Claire Laugier

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.

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.

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.

Genomic Characterization of the Taylorella Genus

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus.

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.

Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains isolated between 1995 and 2008.

The objective of this study was to examine the degree of phenotypic and genotypic diversity between 43 French Taylorella asinigenitalis strains isolated from 22 jacks, two stallions and one mare between 1995 and 2008 by culturing genital swabs obtained during routine diagnosis for contagious equine metritis. This retrospective analysis revealed the existence of T. asinigenitalis species since 1995 and the natural colonization of a mares genital tract in 2001. Despite the presence of 27 different patterns revealed by the combination of API ZYM, antibiogram and 16S rDNA profiles, we show that T. asinigenitalis is a highly homogeneous species. API ZYM diversity only concerns acid phosphatase and naphthol-AS-BI-phosphohydrolase activity. The majority of strains are susceptible to a wide range of antimicrobial agents but most are streptomycin-resistant (95.5%), ampicillin-resistant (88.4%), and four strains are atypical due to a high degree of resistance to at least eight antimicrobial agents. 16S rDNA sequence analysis showed only two clusters and revealed similarity of 99.3-100% between T. asinigenitalis strains. The geographic origin of the 43 isolates correlates to the two 16S rDNA clusters.

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.

Development of a single multi-locus sequence typing scheme for Taylorella equigenitalis and Taylorella asinigenitalis.

We describe here the development of a multilocus sequence typing (MLST) scheme for Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis, a nonpathogenic bacterium. MLST was performed on a set of 163 strains collected in several countries over 35 years (1977-2012). The MLST data were analyzed using START2, MEGA 5.05 and eBURST, and can be accessed at http://pubmlst.org/taylorella/. Our results revealed a clonal population with 39 sequence types (ST) and no common ST between the two Taylorella species. The eBURST analysis grouped the 27 T. equigenitalis STs into four clonal complexes (CC1-4) and five unlinked STs. The 12 T. asinigenitalis STs were grouped into three clonal complexes (CC5-7) and five unlinked STs, among which CC1 (68.1% of the 113 T. equigenitalis) and CC5 (58.0% of the 50 T. asinigenitalis) were dominants. The CC1, still in circulation in France, contains isolates from the first CEM outbreaks that simultaneously emerged in several countries in the late 1970s. The emergence in different countries (e.g. France, Japan, and United Arab Emirates) of STs without any genetic relationship to CC1 suggests the existence of a natural worldwide reservoir that remains to be identified. T. asinigenitalis appears to behave same way since the American, Swedish and French isolates have unrelated STs. This first Taylorella sp. MLST is a powerful tool for further epidemiological investigations and population biology studies of the Taylorella genus.

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.

Rhodococcus equi's extreme resistance to hydrogen peroxide is mainly conferred by one of its four catalase genes.

Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H2O2) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed ΔkatA-, ΔkatB-, ΔkatC-and ΔkatD -deficient mutants to study the ability of R. equi to survive exposure to H2O2 in vitro and within mouse peritoneal macrophages. Results showed that ΔkatA and, to a lesser extent ΔkatC, were affected by 80 mM H2O2. Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H2O2 assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (±122.6) in response to exposure to 50 mM of H2O2 added in the stationary phase, and 3.11 times (±0.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H2O2 resistance capability of R. equi.