Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

Equine Herpesvirus-1 Outbreak During a Show-Jumping Competition: A Clinical and Epidemiological Study.

A total of 752 horses were involved in the CES Valencia Spring Tour 2021. Due to an equine herpesvirus-1 (EHV-1) outbreak, the competition was cancelled and the site was locked down. The objective of this study was to describe epidemiological, clinical, diagnostic, and outcome data of the 160 horses remaining in Valencia. Clinical and quantitative polymerase chain reaction (qPCR) data were analysed for 60 horses in a retrospective case-control observational study. The risk of developing clinical manifestations was explored using a logistic regression approach. EHV-1 was detected by qPCR, genotyped as A2254 (ORF30) and isolated on cell culture. From the 60 horses, 50 (83.3%) showed fever, 30 horses (50%) showed no further signs and 20 (40%) showed neurological signs, with eight horses (16%) hospitalised, of which two died (3%). Stallions and geldings were six times more likely to develop EHV-1 infection compared to mares. Horses older than 9 years, or housed in the middle of the tent were more likely to develop EHV-1 myeloencephalopathy (EHM). These data show that for EHV-1 infection, the risk factor was male sex. For EHM the risk factors were age > 9-year old and location in the middle of the tent. These data highlight the crucial role of stable design, position, and ventilation in EHV-outbreaks. It also showed that PCR testing of the horses was important to manage the quarantine.

Molecular assessment of Theileria equi and Babesia caballi prevalence in horses and ticks on horses in southeastern France.

Equine piroplasmosis (EP) is a tick-borne disease caused by Babesia caballi and Theileria equi that is potentially emerging in non-endemic countries. We conducted a descriptive study to investigate EP prevalence and spatial distribution in an endemic region: the Camargue and the Plain of La Crau in France. In spring 2015 and 2016, we carried out sampling at stables (total n = 46) with a history of horses presenting chronic fever or weight loss. Overall, we collected blood from 632 horses, which were also inspected for ticks; these horses had been housed in the target stables for at least 1 year. We obtained 585 ticks from these horses and described land use around the stables. Real-time PCR was employed to assess T. equi and B. caballi prevalence in the horses and in the ticks found on the horses. For the horses, T. equi and B. caballi prevalence was 68.6% and 6.3%, respectively. For the ticks found on the horses, prevalence was 28.8% for T. equi and 0.85% for B. caballi. The most common tick species were, in order of frequency, Rhipicephalus bursa, R. sanguineus sl., Hyalomma marginatum, Haemaphysalis punctata, and Dermacentor sp. Horses bearing Rhipicephalus ticks occurred in wetter zones, closer to agricultural areas, permanent crops, and ditches, as well as in drier zones, in the more northern countryside. Compared to horses bearing R. bursa, horses bearing R. sanguineus sl. more frequently occurred near the Rhone River. Prevalence of T. equi in the ticks was as follows: Hyalomma marginatum (43%), Dermacentor sp. (40%), R. bursa (33%), R. sanguineus sl. (19%), and Haemaphysalis punctata (17%). In contrast, B. caballi only occurred in Dermacentor sp. (20%) and R. bursa (1%).

Equine Herpesvirus 1 Variant and New Marker for Epidemiologic Surveillance, Europe, 2021.

Equine herpesvirus 1 isolates from a 2021 outbreak of neurologic disease in Europe have a mutation, A713G, in open reading frame 11 not detected in 249 other sequences from equine herpesvirus 1 isolates. This single-nucleotide polymorphism could help identify horses infected with the virus strain linked to this outbreak.

EQUID ALPHAHERPESVIRUS 9 OUTBREAK ASSOCIATED WITH MORTALITY IN A GROUP OF GREVY'S ZEBRA (EQUUS GREVYI) HOUSED IN A MIXED-SPECIES EXHIBIT.

