Combination of an unbiased amplification method and a resequencing microarray for detecting and genotyping equine arteritis virus.

This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.

Survival of taylorellae in the environmental amoeba Acanthamoeba castellanii.

BACKGROUND: Taylorella equigenitalis is the causative agent of contagious equine metritis, a sexually-transmitted infection of Equidae characterised in infected mares by abundant mucopurulent vaginal discharge and a variable degree of vaginitis, cervicitis or endometritis, usually resulting in temporary infertility. The second species of the Taylorella genus, Taylorella asinigenitalis, is considered non-pathogenic, although mares experimentally infected with this bacterium can develop clinical signs of endometritis. To date, little is understood about the basic molecular virulence and persistence mechanisms employed by the Taylorella species. To clarify these points, we investigated whether the host-pathogen interaction model Acanthamoeba castellanii was a suitable model for studying taylorellae. RESULTS: We herein demonstrate that both species of the Taylorella genus are internalised by a mechanism involving the phagocytic capacity of the amoeba and are able to survive for at least one week inside the amoeba. During this one-week incubation period, taylorellae concentrations remain strikingly constant and no overt toxicity to amoeba cells was observed. CONCLUSIONS: This study provides the first evidence of the capacity of taylorellae to survive in a natural environment other than the mammalian genital tract, and shows that the alternative infection model, A. castellanii, constitutes a relevant alternative system to assess host-pathogen interactions of taylorellae. The survival of taylorellae inside the potential environmental reservoir A. castellanii brings new insight, fostering a broader understanding of taylorellae biology and its potential natural ecological niche.

Study of lysozyme resistance in Rhodococcus equi.

Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.

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.

Patterns of variation in equine strongyle community structure across age groups and gut compartments.

BACKGROUND: Equine strongyles encompass more than 64 species of nematode worms that are responsible for growth retardation and the death of animals. The factors underpinning variation in the structure of the equine strongyle community remain unknown. METHODS: Using horse-based strongyle community data collected after horse deworming (48 horses in Poland, 197 horses in Ukraine), we regressed species richness and the Gini-Simpson index upon the horse's age, faecal egg count, sex and operation of origin. Using the Ukrainian observations, we applied a hierarchical diversity partitioning framework to estimate how communities were remodelled across operations, age groups and horses. Lastly, strongyle species counts collected after necropsy (46 horses in France, 150 in Australia) were considered for analysis of their co-occurrences across intestinal compartments using a joint species distribution modelling approach. RESULTS: First, inter-operation variation accounted for > 45% of the variance in species richness or the Gini-Simpson index (which relates to species dominance in communities). Species richness decreased with horse's age (P = 0.01) and showed a mild increase with parasite egg excretion (P < 0.1), but the Gini-Simpson index was neither associated with parasite egg excretion (P = 0.8) nor with horse age (P = 0.37). Second, within-host diversity represented half of the overall diversity across Ukrainian operations. While this is expected to erase species diversity across communities, community dissimilarity between horse age classes was the second most important contributor to overall diversity (25.8%). Third, analysis of species abundance data quantified at necropsy defined a network of positive co-occurrences between the four most prevalent strongyle genera. This pattern was common to necropsies performed in France and Australia. CONCLUSIONS: Taken together, these results show a pattern of ß-diversity maintenance across age classes combined with positive co-occurrences that might be grounded by priority effects between the major species.

Development of a chronic focal equine arteritis virus infection of a male reproductive tract cell line.

