm, and a slightly asymmetric shape with a frictional ratio f / fo = 1.43. As revealed during the purification steps and by immunoprecipitation experiments, the TeqR-like and TeqC-like proteins were not associated forming a heterooligomeric complex, differing from traditional PKA subunits. Co-immunoprecipitation results followed by mass spectrometry sequencing identified two isoforms of the parasite heat-shock protein 70, α-tubulin, and β-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum. • An inhibitor of a Trypanosoma equiperdum PKA catalytic subunit-like protein was identified. • The trypanosome inhibitor corresponds to the PKA regulatory subunit-like protein. • The inhibitor inactivates uncompetitively the PKA catalytic subunit-like protein. • The parasite PKA regulatory and catalytic subunit-like proteins are not associated. • The PKA regulatory subunit-like protein interacts with two HSP70 variants and α/β-tubulin. [ABSTRACT FROM AUTHOR]

• The pathogenesis of neurological clinical signs of dourine has been unclear. • The pathogenesis was investigated by histopathology in four dourine-afflicted horses. • Multifocal radiculoneuropathy was observed in the dourine-afflicted horses. • Neuropathy may be responsible for the neurological clinical signs of dourine. Dourine is an equine protozoan disease caused by Trypanosoma equiperdum. Dourine-afflicted animals die after developing neurological clinical signs, such as unilateral paresis. The disease has been a problem for many years; however, the pathogenesis regarding the neurological clinical signs of dourine has been unclear. In the present study, we conducted a histopathological examination in order to investigate the mechanisms by which dourine-afflicted horses develop the accompanying neurological clinical signs. Four dourine-afflicted horses in Mongolia were evaluated. An apparently healthy horse exhibited multifocal neuritis without axonal or myelin degeneration. The other horses, which had obvious neurological clinical signs, also exhibited multifocal neuritis. In particular, the nerves that innervated areas associated with neurological clinical signs exhibited neuritis with demyelination in the latter horses. Inflamed, non-demyelinating nerves were infiltrated with B lymphocytes and T lymphocytes; while inflamed, demyelinating nerves were infiltrated with mononuclear phagocytes. Our observations revealed lesion progression in the nerves, such that polyradiculoneuropathy could explain the accompanying neurological clinical signs of dourine. To our knowledge, this is the first report to describe a pathogenic mechanism for the development of the neurological clinical signs found in dourine-afflicted horses. [ABSTRACT FROM AUTHOR]

Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07–51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/f o = 1.22–1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed K m values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µ m , respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, β , γ -imidoadenosine 5′-triphosphate and 5′-[ p -(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI- α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum. [ABSTRACT FROM AUTHOR]

• Trypanosomes disseminate to all tissues especially the nervous and reproductive system. • Lesions are predisposed to peripheral nerves, spinal cord and the genital tract. • Similar clinical symptoms of dourine by AI or blood transfusion. • Transmission is possible by semen in the preclinical and post-treatment period. • Pathological changes and venereal transmission of T. equiperdum are clarified. 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. [ABSTRACT FROM AUTHOR]

The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by Trypanosoma evansi. Moreover, T. equiperdum and T. evansi are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the T. equiperdum protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses specifically bind T. equiperdum low molecular weight proteins (from 16 to 35 kDa), which are not recognized by antibodies from uninfected horses. In this work, we tested other 615 sera (7 from naturally infected horses and 608 sera from healthy horses and donkeys): results confirmed the data obtained previously. In addition, six SDS-PAGE bands with molecular weight ranging from 10 to 37 kDa were analyzed by mass spectrometry, in order to identify immunogenic proteins that could be used as biomarkers for the diagnosis of dourine. A total of 167 proteins were identified. Among them, 37 were found unique for T. equiperdum. Twenty-four of them could represent possible candidate diagnostic antigens for the development of serological tests specific for T. equiperdum.

Abstract Background Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia. Methods Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software. Results We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain. Conclusion Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.

Highlights • 8 horses were intravenously infected by T. equiperdum OVI. • 6 horses with parasites in their CSF were treated with melarsomine hydrochloride. • Melarsomine hydrochloride failed to cure horses with T. equiperdum in their CSF. Abstract The aim of this study was to evaluate the ability of melarsomine hydrochloride (Cymelarsan®) to cure horses suffering from a nervous form of dourine, a sexually-transmitted disease caused by Trypanosoma equiperdum. The recently described experimental model for assessing drug efficacy against horse trypanosomosis allowed us to obtain eight horses (Welsh pony mares) infected by T. equiperdum with parasites in their cerebrospinal fluid. The Cymelarsan® treatment evaluated consisted of the daily administration of 0.5 mg/kg of Cymelarsan® over 7 days. Two control horses remained untreated, three horses received the treatment 36 days p.i. and three horses received the treatment 16 days p.i. Following treatment, we observed parasite clearance in blood, stabilization of rectal temperature and a relative improvement in the mean packed cell volume levels for all treated horses. However, live parasites were later observed again in the CSF of all treated horses. Our results indicate the inability of Cymelarsan® to reach Trypanozoon located in the central nervous system of infected horses and thus discourage the use of Cymelarsan® to treat animals suffering from a nervous form of dourine. [ABSTRACT FROM AUTHOR]