A herd of seven captive-born Grevy's zebras (Equus grevyi) experienced an outbreak of nasal discharge and sneezing. Clinical signs, including lethargy and anorexia, were severe and acute in three animals, including a 16-mo-old male that died within 48 h. Treatment of two severely affected zebras included valacyclovir (40 mg/kg PO), meloxicam (0.6 mg/kg IM/PO), and cefquinome (2.5 mg/kg IM q48h). An adult female improved rapidly, and clinical signs resolved within 48 h of treatment. Administration of valacyclovir pellets was very complicated in a 2-mo-old female, and death occurred within 48 h. Histologic examination of the two individuals that died revealed severe fibrinonecrotic interstitial pneumonia with prominent hyaline membranes and type II pneumocyte hyperplasia. Additionally, the 16-mo-old male presented systemic endothelial activation with vascular thrombosis and necrosis and mild nonsuppurative meningoencephalitis. Herpesviral DNA was detected in the lungs of both individuals by nested polymerase chain reaction. The nucleic acid sequence of the amplicons showed 100% similarity with previously published equid alphaherpesvirus 9 sequences. Three additional animals developed mild nasal discharge only and recovered spontaneously. The zebras shared housing facilities with other species, including white rhinoceros (Ceratotherium simum), reticulated giraffe (Giraffa camelopardalis reticulata), and several antelope species. None of these animals showed clinical signs. Additionally, nasal swabs and whole blood samples were collected from cohoused white rhinoceroses (n = 3) and springboks (Antidorcas marsupialis, n = 3) as well as nasal swabs from cohoused reticulated giraffes (n = 4). Nucleic acid sequence from equid herpesviruses was not detected in any of these samples. The source of the infection in the zebras remains unclear.

An Evaluation of Three Different Primary Equine Influenza Vaccination Intervals in Foals.

In order to evaluate the effect of three different primary vaccination intervals on EI vaccine response, 21 unvaccinated thoroughbred foals were randomly divided into three groups of 7 and vaccinated with three different intervals of primary immunization (i.e., with 1, 2 or 3 months intervals between V1 and V2, respectively). The antibody response was measured for up to 1 year after the third immunization V3 (administered 6 months after V2) by single radial hemolysis (SRH) assay. All weanlings had seroconverted and exceeded the clinical protection threshold 2 weeks after V2 and 1 month after V3 until the end of the study. Significant differences were measured at the peak of immunity after V2 and for the duration of the immunity gap between V2 and V3. The group with one month primary vaccination interval had a lower immunity peak after V2 (158.05 ± 6.63 mm2) and a wider immunity gap between V2 and V3 (18 weeks) when compared with other groups (i.e., 174.72 ± 6.86 mm2 and 16 weeks for a two months interval, 221.45 ± 14.48 mm2 and 12 weeks for a 3-month interval). The advantage observed in the group with 1 month primary vaccination interval, which induces an earlier protective immunity, is counterbalance with a lower peak of immunity and a wider immunity gap after V2, when compared with foals vaccinated with 2- and 3-month intervals.

Biosafety Evaluation of Equine Umbilical Cord-Derived Mesenchymal Stromal Cells by Systematic Pathogen Screening in Peripheral Maternal Blood and Paired UC-MSCs.

Background: The growing interest in mesenchymal stromal cells (MSCs) in equine medicine, together with the development of MSC biobanking for allogeneic use, raises concerns about biosafety of such products. MSCs derived from umbilical cord (UC) carry an inherent risk of contamination by environmental conditions and vertical transmission of pathogens from broodmares. There is yet no report in the scientific literature about horses being contaminated by infected MSC products, and no consensus about systematic infectious screening of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to ensure microbiological safety of therapeutic products. Objectives: To develop a standard protocol to ensure UC-MSC microbiological safety and to assess the risk of vertical transmission of common intracellular pathogens from broodmares to paired UC-MSCs. Study Design and Methods: Eighty-four UC and paired peripheral maternal blood (PMB) samples were collected between 2014 and 2016. Sterility was monitored by microbiological control tests. Maternal contamination was tested by systematical PMB PCR screening for 14 pathogens and a Coggins test. In case of a PCR-positive result regarding one or several pathogen(s) in PMB, a PCR analysis for the detected pathogen(s) was then conducted on the associated UC-MSCs. Results: Ten out of 84 UC samples were contaminated upon extraction and 6/84 remained positive in primo culture. The remaining 78/84 paired PMB & UC-MSC samples were evaluated for vertical transmission; 37/78 PMB samples were PCR positive for Equid herpesvirus (EHV)-1, EHV-2, EHV-5, Theileria equi, Babesia caballi, and/or Mycoplasma spp. Hepacivirus was detected in 2/27 cases and Theiler Diseases Associated Virus in 0/27 cases (not performed on all samples due to late addition). All paired UC-MSC samples tested for the specific pathogen(s) detected in PMB were negative (37/37). Main Limitations: More data are needed regarding MSC susceptibility to most pathogens detected in PMB. Conclusions: In-process microbiological controls combined with PMB PCR screening provide a comprehensive assessment of UC-MSC exposure to infectious risk, vertical transmission risk appearing inherently low.

