Antimicrobial resistance and genetic diversity of Klebsiella pneumoniae strains from different clinical sources in horses.

Introduction: Klebsiella pneumoniae is a major cause of infections and reproductive disorders among horses, ranked in recent French studies as the sixth most frequently isolated bacterial pathogen in equine clinical samples. The proportion of multidrug-resistant (MDR) K. pneumoniae is therefore significant in a context where MDR K. pneumoniae strains are considered a major global concern by the World Health Organization. Methods: In this study, we used a genomic approach to characterize a population of 119 equine K. pneumoniae strains collected by two laboratories specialized in animal health in Normandy (France). We describe the main antibiotic resistance profiles and acquired resistance genes, and specify the proportion of virulence-encoding genes carried by these strains. The originality of our panel of strains lies in the broad collection period covered, ranging from 1996 to 2020, and the variety of sample sources: necropsies, suspected bacterial infections (e.g., genital, wound, allantochorion, and umbilical artery samples), and contagious equine metritis analyses. Results: Our results reveal a remarkable level of genomic diversity among the strains studied and we report the presence of 39% MDR and 9% hypervirulent strains (including 5% that are both MDR and hypervirulent). Discussion: These findings clearly emphasize the importance of improving the surveillance of K. pneumoniae in routine equine diagnostic tests to detect high-risk MDR-hypervirulent Klebsiella pneumoniae strains. The circulation of these worrisome strains reveals that they are not being detected by the simple K1, K2, and K5 serotype approach currently implemented in the French horse-breeding sector.

How far away do you keep your equines? Estimation of the equine population's spatial distribution in France.

It is essential to have an accurate picture of the spatial distribution of equines to be able to monitor equine health events effectively. In France, this information is only available for certain categories of live equines kept in professional structures and for dead equines removed by renderers. This limits the surveillance, prevention and control methods able to be used to prevent the spread of equine diseases. Our study aimed to provide a realistic estimate of the spatial distribution of the French equine population at the detailed scale of the French commune (France's smallest administrative unit). For this purpose, we adapted the Bayesian method used by Lo Lacono et al., based on the distance between the owner's location and the location of his/her equines, and on the percentage of urban coverage. To assess whether the location of dead equines could be representative of the location of live equines, the distribution of distances between equines and owners was calculated from a sample of live equines on the one hand, and a sample of dead equines on the other, both accurately located. We also tested two different assignment methods for locating equines: Method 1 assigned to each owner a single holding commune, while Method 2 allowed more variability in holding communes for owners associated with multiple equines. A marked difference was observed between Methods 1 and 2 regardless of the sample used, with only 2.4% and 4.3% respectively of the communes having the same number of equines. Conversely, little difference was observed in the results whether the live or dead equine sample was used, with approximately 45% of the communes having the same number of equines. Regarding differences in spatial distribution, Method 1 based on the live equine sample estimated higher local densities of equines without considering urban areas. In contrast, Method 2 provided more dispersed maps, with low densities in the densest urban areas. In conclusion, dead equines appeared to be representative of live equines and some of our estimates are consistent with the information collected by the French horse and riding institute (IFCE). These results now have to be compared with field data to test their relevance.

Home Sweet Home: New Insights Into the Location of Equine Premises in France and Keeping Habits to Inform Health Prevention and Disease Surveillance.

Identifying and tracking equines are key activities in equine health prevention. France is one of the few European countries with an operational centralized database that records information on equines, owners, and keepers but not on the location and keeping conditions of equines. The objective of our study was to collect information on keeping habits of equines and the relative location of a wide range of equines, owners, and keepers and discuss their implication for surveillance and control of outbreak improvement. A national email survey was conducted among the 1.9% of people registered as owners and 8.2% of people registered as keepers in the French national equine identification database having given their agreement to be contacted by email. It led to the collection of information from 728 owners, 121 keepers, and 2,669 owner-keepers. Most of them housed their equines in a single commune (smallest geographic administrative unit in France) at their home as private individuals. The distance between the communes of residence and of holding was, in most cases (including 79% of owners in the owner survey, 89.5% of the keepers in the keeper survey, and about 94% of the owner-keepers in both surveys), less than 30 km. More than half of the keepers kept a maximum of five equines and the majority with two different uses/destinations together, mostly leisure-retirement, leisure-breeding, leisure-sport, and sport-breeding. The main limitation of the study was that a relatively limited number of people (n = 3518) were reachable due to the low availability of an email address and contact agreement. Nonetheless, the findings provide an overview of how equines are kept by non-professional owners and keepers and complements information usually collected by the French riding institute. Additionally, information collected is very helpful to determine a realistic estimate of the spatial distribution of equines in France. This information is very important for the equine sector, for demographic knowledge and also improvement of surveillance plans and control measures and for the management and monitoring of health events to limit the spread of diseases.

