Julien Cauchard

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

ABSTRACT 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.

The Equine Antimicrobial Peptide eCATH1 Is Effective against the Facultative Intracellular Pathogen Rhodococcus equi in Mice

ABSTRACT 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.

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.

Antimicrobial properties of the equine α-defensin DEFA1 against bacterial horse pathogens

Defensins are small effector molecules of the innate immune system, synthesised by various organisms including plants and animals. The peptides act as endogenous antibiotics with an antimicrobial activity against a broad spectrum of microbes including bacteria, fungi and viruses. alpha-Defensins are a subgroup of the defensin family, their synthesis is limited to some tissues and furthermore to some mammalian species including the horse. Equine DEFA1 is an enteric alpha-defensin exclusively produced in Paneth cells. The peptide showed an activity against a broad spectrum of microbes, but typical pathogens of the horse were not included in the previous antimicrobial studies. Here, we report the antibacterial properties of DEFA1 against clinical isolates of typical horse pathogens including Rhodococcus equi, various streptococci strains, Salmonella choleraesuis, and Pasteurella multocida. The recombinantly expressed DEFA1 peptide exerted potent activity against these pathogenic bacteria. The highest susceptibility showed R. equi. Three genetically different strains of R. equi were killed at low micromolar concentrations, comparable with conventionally used antibiotics.

Genome-wide SNP analysis reveals distinct origins of Trypanosoma evansi and Trypanosoma equiperdum

Abstract 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.

Killing of Trypanozoon parasites by the equine cathelicidin eCATH1

ABSTRACT Trypanozoon parasites infect both humans, causing sleeping sickness, and animals, causing nagana, surra, and dourine. Control of nagana and surra depends to a great extent on chemotherapy. However, drug resistance to several of the front-line drugs is rising. Furthermore, there is no official treatment for dourine. Therefore, there is an urgent need to develop antiparasitic agents with novel modes of action. Host defense peptides have recently gained attention as promising candidates. We have previously reported that one such peptide, the equine antimicrobial peptide eCATH1, is highly active against equine Gram-positive and Gram-negative bacteria, without cytotoxicity against mammalian cells at bacteriolytic concentrations. In the present study, we show that eCATH1 exhibits an in vitro 50% inhibitory concentration (IC50) of 9.5 μM against Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum. Its trypanocidal mechanism involves plasma membrane permeabilization and mitochondrial alteration based on the following data: (i) eCATH1 induces the rapid influx of the vital dye SYTOX Green; (ii) it rapidly disrupts mitochondrial membrane potential, as revealed by immunofluorescence microscopy using the fluorescent dye rhodamine 123; (iii) it severely damages the membrane and intracellular structures of the parasites as early as 15 min after exposure at 9.5 μM and 5 min after exposure at higher concentrations (19 μM), as evidenced by scanning and transmission electron microscopy. We also demonstrate that administration of eCATH1 at a dose of 10 mg/kg to T. equiperdum-infected mice delays mortality. Taken together, our findings suggest that eCATH1 is an interesting template for the development of novel therapeutic agents in the treatment of trypanosome infections.

Assessment of the safety and immunogenicity of Rhodococcus equi-secreted proteins combined with either a liquid nanoparticle (IMS 3012) or a polymeric (PET GEL A) water-based adjuvant in adult horses and foals--identification of promising new candidate antigens.

Rhodococcus equi is the most common infectious cause of mortality in foals between 1 and 6 months of age. Because of an increase in the number of antibiotic-resistant strains, the optimization of a prophylactic strategy is a key factor in the comprehensive management of R. equi pneumonia. The objectives of this study were to assess the safety and immunogenicity of R. equi-secreted proteins (ReSP) co-administered with either the nanoparticular adjuvant Montanide™ IMS 3012 VG, or a new polymeric adjuvant Montanide™ PET GEL A, and to further investigate the most immunogenic proteins for subsequent immunization/challenge experiments in the development of a vaccine against rhodoccocal pneumonia. The approach involved two phases. The first phase aimed to investigate the safety of vaccination in six adult horses. The second phase aimed to determine the safety and immunogenicity of vaccination in twelve 3-week-old foals. We set out to develop a method based on ultrasound measurements for safety assessment in adult horses in order to evaluate any in situ changes at the injection site, in the skin or the underlying muscle, with quantitative and qualitative data revealing that administration of ReSP combined with the Pet Gel A adjuvant led to an increase in local inflammation, associated with 4- to 7-fold higher levels of anti-R. equi IgGa, IgGb and IgGT, compared to administration of ReSP associated with IMS 3012 adjuvant, but without any impact on animal demeanor. Investigations were then performed in foals with serological and clinical follow-up until 6 months of age. Interestingly, we observed in foals a much lower incidence of adverse local tissue reactions at the injection site than in adult horses, with transient and moderate swelling for the group that received ReSP combined with Pet Gel A. Immunized foals with Pet Gel A adjuvant exhibited a similar response in both IgGa and IgGT levels, but a lower response in IgGb levels, compared to adult horses, with a subisotype profile that may however reflect a bias favorable to R. equi resistance. From the crude extract of secreted proteins, dot-blot screening enabled identification of cholesterol oxidase, mycolyl transferase 3, and PSP (probable secreted protein) as the most immunogenic candidates. Taken together, these results are encouraging in developing a vaccine for foals.

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.

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 methods 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.