Success and Limitation of Equine Influenza Vaccination: The First Incursion in a Decade of a Florida Clade 1 Equine Influenza Virus that Shakes Protection Despite High Vaccine Coverage.

Every year, several epizooties of equine influenza (EI) are reported worldwide. However, no EI case has been identified in France between 2015 and late 2018, despite an effective field surveillance of the pathogen and the disease. Vaccination against equine influenza virus (EIV) remains to this day one of the most effective methods to prevent or limit EI outbreaks and the lack of detection of the pathogen could be linked to vaccination coverage. The aim of this study was to evaluate EI immunity and vaccine coverage in France through a large-scale serological study. A total of 3004 archived surplus serums from French horses of all ages, breeds and sexes were selected from four different geographical regions and categories (i.e., sanitary check prior to exportation, sale, breeding protocol or illness diagnosis). EIV-specific antibody response was measured by single radial hemolysis (SRH) and an EIV-nucleoprotein (NP) ELISA (used as a DIVA test). Overall immunity coverage against EIV infection (i.e., titers induced by vaccination and/or natural infection above the clinical protection threshold) reached 87.6%. The EIV NP ELISA results showed that 83% of SRH positive serum samples from young horses (≤3 years old) did not have NP antibodies, which indicates that the SRH antibody response was likely induced by EI vaccination alone (the HA recombinant canarypoxvirus-based EI vaccine is mostly used in France) and supports the absence of EIV circulation in French horse populations between 2015 and late 2018, as reported by the French equine infectious diseases surveillance network (RESPE). Results from this study confirm a strong EI immunity in a large cohort of French horses, which provides an explanation to the lack of clinical EI in France in recent years and highlights the success of vaccination against this disease. However, such EI protection has been challenged since late 2018 by the incursion in the EU of a Florida Clade 1 sub-lineage EIV (undetected in France since 2009), which is also reported here.

Molecular Surveillance of EHV-1 Strains Circulating in France during and after the Major 2009 Outbreak in Normandy Involving Respiratory Infection, Neurological Disorder, and Abortion.

Equine herpesvirus 1 (EHV-1) is an Alphaherpesvirus infecting not only horses but also other equid and non-equid mammals. It can cause respiratory distress, stillbirth and neonatal death, abortion, and neurological disease. The different forms of disease induced by EHV-1 infection can have dramatic consequences on the equine industry, and thus the virus represents a great challenge for the equine and scientific community. This report describes the progress of a major EHV-1 outbreak that took place in Normandy in 2009, during which the three forms of disease were observed. A collection of EHV-1 strains isolated in France and Belgium from 2012 to 2018 were subsequently genetically analysed in order to characterise EHV-1 strain circulation. The open reading frame 30 (ORF30) non-neuropathogenic associated mutation A2254 was the most represented among 148 samples analysed in this study. ORF30 was also sequenced for 14 strains and compared to previously published sequences. Finally, a more global phylogenetic approach was performed based on a recently described Multilocus Sequence Typing (MLST) method. French and Belgian strains were clustered with known strains isolated in United Kingdom and Ireland, with no correlation between the phylogeny and the time of collection or location. This new MLST approach could be a tool to help understand epidemics in stud farms.

Impact of Mixed Equine Influenza Vaccination on Correlate of Protection in Horses.

