Prevalence of Equine Hepacivirus Infections in France and Evidence for Two Viral Subtypes Circulating Worldwide.

Like hepatitis C virus (HCV) in humans, the newly identified equine hepacivirus (NPHV) displays a predominating liver tropism that may evolve into chronic infections. The genomes of the two viruses share several organizational and functional features and are phylogenetically closest amongst the Hepacivirus genus. A limited amount of data is available regarding the spread of hepacivirus infections in horses. In this study, we asked whether in a more representative sample the prevalence and distribution of NPHV infections in France would resemble that reported so far in other countries. A total of 1033 horses sera from stud farms throughout France were analysed by qRT-PCR to determine the prevalence of ongoing NPHV infections and viral loads; in positive samples, partial sequences of NPHV's genome (5'UTR, NS3 and NS5B genes) were determined. Serum concentrations of biliary acids, glutamate dehydrogenase (GLDH) and L-gamma-glutamyl transferase (γ-GT) were measured for most horses. We detected NPHV infections in 6.2% of the horses, a prevalence that reached 8.3% in thoroughbreds and was significantly higher than in other breeds. The presence of circulating virus was neither significantly associated with biological disturbances nor with clinical hepatic impairment. Our phylogenetic analysis was based on both neighbour-joining and maximum-likelihood approaches. Its result shows that, like almost everywhere else in the world so far, two major groups of NPHV strains infect French domestic horses. Based on genetic distances, we propose a classification into two separate NPHV subtypes. Viral loads in the serum of horses infected by the main subtype were, in average, four times higher than in those infected by the second subtype. We hypothesize that amino acid substitutions in the palm domain of NS5B between NPHV subtypes could underlie viral phenotypes that explain this result.

Genetic evolution of equine influenza strains isolated in France from 2005 to 2010.

UNLABELLED: REASON FOR PERFORMING THIS STUDY: Equine influenza virus (EIV) is considered the most economically important equine respiratory pathogen worldwide. The H3N8 subtype, responsible for all outbreaks of equine influenza globally, evolves perpetually. Mutations in the genome of these viruses have the potential to modify their antigenic properties and recognition by pre-existing antibodies. OBJECTIVES: The aim of this study was to determine the genetic evolution of EIV strains in France and to compare it with the evolution of strains isolated globally. Analysis of the sequence data was performed to investigate any possible links between the outbreaks. STUDY DESIGN: Retrospective genetic analysis study of archived material. METHODS: Analyses were performed on the HA1 domain of haemagglutinin H3 of EIV isolated in a previous study carried out from November 2005 to October 2010. The nucleic acid sequence of 41 strains was analysed and translated. The French viruses were compared with 59 Clade 1 strains and 83 Clade 2 strains. Strains were aligned chronologically and on the basis of their geographical origin. RESULTS: The 16 Clade 1 strains are all derived from the outbreak that started in the Grosbois training yard in 2009. The virus genome appears to have been stable during the outbreak. The 25 Clade 2 strains were isolated over the 5-year period during which several mutations had emerged. Some strains incorporate a sporadic mutation, and others a mutation that may occur several times but does not persist. However, all strains are gradually moving towards definitive mutations. CONCLUSION: This study demonstrated that EIVs have evolved in France during this period in a similar manner to EIVs globally. The data lend support to the current World Animal Health Organisation recommendation that the vaccines contain a representative of both Clade 1 and Clade 2 of the Florida sublineage.

Cytokine concentrations in bronchoalveolar lavage fluid from horses with neutrophilic inflammatory airway disease.

BACKGROUND: Multiple cytological patterns occur in bronchoalveolar lavage fluid (BALF) of horses with inflammatory airway disease (IAD). Only few data on BALF cytokine profiles are available for horses with IAD, and are limited to mRNA expression. HYPOTHESIS/OBJECTIVE: Cytological profiles of IAD are associated with different BALF immunological pathways. To investigate BALF cytokine concentrations in a large number of horses with neutrophilic IAD. ANIMALS: One hundred and thirty-eight client-owned Standardbred racehorses in active training. METHODS: Prospective observational study. BALF samples were obtained from left and right lungs. Interleukin (IL)-4, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α concentrations were determined by ELISA. RESULTS: Fourteen horses had normal BALF cytological profiles and 56 exhibited evidence of bilateral neutrophilic IAD. Twenty-four horses showed BALF with, respectively, IAD- and CTL consistent cytology and were excluded; as were 44 horses because of evidence of pulmonary hemorrhage. TNF-α (56 ± 115 pg/mL; P = .034) and IFN-γ concentrations (104 ± 247 pg/mL; P = .044) were significantly higher for IAD horses, compared with controls (respectively 19 ± 41 and 80 ± 116 pg/mL). Horses with 'neutrophil' subtype had significantly higher IFN-γ concentrations (110 ± 154 pg/mL), than 'neutrophil/metachromatic' (56 ± 54 pg/mL; P = .028) and 'neutrophil/metachromatic/eosinophil' subtypes (44 ± 23 pg/mL; P = .012). CONCLUSIONS AND CLINICAL IMPORTANCE: Cytokine concentrations in BALF suggested that neutrophilic IAD is associated with activation of the innate immune system and a possible T-helper (Th)-1 polarized response. This study also suggested that immunological pathways vary according to cytological IAD subtypes.