Highlights • Intravenous injection of T. equiperdum induced a reproducible dourine-like infection. • Cervical sampling is a suitable tool for detecting trypanosomes in equine CSF. • T. equiperdum parasites were detected in equine CSF 12.5 ± 1.6 days post infection. • This model enables evaluation of drug efficacy against T. equiperdum parasites in CSF. Abstract 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 × 104 T. 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. [ABSTRACT FROM AUTHOR]

Highlights • Diagnosis of dourine include serological tests, animal history, clinical, and pathological findings. • Complement Fixation Test is the OIE recommended test for international trade. • Some uninfected equines may give inconsistent or nonspecific reactions to CFT. • An iELISA and an immunoblotting test (cIB) were developed. • iELISA and cIB could be used as supplementary/confirmatory tests in parallel to CFT. Abstract 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. [ABSTRACT FROM AUTHOR]

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi. [ABSTRACT FROM AUTHOR]

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1–18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037–1062 (which contains the predicted 1–18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1–18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1–18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif. [ABSTRACT FROM AUTHOR]

Trypanosoma equiperdum is a protozoan parasite responsible for causing Dourine, a debilitating neglected veterinary disease, found worldwide affecting equids. It is the only pathogenic trypanosome species that does not require an invertebrate vector for transmission, thus being passed from animal to animal via coitus. At present, there is no officially recognized form of chemotherapeutic treatment and therefore all confirmed (or suspected) cases of infected animals must be slaughtered immediately. For many global communities and farming populations, which rely heavily on their animals for their livelihood, such stringent regulations can seriously enhance the socio-economic problems attributing to poverty. Two reference drugs, together with 37 novel diamidine compounds were tested in vitro using a 72 h drug sensitivity assay to determine their efficacy against two axenically adapted T. equiperdum strains. Further in vivo investigations in mouse models of infection against 4 ‘true’ T. equiperdum strains were performed using the 17 most active diamidines. Single bolus doses of 10 mg kg−1, given i.p. were administered to NMRI mice infected with one of the 4 T. equiperdum strains. The results obtained from this study show that experimentally T. equiperdum can indeed be effectively treated with chemotherapy using in vivo mouse models of infection. [ABSTRACT FROM AUTHOR]

Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei , T. evansi and T. equiperdum are widely distributed and limit animal production throughout the world. The infectivity and virulence of clones derived from Trypanosoma evansi and Trypanosoma equiperdum Venezuelan strains were compared in an in vivo mouse model. Primary infectivity and virulence determinants such as survival rates, parasitemia levels, PCV, and changes in body weight and survival rates were monitored for up to 32 days. The T. equiperdum strain was the most virulent, with 100% mortality in mice, with the highest parasitemia levels (7.0 × 10 7 Tryps/ml) and loss of physical condition. The T. evansi strains induced 100% and 20% fatality in mice. Our results show that the homogeneous parasite populations maintain the virulent phenotype of the original T. equiperdum and T. evansi stocks. This is the first comparative study of infectivity and virulence determinants among clonal populations of T. equiperdum and T. evansi . [ABSTRACT FROM AUTHOR]

Trypanosoma equiperdum is the causative agent of dourine, a venereal disease in horses and donkeys. This parasite has a widely distribution, is found in Africa, Asia, Southern and Eastern Europe, Russia, Mexico and Venezuela. The T. equiperdum is morphologically indistinguishable to other Trypanozoon species, however differs from other mammalian trypanosomes due to the fact that it is primarily a tissue parasite, generating cutaneous plaques, swelling of genitalia and neurological signs. The aim of this study was to evaluate the trypanocidal effectiveness of a set of derivatives of thiosemicarbazones on a T. equiperdum ex vivo culture. All compounds appeared to have trypanocidal activity, however one of them shown better solubility and a dose-dependent effect. The median inhibitory concentration (IC 50 ) was 1.2 μM. The selected compound exhibits a greater inhibitory activity than diminazene aceturate, a common drug for animal trypanosomosis treatment. [ABSTRACT FROM AUTHOR]

Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T. equiperdum . An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC 50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC 50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum . The luciferase assay would be suitable for the mass screening of chemical libraries against T. equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Trypanosoma equiperdum possesses a dense coat of a variant surface glycoprotein (VSG) that is used to evade the host immune response by a process known as antigenic variation. Soluble and membrane forms of the predominant VSG from the Venezuelan T. equiperdum TeAp-N/D1 strain (sVSG and mVSG, respectively) were purified to homogeneity; and antibodies against sVSG and mVSG were raised, isolated, and employed to produce anti-idiotypic antibodies that structurally mimic the VSG surface. Prospective VSG-binding partners were initially detected by far-Western blots, and then by immunoblots using the generated anti-idiotypic antibodies. Polypeptides of ~80 and 55 kDa were isolated when anti-idiotypic antibodies–Sepharose affinity matrixes were used as baits. Mass spectrometry sequencing yielded hits with various proteins from Trypanosoma brucei such as heat-shock protein 70, tryparedoxin peroxidase, VSG variants, expression site associated gene product 6, and two hypothetical proteins. In addition, a possible interaction with a protein homologous to the glutamic acid/alanine-rich protein from Trypanosoma congolense was also found. These results indicate that the corresponding orthologous gene products are candidates for VSG-interacting proteins in T. equiperdum. [ABSTRACT FROM AUTHOR]

Trypanosoma equiperdum belongs to the subgenus Trypanozoon , which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca 2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca 2+ homeostasis. Therefore, the plasma membrane Ca 2+ -ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca 2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1–18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1–18 motif. [ABSTRACT FROM AUTHOR]

Identifying a trypanosome isolate is generally based on morphological observations and molecular identification of one of the genes, usually internal transcribed spacer 1 and 2 of ribosomal DNA (ITS1 rDNA, ITS2 rDNA), a variant surface glycoprotein of Rode Trypanozoon antigen type 1.2 (VSG RoTat 1.2), or expression site-associated genes (ESAG). However, this identification is insufficient because these genes cannot distinguish organisms in the subgenus Trypanozoon to the species level. A molecular approach using at least 5 sets of primers is needed, namely, ITS1, ESAG6/7, MINI, RoTat 1.2, and ND5, for stratified selection to obtain more targeted and conclusive results. Using this method to analyze isolates from Indonesia provided unexpected results: 9 isolates previously identified as Trypanozoon were found to have the kDNA maxicircle gene. Nine isolates of Trypanosoma equiperdum were identified for the first time in Indonesia, isolated from bovine (cattle and buffaloes). The identification of T. equiperdum in the 9 isolates was confirmed by analysis of the nucleotide sequence identity of the nad5-kDNA maxicircle gene., (© American Society of Parasitologists 2023.)

In Italia, nel maggio 2011, in seguito a controlli effettuati su uno stallone per la relativa approvazione alla monta, è stato di nuovo segnalato il morbo coitale maligno (MCM). Nel lavoro viene descritto il caso di una cavalla con sintomi clinici della malattia ritenuta verosimilmente all’origine dell’infezione dello stallone.

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified RII-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to theSus scrofaRIIα isoform. In order to evaluate whetherTrypanosoma equiperdumpossessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-RIIIgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinantT. equiperdumPKA R-like protein recognized the PKA RII-subunit purified from porcine heart and the recombinant human PKA RIβ-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector inT. equiperdum. Although the function of theS. scrofaPKA RIIα and theT. equiperdumPKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A modified Baltz's in vitro cultivation system for the propagation of Trypanosoma equiperdum strain OVI was established to develop a replacement for the conventional production procedure of dourine diagnostic antigen in rats. To increase trypanosome yields we designed an optimized culture medium by addition of supplemental compounds. Trypanosomes were adapted to this medium by two succeeding cultivation steps which led to a substantial proliferation rate and an increased cell density tolerance, respectively. As a result, adapted parasites could be propagated to maximum cell densities of >2 × 10 6 cells/ml, facilitating in vitro antigen production in preparative quantities comparable to the conventional method. A panel of 180 horse field sera, previously sent for testing to the German National Reference Laboratory for Dourine, was tested by complement fixation test using culture-derived as well as conventionally produced dourine antigen. Cohen’s kappa values for results obtained with two batches of culture-derived antigen as compared to conventional antigen were 0.91 (95% confidence interval [CI]: 82.2–99.7) and 0.83 (95% CI: 70.3–95.3), respectively. Performance of antigens for diagnostic purposes was characterized in an inter-laboratory comparative study deploying 14 sera from horses with defined dourine statuses. Complement fixation test results from 15 participating European laboratories showed a diagnostic sensitivity of 94.1% (95% CI: 89.4–98.7) and a diagnostic specificity of 96.2% (95% CI: 92.5–99.9) for conventional antigen and a slightly higher diagnostic sensitivity of 96.0% (95% CI: 92.2–99.8) and a diagnostic specificity of 97.1% (95% CI: 94.0–100) for culture-derived antigen. We conclude that our novel approach for dourine antigen production from in vitro -grown trypanosomes described and evaluated herein meets the requirements for the prospective purpose in quantitative and qualitative terms and should be considered by the competent authorities as an alternative for the animal experiment currently prescribed by international standards. [ABSTRACT FROM AUTHOR]

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.