To evaluate the humoral immune response to mixed Equine Influenza vaccination, a common practice in the field, an experimental study was carried out on 42 unvaccinated thoroughbred weanling foals divided into six groups of seven. Three groups were vaccinated using a non-mixed protocol (Equilis® Prequenza-Te, Proteqflu-Te® or Calvenza-03®) and three other groups were vaccinated using a mix of the three vaccines mentioned previously. Each weanling underwent a primary EI vaccination schedule composed of two primary immunisations (V1 and V2) four weeks apart followed by a third boost immunisation (V3) six months later. Antibody responses were monitored until one-year post-V3 by single radial haemolysis (SRH). The results showed similar antibody responses for all groups using mixed EI vaccination and the group exclusively vaccinated with Equilis® Prequenza-TE, which were significantly higher than the other two groups vaccinated with Proteqflu-TE® and Calvenza-03®. All weanlings (100%) failed to seroconvert after V1 and 21% (9/42) still had low or no SRH antibody titres two weeks post-V2. All weanlings had seroconverted and exceeded the clinical protection threshold one month after V3. The poor response to vaccination was primarily observed in groups exclusively vaccinated with Proteqflu-Te® and Calvenza-03®. A large window of susceptibility (3⁻4.5-month duration) usually called immunity gap was observed after V2 and prior to V3 for all groups. The SRH antibody level was maintained above the clinical protection threshold for three months post-V3 for the groups exclusively vaccinated with Proteqflu-Te® and Calvenza-03®, and six months to one year for groups using mixed EI vaccination or exclusively vaccinated with Equilis® Prequenza-Te. This study demonstrates for the first time that the mix of EI vaccines during the primary vaccination schedule has no detrimental impact on the correlate of protection against EIV infection.

Serological investigation of racehorse vaccination against equine influenza in Morocco.

In order to evaluate the vaccination status against equine influenza (EI) in Moroccan racehorses, a serological investigation was carried out on 509 racehorses using three serological tests: an Enzyme-Linked Immunosorbent Assay (ELISA), the Hemagglutination Inhibition (HI) test and the Single Radial Haemolysis (SRH) assay. The serological analysis showed 56% of seropositivity by ELISA, 67% by HI and 89.4% by SRH (with 69.9% above the clinical protection threshold). Using the Kappa test, the SRH and HI assays showed a strong agreement, the SRH and ELISA assays had a moderate agreement and the HI and ELISA assays showed a poor agreement. Seropositivity was positively correlated with the age of horses and the number of immunisation received. EI vaccines used during the last immunisation before the study had a weak influence on the serological status. This effect was observed when the vaccines Calvenza and Fluvac Innovator® were used, with 94.1% and 100% of seropositivity when measured by HI, and with 100% and 94.7% exceeding the clinical protection threshold when measured by SRH, respectively. No effect was found when other EI vaccines, including Prequenza-Te® (67% coverage (342/509) and Proteqflu-Te® (22% coverage (114/509) were used; with 64% and 67.5% seropositivity (HI) and with 66.4% and 72.8% above the clinical threshold (SRH), respectively. The location and the time since last vaccination have no influence on the serological result. Overall, levels of protective antibody against EI in Moroccan racehorses remain a concern despite mandatory vaccination.

Seroprevalence of horses to Coxiella burnetii in an Q fever endemic area.

Coxiella burnetii can infect many animal species, but its circulation dynamics in and through horses is still unclear. This study evaluated horse exposure in an area known to be endemic for ruminants and humans. We assessed antibody prevalence in horse serum by ELISA, and screened by qPCR horse blood, ticks found on horses and dust from stables. Horse seroprevalence was 4% (n = 335, 37 stables) in 2015 and 12% (n = 294, 39 stables) in 2016. Of 199 horses sampled both years, 13 seroconverted, eight remained seropositive, and one seroreverted. Seropositive horses were located close to reported human cases, yet none displayed Q fever-compatible syndromes. Coxiella DNA was detected in almost 40% of collected ticks (n = 59/148 in 2015; n = 103/305 in 2016), occasionally in dust (n = 3/46 in 2015; n = 1/14 in 2016) but never in horse blood. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.

Refinement of the equine influenza model in the natural host: A meta-analysis to determine the benefits of individual nebulisation for experimental infection and vaccine evaluation in the face of decreased strain pathogenicity.

