Search

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. [ABSTRACT FROM AUTHOR]

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. The Summary is available in Chinese - see . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. The Summary is available in Chinese - see Supporting information. [ABSTRACT FROM AUTHOR]

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 (G2254) was isolated during the current outbreak; Ct 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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

Summary 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 >105 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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to several articles on the role of serum cytotoxicity in the diagnosis of equine viral arteritis that are published in the periodical in 2003 and in 2004.

Introduction Exercise-induced pulmonary haemorrhage (EIPH) is a highly prevalent airway disorder responsible for poor-performance in horses. While various methodologies are available for diagnosing EIPH, no readily available systemic biomarker has to date been definitively validated. A recent study demonstrated that angiotensin-converting enzyme (ACE) activity in plasma was increased in horses with higher degrees of haemorrhage, as assessed by post exercise tracheal endoscopy. Aims To investigate plasma ACE activity in horses suffering previous episodes of EIPH and controls. Methods Venous blood samples were collected at rest from 121 client-owned, racing or race-fit Standardbred racehorses. Bronchoalveolar lavage fluid (BALF) samples were collected from right and left lung, cytocentrifuged and stained with Perl's Prussian blue. Total haemosiderin scores (THS) were calculated for detecting EIPH. According to THS, 36 horses were diagnosed with EIPH and 85 were assigned as controls. The ACE levels were determined on an automated spectrophotometer, using FAPGG as a substrate. Results Plasma ACE levels in EIPH-affected horses (85.1 ± 24.6 u/l) were lower than those of control horses (94.4 ± 24.0 u/l), even if the tendency was not statistically significant (P = 0.056). Within each group of horse (EIPH and control), no significant difference was found for THS between lungs; a significant correlation between right and left BALF being furthermore observed (R = 0.643; P<0.001). No significant correlation was however found between plasma ACE levels and THS of BALF from either left or right lung. Conclusions and practical significance This is the first study determining plasma ACE activity in a large population of racehorses at training. Apart from acute pulmonary haemorrhage, these results highlight that previous episodes of EIPH might be associated with a moderate decrease of circulating ACE levels. Further studies are warranted to establish the precise implication of ACE in the physiopathology of EIPH and determine its relevance as a systemic biomarker. Ethical animal research This study has been approved by the Regional Animal Ethic Committee and all owners gave consent. Sources of funding: I.F.C.E. (French National Studs) and S.E.C.F. (French Trotters Racing Association). Competing interests: None. [ABSTRACT FROM AUTHOR]

Native bovine serum amine oxidase (BSAO) and poly(ethylene glycol) (PEG)-treated ('PEGylated') BSAO were immobilized into a hydrogel during its synthesis. The hydrogel was obtained by cross-linking of BSA with PEG di-nitrophenyl carbonates with a molecular mass of 10 kDa. Approx. 60% of the amino groups at the surface of BSAO were modified by monomethoxy-PEG with a molecular mass of 5 kDa when the reaction was carried out for 5 h in borate buffer, pH 9. The number of anchorage points of BSAO in the matrix, which was determined as minimal when PEGylated BSAO was used or maximal when native BSAO was used, did not influence the apparent K(m) and V(max) values of the different preparations. The apparent K(m) values of both forms of the enzyme were decreased due to preconcentration of benzylamine substrate by the negatively charged hydrogel. V(max) values were generally lower upon immobilization. We can therefore conclude that the hydrogel swelling has no significant effect on the enzyme's structure. The operational stability, evaluated in the presence of substrate, was generally increased upon enzyme immobilization into the hydrogel. Enzymic hydrogels were very stable during storage in solution at 4 degrees C, maintaining a high activity even after several weeks. The immobilization of both forms of BSAO did not improve their thermostability at 65 degrees C. The BSA-PEG hydrogel is a good matrix for immobilization of enzymes with therapeutic potential such as BSAO.

The L-asparaginase of Escherichia coli (ASNase) is currently used in combination with antineoplastic drugs to treat various lymphoblastic leukaemias. However, its use is limited by severe immunological reactions and the short serum half-life associated with the enzyme. Immobilization of ASNase into a biocompatible matrix can greatly decrease the immunogenicity of the enzyme, increase its half-life in vivo and its therapeutic index. Thus the E. coli ASNase was immobilized in a biocompatible hydrogel made of rat serum albumin and poly(ethylene glycol) (PEG; molecular mass 10 kDa). The effectiveness of this enzymic bioreactor to deplete serum L-asparagine was evaluated after its peritoneal implantation in rats. Seven units of immobilized ASNase/rat depleted serum asparagine to an undetectable level (< 1 microM) during 6 days, while 5 units of immobilized ASNase/rat decreased the level of serum asparagine by 85-90% during at least 2 days. Under both conditions asparagine levels returned to normal about 10 days after surgery, and hydrogels still retained 80% of their enzymic activity when assayed in vitro. After 10-14 days in vivo, hydrogels became opaque and surrounded by a fibrotic capsule with a few inflammatory sites. Nevertheless, the enzymic hydrogel showed great stability in vivo, and, after 4 months of implantation, 12% of the initial ASNase activity was still present. At 6 months, histological analysis showed stabilization of the fibrotic capsule thickness. Assays on the levels of ASNase and asparagine synthetase indicated an induction of the latter activity, mainly in the pancreas when compared with the level observed in spleen or liver. ELISA tests at 28 days and 120 days showed the presence of anti-ASNase (and, in lower amounts, anti-PEG) antibodies in sera of implanted rats. As observed with other enzyme-immobilization systems used in vivo, the formation of fibroblast-like cell layers around the implant, which block the translocation of the substrate into the enzymic matrix, is the major factor affecting the performance and longevity of the bioreactor.

The feasibility of the immobilization of Escherichia coli L-asparaginase into a hydrogel matrix made of poly-(ethylene glycol) (PEG) and BSA was demonstrated. After immobilization a 200-fold increase in the Km value was observed. The use of an L-aspartic acid analogue, carbobenzoxy-L-aspartic acid and surface modification by methoxy-PEG of molecular mass 5 kDa cause a only a slight gain in affinity of the enzyme for its natural substrate. The immobilized L-asparaginase has an optimal activity over a larger range of pH than the native enzyme, owing to the effect of the matrix. At a physiological pH of 7.3, the immobilized enzyme retained 90% of its activity compared with only 43% for the native form. The immobilized enzyme retained a high proportion of its initial activity, more than 90% after 50 days of incubation at 37 degrees C, even in the presence of its substrate. This may be compared with a half-life of 2 days observed for native enzyme incubated under the same conditions. These results suggest that the BSA-PEG matrix can be very useful for enzyme immobilization and, taking into account the good biocompatibility of the matrix, one can expect that this matrix will provide a functional bioreactor for use in vivo.