Background: Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia. Methods: Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software. Results: We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain. Conclusion: Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America. [ABSTRACT FROM AUTHOR]

A summary of the goals and activities of the ad hoc group on Non Tsetse Transmitted Animal Trypanosomosis belonging to the Office International des Epizooties is presented.

To evaluate the reproducibility of routine serological methods to detect Trypanosoma equiperdum antibodies in equine sera, two inter-laboratory ring trials were organized involving 22 European and 4 non-European reference laboratories for dourine. The serological methods were the complement fixation test (CFT; 25 laboratories) and the indirect fluorescent antibody test (IFAT; 4 laboratories). Three of the laboratories applied both these methods. The sample panels were composed of sera that were negative, positive or suspected for dourine. Of the negative sera, one was from a donkey naturally infected with Trypanosoma evansi . This study confirmed the reliability of CFT and highlighted its inter-laboratory reproducibility for known T. equiperdum positive and negative sera. However the reproducibility was less good for sera positive for T. evansi or of unknown status, e.i. nine out of 22 laboratories observed a false-positive result with the T. evansi -positive serum, whether by CFT or IFAT. This interesting result suggests that the specificity of dourine serodiagnosis may be improved by standardizing the critical reagents, including antigens and by developing a standard T. equiperdum serum which could be used calibrate test systems across multiple laboratories. Trial data confirmed seropositivity in one of the three horses suspected of dourine. It may be beneficial to generalize the use of a suitable low-titer serum control, derived from a standard serum in order to standardize the method's detection limit. [ABSTRACT FROM AUTHOR]

Los tripanosomas africanos T. equiperdum y T. evansi son dos especies patógenas del caballo con una amplia distribución en los países en áreas tropicales y subtropicales de ífrica, Asia y parte de Europa para los primeros y en ífrica, Asia y América para los segundos. Estas especies son los agentes etiológicos de la Dourina o Morbo Coital Maligno y de la Surra o Derrengadera respectivamente, están morfológica y antigénicamente relacionadas. Sólo es posible diferenciar las dos especies por la sintomatología clínica y el mecanismo de transmisión de la infección, la cual es por vía coital la primera y por picadura de insectos hematófagos la segunda. Se prepararon hibridomas secretantes de anticuerpos monoclonales contra T. equiperdum y T. evansi. Se usó como antígenos hidrolizados totales de ambas especies y se inocularon intraperitonealmente en ratones hembras Balb/c, sin adjuvante. De la preparación de los hibridomas se clonaron y subclonaron siete hibridomas positivos para cada una de las especies de tripanosomas en estudio. Los anticuerpos monoclonales derivados de los hibridomas para ambas especies mostraron tener relativamente bajos títulos, (1:80), ser de la clase IgM, tener una alta reactividad cruzada con antígenos de ambas especies y ser incapaces de reaccionar frente antígenos separados por electroforesis en geles de poliacrilamida en condiciones desnaturantes. Mostraron una adecuada capacidad de reacción frente antígenos acoplados a placas de microtitulación en ensayos de ELISA. Nuestros resultados demuestran y reafirman que ambas especies son antigénicamente semejantes, probablemente relacionada con la estructura de la parte glicosídica de las proteínas variables de superficie. Estos anticuerpos monoclonales pueden ser útiles como herramientas para la identificación y detección de estas especies de tripanosomas donde ambas especies no se encuentran en infecciones mixtas (Europa del Este y América).

Abstract: In 2011, Trypanosoma equiperdum reemerged in Italy, almost 10 years after its last appearance. A total of eight infected horses have been observed to date. Six horses were affected by natural outbreaks of the disease, whereas two were infected experimentally. The aim of this study was to offer a recent perspective on clinical cases of dourine in Europe. Investigation of the clinical aspects confirmed the three stages reported in the literature: stage 1 (genital lesions), stage 2 (cutaneous signs), and stage 3 (nervous signs). The most common signs in the horses under study were notable weight loss, edematous skin eruptions and oedemas of the abdomen, mammary glands and hind legs. Three animals presented neurological signs (lip ptosis of lower lip and ataxia). Infections were paucisymptomatic or asymptomatic in some animals. Hyperthermia was not reported in infected animals and considerable anemia was observed. High antibody titers did not always correspond to clinical signs. Positive polymerase chain reaction test results of blood or tissue (skin, eye swab) often correspond to an advanced stage of the disease. Dourine is a variable disease; owing to its low prevalence and chronic manifestation, it can be difficult to make a quick diagnosis when facing a Dourine-positive horse. [Copyright &y& Elsevier]

Summary: 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. [Copyright &y& Elsevier]