Equine Influenza (EI) is an important respiratory disease of horses caused by H3N8 equine influenza viruses (EIV). Vaccination is a key strategy to prevent or control this disease. However, EIV undergoes continuous antigenic drift and whilst numerous EI vaccines are commercially available worldwide, an accurate evaluation of their efficacy is frequently required through clinical trials conducted in the natural host. Room nebulisation is one of the chosen methods to challenge horses during EI vaccine studies. A potential decreased pathogenicity observed with recent Florida Clade 2 (FC2) EIV isolates have increased the heterogeneity of the clinical response and virus shedding measured after infection by room nebulisation, which reduced the statistical power of studies. Our objectives were to compare clinical and virological parameters following experimental infection with several different EIV strains and to confirm that individual nebulisation is a model refinement that prevents an increase of the number of animals per group. This study is a retrospective comparison and meta-analysis of clinical and virological results collected from 9 independent EIV infection studies in the natural host. Naïve Welsh mountain ponies were experimentally infected by room or individual nebulisation with FC2 EIV strains, including A/equine/Richmond/1/07 (R/07), A/equine/East Renfrewshire/11 (ER/11), A/equine/Cambremer/1/2012 (C/12) and A/equine/Northamptonshire/1/13 (N/1/13). The retrospective meta-analysis confirmed a decreased pathogenicity of the EIV ER/11 and C/12 strains when compared with R/07. Experimental infection by individual nebulisation improved the clinical and virological parameters induced by recent FC2 strains, when compared with conventional room nebulisation. In conclusion, individual nebulisation offers a better control of the challenge dose administered and a greater homogeneity of the response measured in control animals. This in turn, helps maintain the number of animals per group to the minimum necessary required to obtain meaningful results in vaccine efficacy studies, which adheres to the 3Rs (Replacement, Reduction and Refinement) principles.

Genetic evolution of equine influenza virus strains (H3N8) isolated in France from 1967 to 2015 and the implications of several potential pathogenic factors.

Equine influenza virus (EIV) is a major respiratory pathogen of horses despite the availability of equine influenza vaccines. This study aimed to determine genetic evolution of EIV strains in France between 1967 to present. A whole genome comparative analysis was also conducted on recent French strains in order to identify potential factors of pathogenicity. Comparison of French EIV sequences with vaccine and worldwide epidemic strains revealed amino acid substitutions in both haemagglutinin (HA) and neuraminidase, especially within the antigenic sites and/or close to receptor binding sites (HA). Amino acid substitutions were also identified in other genes, mainly the polymerase complex proteins and PB1-F2. Viruses belonging to Eurasian and American lineages have circulated until 2003 and Florida sub-lineage Clade 2 strains predominates since 2005. The last French strain (2015) displayed several specificities in HA suggesting the occurrence of antigenic drift with presence of pathogenic markers in the PA and PB1-F2 genes.

Influential factors inducing suboptimal humoral response to vector-based influenza immunisation in Thoroughbred foals.

CONTEXT: Numerous equine influenza (EI) epizooties are reported worldwide. EI vaccination is the most efficient methods of prevention. However, not all horses develop protective immunity after immunisation, increasing the risk of infection and transmission. OBJECTIVES: This field study aimed to understand the poor response to primary EI vaccination. STUDY DESIGN: The EI antibody response was measured in 174 Thoroughbred foals set in 3 stud farms (SF#1 to SF#3) over a 2years period. All foals were immunised with a commercial recombinant canarypox-based EI vaccine. Sera were tested by single radial haemolysis against the A/equine/Jouars/4/06 EIV strain (H3N8) at the time of the first vaccination (V1), 2weeks and 3months after the second immunisation (V2), 2days and 3months after the third immunisation (V3). RESULTS: The frequency of poor-responders (no detectable antibody titres) was surprisingly elevated after V2 (56.8%), increased to 81.7% at V2+3months and reached 98.6% at V3. The frequency of poor-responder was still 19.2%, 3months after V3. Two independent influential factors were identified. The short (V2+2weeks) and mid-term (V2+3months, V3+3months) antibody levels were positively correlated to the age at V1 (p-value=0.003, 0.031 and 0.0038, respectively). Presence of maternally-derived antibodies (MDA) at V1 was negatively correlated with antibody levels after V3 only (p-value=0.0056). Given that SF#1 antibody response was below clinical protective levels at all-time points studied, the annual boost immunisation (V4) was brought forward by 7.0±1.1months. V1 was delayed by 7weeks the following year, which significantly increased short- and mid-term antibody titres (p-value=9.9e-07 and 2.31e-07, respectively). CONCLUSION: The age and MDA at first immunisation with the canarypox-based IE vaccine play an independent role in the establishment of antibody levels. This study also highlights the benefit provided by serological surveillance to evaluate herd immunity and to implement corrective management/vaccination measures.

