Tour d'horizon des tests de diagnostic recommandés dans le Manuel des tests de diagnostic et des vaccins pour les animaux terrestres de l'Organisation mondiale de la santé animale.

To provide a standardised approach to the diagnosis of diseases and to facilitate health certification for trade, the World Organisation for Animal Health (OIE) standards, described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual), include internationally agreed laboratory diagnostic techniques. This review examines the type of tests recommended in the disease-specific chapters of the Terrestrial Manual for the six most common purposes of diagnostic techniques, including certification for movement, confirmation of clinical cases and disease surveillance. The most frequently recommended tests for all six purposes are enzyme-linked immunosorbent assay and/or polymerase chain reaction, for which there are detailed validation guidelines in the OIE Terrestrial Manual. This is true for all species and no species-specific barriers to validation related to test type were identified. Classical techniques continue to be well represented in the Terrestrial Manual recommendations whereas novel technologies are slow to gain acceptance. These classical tests can present challenges for validation as there may be a dearth of international standard reagents and harmonised protocols.

Le normes de l'Organisation mondiale de la santé animale (OIE) décrites dans le Manuel des tests de diagnostic et des vaccins pour les animaux terrestres (Manuel terrestre) comprennent notamment des techniques de diagnostic de laboratoire acceptées au niveau international, destinées à fournir un cadre normalisé pour le diagnostic des maladies et à faciliter ainsi la certification sanitaire des échanges internationaux d'animaux et de produits d'origine animale. Les auteurs examinent les différentes catégories de tests recommandés dans les chapitres du Manuel terrestre dédiés à des maladies particulières pour les six principaux emplois assignés aux techniques diagnostiques, dont la certification sanitaire à des fins de déplacement, le diagnostic de confirmation des cas cliniques et la surveillance sanitaire. Les tests recommandés les plus courants pour chacun des six emplois sont l'épreuve immuno-enzymatique et/ou l'amplification en chaîne par polymérase, épreuves pour lesquelles le Manuel terrestre fournit des directives de validation détaillées. Ces directives s'appliquent à toutes les espèces, et aucun obstacle à la validation en lien avec une espèce particulière n'a été identifié pour l'une ou l'autre catégorie de tests. Les techniques classiques figurent toujours en bonne place dans les recommandations du Manuel terrestre, tandis que les technologies innovantes sont plus lentes à être acceptées. Les tests classiques peuvent poser des difficultés lors des essais de validation en raison du risque de pénurie de réactifs de référence internationaux et de l'absence de protocoles harmonisés.

Con objeto de instaurar métodos normalizados de diagnóstico de enfermedades y de facilitar la expedición de certificados sanitarios para el comercio, la Organización Mundial de Sanidad Animal (OIE), en las normas que establece en su Manual de las Pruebas de Diagnóstico y de las Vacunas para los Animales Terrestres (el Manual Terrestre), incluye técnicas de diagnóstico en laboratorio que suscitan consenso a nivel internacional. Los autores pasan revista a los tipos de prueba recomendados en los capítulos del Manual Terrestre relativos específicamente a una determinada enfermedad en relación con los seis propósitos con los que más comúnmente se utilizan las técnicas de diagnóstico, entre ellos la expedición de certificados para el desplazamiento de animales, la confirmación de casos clínicos y la vigilancia de enfermedades. Las pruebas recomendadas con más frecuencia para este conjunto de seis propósitos son el ensayo inmunoenzimático y/o la reacción en cadena de la polimerasa, para cuya validación se ofrecen detalladas indicaciones en el Manual Terrestre de la OIE. Esto se aplica a todas las especies, pues no se ha observado ninguna barrera a la validación asociada a una u otra especie que tenga que ver con el tipo de prueba. En las recomendaciones del Manual Terrestre siguen estando bien representadas las técnicas clásicas, a la par que las tecnologías novedosas van cobrando aceptación con lentitud. A veces la validación de estas pruebas clásicas presenta dificultades por la escasez de protocolos armonizados y de reactivos de referencia a nivel internacional.

Measurement of Typhi Vi antibodies can be used to assess adaptive immunity in patients with immunodeficiency.

Vaccine-specific antibody responses are essential in the diagnosis of antibody deficiencies. Responses to Pneumovax II are used to assess the response to polysaccharide antigens, but interpretation may be complicated. Typhim Vi® , a polysaccharide vaccine for Salmonella typhoid fever, may be an additional option for assessing humoral responses in patients suspected of having an immunodeficiency. Here we report a UK multi-centre study describing the analytical and clinical performance of a Typhi Vi immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) calibrated to an affinity-purified Typhi Vi IgG preparation. Intra- and interassay imprecision was low and the assay was linear, between 7·4 and 574 U/ml (slope = 0·99-1·00; R2  > 0·99); 71% of blood donors had undetectable Typhi Vi IgG antibody concentrations. Of those with antibody concentrations  > 7·4 U/ml, the concentration range was 7·7-167 U/ml. In antibody-deficient patients receiving antibody replacement therapy the median Typhi Vi IgG antibody concentrations were  < 25 U/ml. In vaccinated normal healthy volunteers, the median concentration post-vaccination was 107 U/ml (range 31-542 U/ml). Eight of eight patients (100%) had post-vaccination concentration increases of at least threefold and six of eight (75%) of at least 10-fold. In an antibody-deficient population (n = 23), only 30% had post-vaccination concentration increases of at least threefold and 10% of at least 10-fold. The antibody responses to Pneumovax II and Typhim Vi® correlated. We conclude that IgG responses to Typhim Vi® vaccination can be measured using the VaccZyme Salmonella typhi Vi IgG ELISA, and that measurement of these antibodies maybe a useful additional test to accompany Pneumovax II responses for the assessment of antibody deficiencies.

