Melarsomine hydrochloride (Cymelarsan®) fails to cure horses with Trypanosoma equiperdum OVI parasites in their cerebrospinal fluid.

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.

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.

Genome-Wide SNP Analysis Reveals Distinct Origins of Trypanosoma evansi and Trypanosoma equiperdum.

Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission.

First Draft Genome Sequence of the Dourine Causative Agent: Trypanosoma Equiperdum Strain OVI.

Trypanosoma equiperdum is the causative agent of dourine, a sexually-transmitted infection of horses. This parasite belongs to the subgenus Trypanozoon that also includes the agent of sleeping sickness (Trypanosoma brucei) and surra (Trypanosoma evansi). We herein report the genome sequence of a T. equiperdum strain OVI, isolated from a horse in South-Africa in 1976. This is the first genome sequence of the T. equiperdum species, and its availability will provide important insights for future studies on genetic classification of the subgenus Trypanozoon.

Inter-laboratory ring trials to evaluate serological methods for dourine diagnosis.

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.

In vitro effectiveness of the antimicrobial peptide eCATH1 against antibiotic-resistant bacterial pathogens of horses.

The equine antimicrobial peptide eCATH1 previously has been shown to have in vitro activity against antibiotic-susceptible reference strains of Rhodococcus equi and common respiratory bacterial pathogens of foals. Interestingly, eCATH1 was also found to be effective in the treatment of R. equi infection induced in mice. The aim of this study was to assess the in vitro activity of eCATH1 against equine isolates of Gram-negative (Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Pseudomonas spp.) and Gram-positive (R. equi, Staphylococcus aureus) bacteria resistant to multiple classes of conventional antibiotics. A modified microdilution method was used to evaluate the minimum inhibitory concentrations (MICs) of the antimicrobial peptide. The study revealed that eCATH1 was active against all equine isolates of E. coli, S. enterica, K. pneumoniae, Pseudomonas spp. and R. equi tested, with MICs of 0.5-16 µg mL(-1), but was not active against most isolates of S. aureus. In conclusion, the activity of the equine antimicrobial peptide eCATH1 appears to not be hampered by the antibiotic resistance of clinical isolates. Thus, the data suggest that eCATH1 could be useful, not only in the treatment of R. equi infections, but also of infections caused by multidrug-resistant Gram-negative pathogens.

The equine antimicrobial peptide eCATH1 is effective against the facultative intracellular pathogen Rhodococcus equi in mice.

Rhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivo is limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitro and ex vivo experiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivo toxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivo at doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivo and in vivo experiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis.

In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment.

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.

Immune response to Rhodococcus equi ATCC 33701-secreted proteins in mice and identification of immunogenic recombinant proteins by dot-blotting.

Rhodococcus equi remains a significant pathogen, causing severe pneumonia in foals. The development of vaccines and serologic diagnosis could be greatly facilitated by studying the humoral immune response to this equine pathogen. In this study, a crude extract of R. equi ATCC 33701-secreted proteins combined with the Montanide® ISA70 adjuvant was found to be highly immunogenic in mice with the highest titer of 99,000 on day 42 after the first subcutaneous immunization. This immune response was dependent on the quantity of proteins injected and the presence of adjuvant. By dot-blotting, eight recombinant secreted proteins were identified to react strongly with sera from immunized mice. Of these eight proteins, four were detected as immunogenic only when administered in conjunction with adjuvant. This screening strategy led to the identification of promising new candidates for vaccine development.

Viability of Rhodococcus equi and Parascaris equorum eggs exposed to high temperatures.

There is great concern about the potential pathogen contamination of horse manure compost spread in the same fields horses graze in. To ensure that pathogen destruction occurs, temperatures need to be sufficiently high during composting. Here, we investigated the survival rate of two marker organisms, Rhodococcus equi and Parascaris equorum eggs, exposed to temperatures potentially encountered during horse manure composting. Our results show that the time required to achieve a 1 log10 reduction in R. equi population (D-value) are 17.1 h (+/-1.47) at 45 degrees C, 8.6 h (+/-0.28) at 50 degrees C, 2.9 h (+/-0.04) at 55 degrees C and 0.7 h (+/-0.04) at 60 degrees C. For P. equorum eggs we show that at 45 and 50 degrees C, 2 log10 reduction of viability is reached between 8 and 24 h of incubation and that it takes less than 2 h at 55 and 60 degrees C to achieve a viability reduction of 2 log10. These results are useful for identifying composting conditions that will reduce the risk of environmental contamination by R. equi and P. equorum eggs.

Immunogenicity of synthetic Rhodococcus equi virulence-associated protein peptides in neonate foals.

Rhodococcus equi infection is considered the most common cause of pneumonia in foals less than 6 months of age. Immunization of foals and/or mares may become a procedure of choice for prevention. The present work documents the antibody response of neonate foals to R. equi virulence-associated protein (Vap) vaccine candidate peptides. A mixture of 4 R. equi (ATCC 33701) Vap peptides was selected based on their hydrophilicity and recognition by naturally acquired IgG antibodies from 13 adult horses and 33 neonate foals from France and Japan. They were combined with a water-based nanoparticular adjuvant to promote a protective immune response including both Th1 cytokine pattern and antibody response. A single intramuscular injection resulted in an IgG antibody response 30 days later, although inconsistently. In responding animals, no bias in IgG subclass distribution was observed, and antibody response was associated with enhanced serum opsonic activity. In conclusion, data indicate that synthetic Vap peptides combined with nanoparticular adjuvant were immunogenic and resulted in a significant increase in IgG antibodies against the corresponding virulent R. equi strain in a majority of foals.

Foal IgG and opsonizing anti-Rhodococcus equi antibodies after immunization of pregnant mares with a protective VapA candidate vaccine.

The aim of this study was to evaluate serum IgG antibody levels and opsonizing activity in foals from pregnant mares immunized with either proteins from an R. equi strain containing virulence-associated protein A (VapA), an immunodominant surface-expressed lipoprotein encoded by a virulence plasmid crucial for virulence in foals, or a whole killed virulent R. equi preparation. Forty-eight pregnant mares were distributed into three groups, i.e. 24 immunized with R. equi VapA protein antigen associated with a water-based nanoparticle adjuvant (Montanide IMS 3012), 8 immunized with whole killed R. equi, and 16 non-immunized as control. Serum IgG and opsonizing capacity were evaluated during pregnancy in mares, and up to day 45 post-delivery in foals in which R. equi infections were recorded in the first 6 months of life. Pregnant mares immunized with virulent R. equi proteins developed higher serum IgG and opsonic activity which were transferred to the foals than either in the whole R. equi immunized or the control group. Four foals developed pneumonia in the control group while none in immunized groups. Results support further evaluation of VapA protein antigen associated with a water-based nanoparticle adjuvant as a candidate vaccine for immunization of pregnant mares resulting in passive antibody-mediated protection of foals.