Surveillance of equine influenza viruses through the RESPE network in France from November 2005 to October 2010.

REASONS FOR PERFORMING THE STUDY: The Réseau d'Epidémio-Surveillance en Pathologie Equine (RESPE, the French epidemiological network for equine diseases) is a network for epidemio-surveillance of major equine diseases based around sentry veterinarians in France. OBJECTIVE: The aim of this study was to evaluate the contribution of RESPE to efficient surveillance of equine influenza virus (EIV) in France. STUDY DESIGN: Retrospective cross-sectional study. METHODS: From November 2005 to October 2010, epidemiological and phylogenetic studies were performed on 1426 nasopharyngeal swabs received at the Frank Duncombe Laboratory. Detection was performed by real-time reverse transcription polymerase chain reaction using original primers and probes designed in the matrix protein gene. Phylogenetic analysis was carried out on the HA1 part of haemagglutinin gene amplified from 47 positive-testing samples. Epidemiological information was provided with the majority of samples submitted through RESPE. RESULTS: Of the 920 samples submitted by RESPE-associated veterinarians, 121 (13.1%) from 42 premises were positive for EIV, compared to 26 (5.1%) of the 607 samples received from non-RESPE associated veterinarians. The most extensive outbreak was observed between February and May 2009, affecting 70 horses on 23 premises, 15 of which were managed by RESPE-associated veterinarians. All strains belonged to the American lineage, Florida sublineage, Clade 1 and Clade 2. Clade 1 was identified only during the Grosbois episode. CONCLUSION: RESPE improved detection of EIV in France, enabled characterisation of the virus strains, yielded valuable information relating to the epidemiology of the disease and identified vaccine breakdown. POTENTIAL RELEVANCE: Implementation of a similar surveillance network in other countries may reduce the economic losses associated with outbreaks of EIV.

Outbreak of equine herpesvirus myeloencephalopathy in France: a clinical and molecular investigation.

Equid herpesvirus 1 (EHV-1)-associated myeloencephalopathy (EHM) is a disease affecting the central nervous system of horses. Despite the constantly increasing interest about this syndrome, epidemiological data are limited especially when related to the description of large outbreaks. The aim of this article is to describe clinical, virological and molecular data obtained throughout a severe outbreak of EHM, with emphasis on laboratory diagnostic methods. The epidemic disease concerned a riding school in France where 7/66 horses aged 12-22 years developed signs of neurological disease in July 2009. Diagnosis of EHM was supported by EHV-1 detection using both real-time PCR and virus culture, and SNP-PCR test for viral strain characterization. EHM morbidity was 10.6% (7/66), mortality was 7.5% (5/66) and case fatality rate was 71.4% (5/7). Clinical presentation of the disease was characterized by the fact that fever was systematically present within 2 days before the severe neurological signs were noted. EHV-1 was detected by PCR in each available blood and nasal swab samples. Neuropathogenic strain only (G(2254) ) was isolated during the current outbreak; C(t) values, used as an indicative level of the viral load, ranged 26.0-37.0 among the six sampled horses. The amount of virus in biological samples was not systematically related to the intensity of the clinical signs being observed. In conclusion, this article described a severe outbreak of EHM while limited in time and restricted to one premise. Molecular data strongly suggested taking into account any low viral load as being a potential risk factor for neurological manifestations.

Serum concentration of surfactant protein D in horses with lower airway inflammation.

REASONS FOR PERFORMING STUDY: Surfactant protein D (SP-D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP-D are routinely used as biomarkers for pulmonary injury. To date, serum SP-D levels have only been investigated in horses in an experimental model of bacterial airway infection. OBJECTIVES: To compare serum SP-D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). METHODS: Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD-affected and 20 classified as controls. Serum SP-D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. RESULTS: Serum concentrations of SP-D in IAD-affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD-affected group, no significant correlation was found between serum SP-D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP-D levels. CONCLUSIONS: This is the first study determining serum SP-D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP-D levels. POTENTIAL RELEVANCE: Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.

Description of the first recorded major occurrence of equine viral arteritis in France.