Equine arteritis virus (EAV) is an Alphaarterivirus (family Arteriviridae, order Nidovirales) that frequently causes an influenza-like illness in adult horses, but can also cause the abortions in mares and death of newborn foals. Once primary infection has been established, EAV can persist in the reproductive tract of some stallions. However, the mechanisms enabling this persistence, which depends on testosterone, remain largely unknown. We aimed to establish an in vitro model of non-cytopathic EAV infection to study viral persistence. In this work, we infected several cell lines originating from the male reproductive tract of different species. EAV infection was fully cytopathic for 92BR (donkey cells) and DDT1 MF-2 (hamster cells) cells, and less cytopathic for PC-3 cells (human cells); ST cells (porcine cells) seemed to eliminate the virus; LNCaP (human cells) and GC-1 spg (murine cells) cells were not permissive to EAV infection; finally, TM3 cells (murine cells) were permissive to EAV infection without any overt cytopathic effects. Infected TM3 cells can be maintained at least 7 days in culture without any subculture. They can also be subcultured over 39 days (subculturing them at 1:2 the first time at 5 dpi and then every 2-3 days), but in this case, the percentage of infected cells remains low. Infected TM3 cells may therefore provide a new model to study the host-pathogen interactions and to help determine the mechanisms involved in EAV persistence in stallion reproductive tract.

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.

Genome sequence of Taylorella equigenitalis MCE9, the causative agent of contagious equine metritis.

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We herein report the genome sequence of T. equigenitalis strain MCE9, isolated in 2005 from the urethral fossa of a 4-year-old stallion in France.

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.

Retrospective study of necropsy-associated coagulase-positive staphylococci in horses.

Coagulase-positive staphylococci (CoPS) are potential causative agents of equine infections, but they are rarely responsible for the death of the animal. In the current study, staphylococci implicated in the death or euthanasia of horses were retrospectively studied in 3,457 necropsies performed over a decade (1995-2006). Morbidity associated with CoPS was 1.7%, representing 60 isolates of CoPS, which were identified as Staphylococcus aureus (59) and Staphylococcus pseudintermedius (1). Coagulase-positive staphylococci (alone or in association with another bacterial species) were associated with the death or euthanasia of 90% of the cases (54/60). Proportions of antibiotic resistance to penicillin G and tetracycline reached 62.7% and 23.7%, respectively. Virulence genes were detected in 91.7% of the strains, with a majority of seh or sei enterotoxin genes. Finally, 3 methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to the t064 spa-type were identified. One strain was isolated in 2003 and might thus be one of the first cases of equine MRSA in France.

Relationship between rifampicin resistance and RpoB substitutions of Rhodococcus equi strains isolated in France.

OBJECTIVES: Study of the rifampicin resistance of Rhodococcus equi strains isolated from French horses over a 20-year period. METHODS: Rifampicin susceptibility was tested by disk diffusion (DD) and broth macrodilution methods, and rpoB gene sequencing and MLST were performed on 40 R. equi strains, 50.0% of which were non-susceptible to rifampicin. RESULTS: Consistency of results was observed between rifampicin susceptibility testing and rpoB sequencing. Strains non-susceptible to rifampicin by DD had a substitution at one of the sites (Asp516, His526 and Ser531) frequently encountered and conferring rifampicin resistance. High-level resistance was correlated with His526Asp or Ser531Leu substitutions; low-level resistance was correlated with Asp516Tyr substitution, a novel substitution for R. equi. Strains susceptible to rifampicin by DD showed no substitution in the three sites, except for two strains carrying, respectively, the His526Asn and Asp516Val substitutions (previously correlated with low-level rifampicin resistance). Both strains were isolated from an animal from which ten other strains were also isolated and found to be rifampicin-non-susceptible by DD. MLST showed the presence of 10 STs (including the novel ST43), but no association was observed with rifampicin resistance. CONCLUSIONS: This study confirms that certain substitutions in RpoB are more likely to confer high- or low-level rifampicin resistance, describes a new substitution conferring rifampicin resistance in R. equi and suggests non-clonal dissemination of rifampicin-resistant strains in France. Standard DD may miss strains with a low-level rifampicin-resistant substitution; further studies are needed to remedy the absence of R. equi-specific clinical breakpoints.

Proteomic analysis and immunogenicity of secreted proteins from Rhodococcus equi ATCC 33701.

Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 degrees C versus 37 degrees C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.

Isolation and comparison of Arcanobacterium hippocoleae isolates from the genital tract of 15 mares.

