Epidemiology and molecular detection of equine herpesviruses in western Algeria in 2011.

An episode of acute equine respiratory infection was reported in western Algeria (Tiaret province) between February and March 2011, affecting a large population of horses. Nasal swabs (n=100) were taken from horses aged between 1 and 27 years, presenting with cough and mucopurulent nasal discharge. The prevalence of equine respiratory virus infections was examined using quantitative polymerase chain reaction (qPCR). One, or more, of four equine respiratory viruses were detected in the nasal swabs of 90 of 100 horses (90%) and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 2 (EHV-2) and equine herpesvirus type 5 (EHV-5) were 2%, 14%, 90% and 75%, respectively. Equine influenza virus and equine arteritis virus were not detected in any samples. Among the 90 infected horses, 70 were co-infected with EHV-2 and EHV-5 and 14 others were co-infected with EHV-4, EHV-2 and EHV-5. The present study shows a positivity rate of 97.3% for EHV-5 in young horses aged <3years; a finding which decreased with age. Viral load of EHV-5 was significantly higher in <3years whereas no effect of age was observed with EHV-2. The study shows that equine herpesviruses 1, 2, 4 and 5 are endemic in horse populations from Algeria as detected for the first time by qPCR.

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.

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.

Detection of equine herpesviruses in aborted foetuses by consensus PCR.

The major role of EHV-1 in equine abortion is widely reported in the literature but the contribution of EHV-2, EHV-3, EHV-4 or EHV-5 remains less well documented. The objective of this study is to evaluate the contribution of these five different EHVs to equine abortion in a variety of biological tissues using a consensus polymerase chain reaction (PCR). The test was validated for specificity and sensitivity in horses before screening specimens from 407 foetuses, stillbirths and premature foals collected over a 2.5-year interval. Positive results obtained with this assay were compared to other EHV type-specific PCR or by sequencing. EHV-1 was identified as the major cause of abortion in French mares (59/407 cases). However, there was evidence to suggest some variation in the potential of EHV-1 strains to induce abortion. Indeed, DNA samples from EHV-2 (in three cases) and EHV-5 (in one case) inferred a role of these viruses in abortion. The presence of viral DNA from EHV-3 or EHV-4 strains was not detected in the specimens studied. The data obtained suggest that the consensus herpesvirus PCR is an efficient screening tool. In association with a specific PCR, the test provides a rapid identification of the type of herpesvirus involved in abortion and is useful for routine diagnostic tests as it allows the identification of herpesviruses other than the EHV-1 strain.

Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus.

Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001-2004) were determined by sequencing open reading frames (ORFs) 2a-7. The ORFs 2a-7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296-11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France.

Identification of Taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase chain reaction.

A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosomal DNA product only in all T. equigenitalis.

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).

Detection and isolation of equine herpesviruses 1 and 4 from horses in Normandy: an autopsy study of tissue distribution in relation to vaccination status.

Equine herpesviruses type 1 and 4 (EHV-1 and EHV-4) are ubiquitous in the equine population. One of their main properties is their ability to establish life-long latent infections in their hosts even in those with natural or vaccine-induced immunity. However, effect of vaccination status on prevalence and tissue tropism was not established. In this study, EHV-1 and EHV-4 were detected by polymerase chain reaction and by classical virus isolation from neural, epithelial and lymphoid tissues collected from unvaccinated (33) or vaccinated (23) horses. The percentage of EHV-1- and EHV-4-positive horses between vaccinates and unvaccinates was similar. Both viruses were detected in all tissues of both groups; in particular, lymph nodes draining the respiratory tract, nasal epithelium and nervous ganglia [i.e. trigeminal ganglia (TG)], which represent the main positive sites for EHV-1 and EHV-4. In vaccinated animals, the nervous ganglia (i.e. TG) were less frequently positive than in unvaccinated animals. Detection of positive TG was strongly correlated to the presence of EHV-1 in nasal epithelium.

Physiological measurements and upper and lower respiratory tract evaluation in French Standardbred Trotters during a standardised exercise test on the treadmill.

There are a variety of reasons for poor performance in racehorses. Exercise intolerance has often been associated with subclinical respiratory abnormalities, and diagnostic aids are therefore used to enhance clinical detection. Physiological variables can also be measured in order to evaluate the metabolic reponse to exercise. This study evaluated the relationship between physiological measurements and upper airway videoendoscopy during a standardised treadmill exercise test and bronchoalveolar lavage (BAL) cytology in control horses (good racing performance, n = 14) and poor performers (n = 27). The poor performers were divided into 2 groups: Group 1 = both upper and lower respiratory airway abnormal findings (n = 10); Group 2 = lower respiratory airway abnormal findings (n = 17). Horses in Group 2 were divided into 3 categories: Group 2A = exercise-induced pulmonary haemorrhage (EIPH ; n = 5); Group 2B = small airway inflammation (SAI +/- EIPH; n = 7) and Group 2C = other (n = 5). During exercise, the poor performers had significantly lower arterial PaO2 and higher HR and blood lactate concentrations compared to controls. Total nucleated cell count of BAL fluid collected from poor racing performers was significantly higher than in controls; also, epithelial cells and haemosiderophage percentage collected from poor racing performers were significantly higher than in controls. Eight horses with dorsal displacement of the soft palate also had cytological evidence of lower respiratory airway disease. The results of this study suggest that there is a significantly different metabolic response (HR, blood lactate, PaO2) to exercise in poor compared to good performers. As both upper and/or lower respiratory problems can be associated with poor racing performance, a detailed examination of the upper and lower respiratory tracts at rest, during and after exercise is advised.

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.

Characterization of mutations in the rpoB gene associated with rifampin resistance in Rhodococcus equi isolated from foals.

Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rif(r))-determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 microg/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 microg/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in R. equi, and the resistance levels are dependent on both the location and the nature of the substitution.