REASONS FOR PERFORMING STUDY: The vast majority of equine arteritis virus (EAV) infections are inapparent or relatively mild, but may occasionally cause outbreaks of equine viral arteritis. The event observed in France during the summer of 2007 was the most important seen in the country, with mortality and disruption of economic activity. OBJECTIVES: To describe the different stages seen during the outbreak and to show how molecular tools were used for both the detection and management of the crisis. METHODS: EAV detection was performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in blood, nasal swabs, semen or organ samples. Characterisation of EAV strains was performed by sequencing the ORF5 fragment. RESULTS: The outbreak affected 18 premises in 5 counties in western France, which represented the index, 8 primary and 9 secondary premises. Artificial insemination in draught horses was responsible for the virus spread. Eight mortality cases were observed, including one fetus, 5 young foals and 2 mature horses. Forty-three individuals had positive results by real-time RT-PCR. The range of measured cycle threshold (Ct) values varied from 19.8 to 40.4 depending on the biological samples. Phylogenetic analysis revealed that the 33 isolated strains all clustered within the EU-2 subgroup. CONCLUSIONS: The mortality rate attests to the virulence of the strain involved in this outbreak. Real-time RT-PCR was used for the first time in order to follow-up an epidemic disease in horses. POTENTIAL RELEVANCE: The early detection of 3 signals with high Ct values attest the importance of taking low signals into account in field conditions.

Physiological measurements and prevalence of lower airway diseases in Trotters with dorsal displacement of the soft palate.

REASONS FOR PERFORMING STUDY: Dorsal displacement of the soft palate (DDSP) is one of the most common obstructive conditions of the upper respiratory tract in the racehorse. This condition has a complex aetiology which may be caused or exacerbated by pharyngeal inflammation. Additionally, lower respiratory airway diseases may be associated with DDSP thereby contributing to exercise intolerance in these horses. OBJECTIVE: The aim of this study was to measure physiological variables during a standardised exercise test and to assess the prevalence and consequences of lower respiratory airway disease in horses with DDSP. METHODS: A total of 46 horses were included in this study: 22 in the control and 24 in the DDSP groups. All horses performed a SET with measurement of heart rate (HR) and blood lactate concentration. One hour post exercise, respiratory samples were collected for cytological and bacteriological analysis. RESULTS: During exercise, the DDSP group had higher blood lactate concentration than the control group. According to BAL results, 50 and 63% of control and DDSP group horses, respectively, had evidence of inflammatory airway disease (IAD). In the DDSP group, 42% of horses had a syndrome of tracheal inflammation (STI) with 71% of this group having bacteria isolated at >10(5) CFU/ml. CONCLUSIONS: Horses with DDSP showed evidence of a high prevalence of IAD and STI with an associated positive bacteriology in 55% of the cases. Even if DDSP is treated by surgery, the authors' recommendation would be to investigate the possibility of lower respiratory airway problems which may also be impacting the horse's performance and/or surgery efficiency.

[An epidemiological survey of equine anaplasmosis (Anaplasma phagocytophilum) in southern France]. / Enquête epidémiologique sur l'anaplasmose equine (Anaplasma phagocytophilum) dans le Sud de la France.

Anaplasmosis is caused by the bacterium Anaplasma phagocytophilum and transmitted by Ixodes spp. ticks. According to some reports the disease can be introduced into disease-free zones by migrating birds. The purpose of this study was to evaluate the seroprevalence of A. phagocytophilum in horses in the Camargue. Data concerning 424 horses were gathered and the sera were tested for A. phagocytophilum and for piroplasmoses using an enzyme-linked immunosorbent assay and a complement fixation test, respectively. The seroprevalence rates were 11.3 % for A. phagocytophilum, 64.4 % for Theileria equi and 19.7% for Babesia caballi. Stallions were less likely to produce antibodies against A. phagocytophilumthan were females or geldings (odds ratio [OR] = 0.3; p = 0.021). The presence of swallows increased the risk of infections in stables (OR = 5.18; p = 0.011). Spatial analysis showed the existence of groups of infected stables along canals and rivers (p = 0.008). These results suggest an emergence of A. phagocytophilum in the Camargue.

Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases.

Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or stayed in the US exhibited titers over 50, a higher figure than in the group which did not travel out of France or stayed in an other European country. No correlation was found between anti-S. neurona and anti-Neospora sp. antibody titers. Data prompt further study of significance of anti-S. neurona antibodies in clinically healthy or diseased European horses, and identification of putative immunizing parasite(s) and their host(s).

Neosporosis in bovine dairy herds from the west of France: detection of Neospora caninum DNA in aborted fetuses, seroepidemiology of N. caninum in cattle and dogs.

Neospora caninum is considered one of the major causes of abortion in cattle in most parts of the world. In this study, the role of N. caninum was investigated in groups of aborted cattle and dairy herds from the west of France. Good correlation was found between parasite DNA detection in fetuses and serologic statuses of dams. In groups with documented abortion status and no antibodies to other pathogens, 17-45% of aborted animals were seropositive for N. caninum, and significant relationship between prevalence of Neospora antibodies and frequency of abortions was found. Neospora-associated abortions were observed all the year round, with a peak in summer. Higher ratios of seropositive abortions were found before the 6th month of gestation. In 12 herds studied in the field, serologic prevalence ranged 6-47%. No difference in age was found between seropositive and seronegative cows. Results indicate that N. caninum is an important and stable cause of abortion in cattle in France.