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Multilocus sequence analysis of Anaplasma phagocytophilum reveals three distinct lineages with different host ranges in clinically ill French cattle.

Molecular epidemiology represents a powerful approach to elucidate the complex epidemiological cycles of multi-host pathogens, such as Anaplasma phagocytophilum. A. phagocytophilum is a tick-borne bacterium that affects a wide range of wild and domesticated animals. Here, we characterized its genetic diversity in populations of French cattle; we then compared the observed genotypes with those found in horses, dogs, and roe deer to determine whether genotypes of A. phagocytophilum are shared among different hosts. We sampled 120 domesticated animals (104 cattle, 13 horses, and 3 dogs) and 40 wild animals (roe deer) and used multilocus sequence analysis on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA, and an intergenic region) to characterize the genotypes of A. phagocytophilum present. Phylogenic analysis revealed three genetic clusters of bacterial variants in domesticated animals. The two principal clusters included 98% of the bacterial genotypes found in cattle, which were only distantly related to those in roe deer. One cluster comprised only cattle genotypes, while the second contained genotypes from cattle, horses, and dogs. The third contained all roe deer genotypes and three cattle genotypes. Geographical factors could not explain this clustering pattern. These results suggest that roe deer do not contribute to the spread of A. phagocytophilum in cattle in France. Further studies should explore if these different clusters are associated with differing disease severity in domesticated hosts. Additionally, it remains to be seen if the three clusters of A. phagocytophilum genotypes in cattle correspond to distinct epidemiological cycles, potentially involving different reservoir hosts.

Using Bayes' rule to define the value of evidence from syndromic surveillance.

In this work we propose the adoption of a statistical framework used in the evaluation of forensic evidence as a tool for evaluating and presenting circumstantial "evidence" of a disease outbreak from syndromic surveillance. The basic idea is to exploit the predicted distributions of reported cases to calculate the ratio of the likelihood of observing n cases given an ongoing outbreak over the likelihood of observing n cases given no outbreak. The likelihood ratio defines the Value of Evidence (V). Using Bayes' rule, the prior odds for an ongoing outbreak are multiplied by V to obtain the posterior odds. This approach was applied to time series on the number of horses showing clinical respiratory symptoms or neurological symptoms. The separation between prior beliefs about the probability of an outbreak and the strength of evidence from syndromic surveillance offers a transparent reasoning process suitable for supporting decision makers. The value of evidence can be translated into a verbal statement, as often done in forensics or used for the production of risk maps. Furthermore, a Bayesian approach offers seamless integration of data from syndromic surveillance with results from predictive modeling and with information from other sources such as disease introduction risk assessments.

Detection and quantitation of equid gammaherpesviruses (EHV-2, EHV-5) in nasal swabs using an accredited standardised quantitative PCR method.

Equid gammaherpesviruses-2 and -5 are involved in respiratory problems, with potential clinical manifestations such as nasal discharge, pharyngitis and swollen lymph nodes. These viruses are sometimes associated with a poor-performance syndrome, which may result in a significant and negative economic impact for the horse industry. The aim of the present study was to develop and validate quantitative PCR methods for the detection and quantitation of EHV-2 and EHV-5 in equine respiratory fluids. Two distinct tests were characterised: (a) for the qPCR alone and (b) for the whole method (extraction and qPCR) according to the standard model AFNOR XP U47-600-2 (viz., specificity, quantifiable sensibility, linearity, accuracy, range of application, trueness, precision, repeatability and precision of reproducibility). EHV-2 and EHV-5 detection were performed on nasal swabs collected from 172 horses, all of which exhibited clinical signs of respiratory disease. The data revealed a high rate of EHV-2/EHV-5 co-detection that was correlated significantly with age. Viral load of EHV-2 was significantly higher in young horses whereas viral load of EHV-5 was not significantly different with age.