Is the French SIRE equine information system a good basis for surveillance and epidemiological research? Quality assessment using two surveys.

Accurate demographic knowledge of the equine population is needed to assess and model equine health events. France is one of the few European countries with an operational centralized database (SIRE) recording individual data on all declared equines living in France and on their owners and keepers. Our study aimed to assess SIRE database quality concerning the updating of information by equine owners and keepers with a view to its improvement and use in surveillance and research. Two online surveys were conducted with the participation of 6244 registered keepers and 13,869 owners. Results showed some inconsistencies between SIRE records and survey responses. The inconsistency rate for equines whose castration and death were not registered in the database was 28.7% and 5.9% respectively. Concerning owners, 11% of respondents did not own the reference equine selected considered by the survey, 33% had changed address without updating it in the SIRE. Concerning premises hosting equines, the keeper survey's inconsistency rate was 7.3%, of which 57 respondents had closed and 32 had opened premises without reporting it. Comparatively, the owner survey's inconsistency rate was 40.7% including respondents who owned and hosted an equine without reporting these equine premises, and owners who did not keep any equines on their premises. In conclusion, the SIRE database proved to be a valuable and reliable source for epidemiological research as long as some bias is taken into account. On the contrary, its use in surveillance is currently limited due some shortcomings in updating and/or reporting by owners and keepers.

Contrasted Epidemiological Patterns of West Nile Virus Lineages 1 and 2 Infections in France from 2015 to 2019.

Since 2015, annual West Nile virus (WNV) outbreaks of varying intensities have been reported in France. Recent intensification of enzootic WNV circulation was observed in the South of France with most horse cases detected in 2015 (n = 49), 2018 (n = 13), and 2019 (n = 13). A WNV lineage 1 strain was isolated from a horse suffering from West Nile neuro-invasive disease (WNND) during the 2015 episode in the Camargue area. A breaking point in WNV epidemiology was achieved in 2018, when WNV lineage 2 emerged in Southeastern areas. This virus most probably originated from WNV spread from Northern Italy and caused WNND in humans and the death of diurnal raptors. WNV lineage 2 emergence was associated with the most important human WNV epidemics identified so far in France (n = 26, including seven WNND cases and two infections in blood and organ donors). Two other major findings were the detection of WNV in areas with no or limited history of WNV circulation (Alpes-Maritimes in 2018, Corsica in 2018-2019, and Var in 2019) and distinct spatial distribution of human and horse WNV cases. These new data reinforce the necessity to enhance French WNV surveillance to better anticipate future WNV epidemics and epizootics and to improve the safety of blood and organ donations.

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.

Further Evidence for in Utero Transmission of Equine Hepacivirus to Foals.

(1) Background: Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses-and dogs in some instances-and is closely related to hepatitis C virus (HCV) that has infected up to 3% of the world's human population, causing an epidemic of liver cirrhosis and cancer. EqHV also chronically infects the liver of horses, but does not appear to cause serious liver damages. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. (2) Methods: In this retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. (3) Results: Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. (4) Conclusions: As seemingly benign viruses could turn deadly (e.g., Zika flavivirus) and EqHV happens to have infected a significant proportion of the world's horse herds, EqHV infectious cycle should be further clarified.

Resumeq: A Novel Way of Monitoring Equine Diseases Through the Centralization of Necropsy Data.

The French surveillance network for causes of equine mortality (Resumeq) was created in 2015 for the qualitative surveillance of equine mortality through the centralization in a national database of necropsy data and their subsequent epidemiological analysis. It was designed to identify the causes of equine mortality, monitor their evolution over time and space, and detect emerging diseases as early as possible. Resumeq is an event-based surveillance system involving various players and structures. It is organized around a steering body, a scientific and technical support committee and a coordination unit. Different tools have been developed specifically for Resumeq. These include standardized necropsy protocols, a thesaurus for the anatomopathological terms and the causes of equine death, and an interactive web application so that network contributors can display data analysis results. The four French veterinary schools, seventeen veterinary laboratories, and ten veterinary clinics already contribute to the production and centralization of standardized data. To date, the data from around 1,000 equine necropsies have been centralized. While most deaths were located in western France, the geographic coverage is gradually improving. Data analysis allows the main causes of death to be ranked and major threats identified on a local, regional or national level. Initial results demonstrate the feasibility and benefits of this national surveillance tool. Moreover, in the future, this surveillance could take an international dimension if several countries decided to jointly capitalize on their necropsy data.

Utility of examining fallen stock data to monitor health-related events in equids: Application to an outbreak of West Nile Virus in France in 2015.