The present study reports the isolation of A. hippocoleae from genital swabs of 15 apparently healthy mares (at least one had an abortion one month earlier) and describes the genotypic and phenotypic characterisation of these strains. The mares were of eight different breeds with a thoroughbred dominance and came from 11 breeding farms located in the French region of Brittany. 16S rRNA gene sequencing confirmed the species' identification by comparing it with reference strain A. hippocoleae CIP 106850T. Some degree of natural divergence within A. hippocoleae was observed by 16S rRNA sequencing (two 1,002-pb sequences), MALDI-TOF MS typing (two groups), a CAMP test (three different intensities of haemolysis from CAMP-positive results) and API® Coryne system (five profiles). The strains were all susceptible to the antimicrobials tested. A national prevalence survey would be required to estimate the frequency of A. hippocoleae carriage in mares and stallions and to verify the presence of A. hippocoleae outside the French region of Brittany, which is the only one found to be affected in the current study, probably because the isolates were recovered from a single field laboratory in this region.

Towards European harmonisation of contagious equine metritis diagnosis through interlaboratory trials.

The performance of culture and PCR methods routinely used to diagnose contagious equine metritis (CEM) was evaluated and compared by two interlaboratory trials involving a total of 24 European laboratories, including 22 National Reference Laboratories for CEM. Samples were swab specimens artificially contaminated with bacteria present in the genital tract of Equidae, some with and some without Taylorella equigenitalis, the causative agent of CEM, and T asinigenitalis, responsible for possible misidentification as T equigenitalis Throughout both interlaboratory trials, PCR performed better in terms of specificity and sensitivity than the culture method, supporting the assertion that PCR should be accepted for CEM diagnosis. However, the culture performance during the second interlaboratory trial was better than during the first one, suggesting that the expertise of participants improved. This reveals the advantage of regular interlaboratory trials to constantly improve the expertise of laboratories. It also shows the need to develop new culture media that are more selective and/or better geared to the metabolism of T equigenitalis in order to improve the bacteriological diagnosis of CEM.

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.

Myocardial fibrosis in a horse with polymorphic ventricular tachycardia observed during general anesthesia.

Ventricular dysrhythmias are more commonly associated with myocardial disease than are supraventricular dysrhythmias. Management of arrhythmias under general anesthesia is difficult because of the dysrhythmogenic effects of the anesthetic drugs. This report describes a severe ventricular dysrhythmia observed in a pony under general anesthesia, with a severe and old myocardial fibrosis found on postmortem examination.

Development of a multilocus sequence typing scheme for Rhodococcus equi.

Rhodococcus equi causes pulmonary and extrapulmonary infections in animals and humans, with endemic situations and significant young foal mortality in stud farms worldwide. Despite its economic impact in the horse-breeding industry, the broad geographic and host distribution, global diversity and population structure of R. equi remain poorly characterised. In this context, we developed a multilocus sequence typing (MLST) scheme using 89 clinical and environmental R. equi of various origins and eight Rhodococcus sp. Data can be accessed at http://pubmlst.org/rhodococcus/. A clonal R. equi population was observed with 16 out of 37 sequence types (STs) grouped into six clonal complexes (CC) based on single-locus variants. One of the six CCs (CC3) is not host-specific, suggesting potential exchanges between different R. equi reservoirs. Most of the virulent equine R. equi CCs/unlinked STs were plasmid-type-specific. Despite this, marked genetic variability with the circulation of multiple R. equi genotypes was generally observed even within the same animal. Focusing on outbreaks, data indicated (i) the potential contagious transmission of R. equi during the 2012-Mayotte equine outbreak because of the poor genotype diversity of clinical strains; (ii) a potential porcine outbreak among the 30 Belgian farms investigated in 2013. This first Rhodococcus equi MLST is a powerful tool for further epidemiological investigations and population biology studies of R. equi isolates.

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.

Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis.

Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 (hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin-Starry-stained tissue sections. Results of PCR analysis and histologic examination suggested a leptospiral infection in the newborn foal. At the moment of death, the infection coexisted with a streptococcal-associated aspiration bronchopneumonia and postpartum septicemia. These findings indicate that the PCR assay based on the amplification of the hap1 gene represents a useful tool for specific detection of pathogenic leptospira in field samples taken from horses.