Few studies about the use of quantitative equine mortality data for monitoring purposes are available. Our study evaluated the utility of monitoring emerging equine diseases using mortality data collected by rendering plants. We used approaches involving modelling of historical mortality fluctuations and detection algorithm methods to analyse changes in equine mortality in connection with the West Nile Virus (WNV) outbreak that occurred between July and September 2015 along the Mediterranean coast of France. Two weeks after the first equine WNV case was detected by clinical surveillance, detection algorithms identified excess mortality. The temporal distribution of this excess mortality suggested that it was related to the WNV outbreak, which may helped to assess the impact of the WNV epizootic on equine mortality. The results suggest that real-time follow-up of mortality could be a useful tool for equine health surveillance.

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.

Evaluation of a Multivariate Syndromic Surveillance System for West Nile Virus.

BACKGROUND: Various methods are currently used for the early detection of West Nile virus (WNV) but their outputs are not quantitative and/or do not take into account all available information. Our study aimed to test a multivariate syndromic surveillance system to evaluate if the sensitivity and the specificity of detection of WNV could be improved. METHODS: Weekly time series data on nervous syndromes in horses and mortality in both horses and wild birds were used. Baselines were fitted to the three time series and used to simulate 100 years of surveillance data. WNV outbreaks were simulated and inserted into the baselines based on historical data and expert opinion. Univariate and multivariate syndromic surveillance systems were tested to gauge how well they detected the outbreaks; detection was based on an empirical Bayesian approach. The systems' performances were compared using measures of sensitivity, specificity, and area under receiver operating characteristic curve (AUC). RESULTS: When data sources were considered separately (i.e., univariate systems), the best detection performance was obtained using the data set of nervous symptoms in horses compared to those of bird and horse mortality (AUCs equal to 0.80, 0.75, and 0.50, respectively). A multivariate outbreak detection system that used nervous symptoms in horses and bird mortality generated the best performance (AUC = 0.87). CONCLUSIONS: The proposed approach is suitable for performing multivariate syndromic surveillance of WNV outbreaks. This is particularly relevant, given that a multivariate surveillance system performed better than a univariate approach. Such a surveillance system could be especially useful in serving as an alert for the possibility of human viral infections. This approach can be also used for other diseases for which multiple sources of evidence are available.

Assessment of fallen equine data in France and their usefulness for epidemiological investigations.

Quantitative information about equine mortality is relatively scarce, yet it could be of great value for epidemiology purposes. Several European projects based on the exploitation of data from rendering plants have been developed to improve livestock surveillance. Similar data are available for equines in France but have never been studied to date. The objective of this research was to evaluate the potential of the French Ministry of Agriculture's Fallen Stock Data Interchange (FSDI) database to provide quantitative mortality information on the French equine population. The quality of FSDI equine data from 2011 to 2014 was assessed using complementary data registered in the French equine census database, SIRE. Despite a perfectible quality, the FSDI database proved to be a valuable source for studying the basal patterns of mortality over time in the French equine population as illustrated by the spatial representation of the number of deaths. However, improvements in the FSDI database are needed, in particular regarding the registration of animal identification numbers, in order to detail equine mortality for epidemiology purposes.

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.

Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid.

To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene.

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.

Ultrasound-guided atlanto-occipital puncture for myelography in the horse.

Complications of cervical myelography arising from the puncture of the subarachnoid space to collect the cerebrospinal fluid and to inject the contrast medium have been described in humans and animals. In this study, 2 ultrasound-guided procedures were developed for puncture of the atlanto-occipital subarachnoid space, collection of cerebrospinal fluid, and injection of contrast medium. Myelography was performed on 6 ataxic horses using these procedures. The first attempt to puncture the subarachnoid space was successful in 5 horses and in one horse, a second attempt was necessary. Collection of cerebrospinal fluid and injection of contrast medium were achieved without difficulty. Ultrasound-guided myelography allowed reduction of potential complications associated with blind percutaneous puncture of the subarachnoid space. Methods described in this study should be tried-at least initially in an experimental setting--to collect cerebrospinal fluid from the atlanto-occipital site in standing horses where it may represent an alternative method when lumbosacral cerebrospinal fluid collection has been unsuccessful or contaminated with blood.

Magnetic resonance imaging for the diagnosis of stress fractures in a horse.

In humans, magnetic resonance (MR) imaging is the method of choice for the diagnosis of stress fractures. In this paper, bilateral stress fracture of the lateral condyle of the third metacarpal bone in a French trotter is described. Results of the radiographic, MR imaging, and histologic examinations are presented, with a focus on the MR signal abnormalities found. Based on this patient, the potential use of MR imaging for the diagnosis of stress fractures in horses is discussed.