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.

The impact of different equine influenza vaccine products and other factors on equine influenza antibody levels in Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: More knowledge of equine influenza (EI) vaccine usage in training yards and the factors that influence serological response to vaccination are required to determine evidence-based vaccination strategies. OBJECTIVES: The aim of this study was to ascertain the vaccination history of a population of Thoroughbred racehorses and identify factors that impacted on their antibody titres against EI. STUDY DESIGN: Observational field study. METHODS: The study population consisted of 102 vaccinated Thoroughbred horses in training on a single premises. The vaccination histories recorded in their official passports were analysed. Blood samples for serological testing were collected by the veterinary surgeon one month after booster vaccination with ProteqFlu-Te. Antibodies against EI were measured by single radial haemolysis (SRH). Multivariate statistical analysis was undertaken to determine the predictors of SRH antibody titres. RESULTS: There was a strong correlation between age and number of vaccine doses received. Over 70% of horses received their first vaccine dose between ages 6 and 12 months. On average, horses had received 6 vaccine doses and the mean interval between booster vaccinations was 7.7 months. The majority of horses (95%) received more than one influenza vaccine product while 32% had received 3 vaccine products. Significantly higher antibody levels were observed in females than males and there was a significant association between the number of vaccine products administered and antibody levels. In contrast, a negative association between number of vaccine doses and SRH antibody level was demonstrated. CONCLUSIONS: Important predictors of EI antibody titres in racehorses were sex, number of vaccine doses received and number of different vaccine products administered.

The potential impact of a single amino-acid substitution on the efficacy of equine influenza vaccines.

REASONS FOR PERFORMING STUDY: The protection induced by an equine influenza (EI) vaccine strain depends on its antigenic relatedness to the challenge virus. Although the World Organisation for Animal Health (OIE) recommend that both Florida sublineage clade 1 (Fc1) and clade 2 (Fc2) viruses should be included in EI vaccines, Japanese EI vaccines have not, thus far, been updated to include a Fc2 virus. OBJECTIVES: To evaluate the efficacy of antibodies raised against Japanese EI vaccine strains in the neutralisation of recent Fc2 viruses. STUDY DESIGN: Antigenic analysis. METHODS: Virus neutralisation tests were performed using antisera from experimentally infected horses and from horses that had received a primary course of the currently available vaccines. RESULTS: Antiserum raised against the Japanese EI vaccine strain, A/equine/La Plata/1993, exhibited poor cross-neutralising activity against the Fc2 viruses isolated recently in Ireland and the UK, which have the substitution of alanine to valine at position 144 in antigenic site A of the haemagglutinin gene. In contrast, the antiserum exhibited good cross-neutralising activity against the Fc2 viruses without the substitution. This finding was supported in experiments with antisera collected from vaccinated horses. CONCLUSIONS: This suggests that the efficacy of the Japanese EI vaccine for some of the recent Fc2 viruses is suboptimal and that vaccines should be updated in accordance with the OIE recommendations.

Comparison of primary vaccination regimes for equine influenza: working towards an evidence-based regime.

REASONS FOR PERFORMING STUDY: Vaccination is crucial to the control of equine influenza (EI). The study was conducted in an effort to lay the groundwork for achieving international harmonisation of regulatory requirements based on scientific evidence of performance of different vaccination regimes. OBJECTIVES: To evaluate the effectiveness of 3 different primary vaccination regimes: vaccination with the minimal intervals permitted by the racing authorities; vaccination in accordance with the manufacturer's instructions and vaccination with the longest intervals permitted by the racing authorities. STUDY DESIGN: Randomised, prospective clinical trial. METHODS: The 55 seronegative unvaccinated horses in this study were subdivided by age and randomly allocated one of the 3 vaccination regimes. All groups were sampled each time a group was vaccinated and 3-5 weeks post vaccination. Horses were vaccinated with a subunit immune stimulating complex-based vaccine (Equip FT). Antibodies against EI were measured by single radial haemolysis. RESULTS: Lengthening the vaccination intervals increased the immunity gaps between first (V1) and second (V2) doses, and V2 and third dose (V3) but did not inhibit the response to V2 and V3. The response to V2 and V3 was similar irrespective of the regime. Poor responders to V1 were identified in all age groups included in this study but the greatest number of poor responders was among the yearlings. The 2- and 3-year-old horses responded better to vaccination than the weanlings or yearlings. CONCLUSIONS: Longer vaccination intervals permitted by racing authorities increase the periods of susceptibility to EI but they may facilitate strategic vaccination prior to times of increased risk of exposure to virus. The study provides the type of evidence-based data necessary to commence meaningful discussion of international harmonisation of EI vaccination requirements.

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.

Equine influenza--a global perspective.

To date, equine influenza outbreaks have been reported all over the world with the exception of a small number of island nations including New Zealand and Iceland. Influenza is endemic in Europe and North America and is considered to be of potentially major economic significance to the equine industry worldwide. The importation of subclinically infected vaccinated horses, and inadequate quarantine procedures have resulted in several major outbreaks in susceptible populations for example, in Australia (2007) when more than 76,000 horses on over 10,000 properties were reported as infected. This review summarises the current understanding of, and recent research on, equine influenza, including epidemiology, pathogenesis, clinical characteristics, laboratory diagnosis, management and prevention. Recent advances in diagnostic techniques are discussed as are the merits of different vaccination regimes.

The molecular epidemiology of equine influenza in Ireland from 2007-2010 and its international significance.

REASONS FOR PERFORMING THE STUDY: Antigenic and genetic drift of equine influenza (EI) virus is monitored annually by the Expert Surveillance Panel (ESP), which make recommendations on the need to update vaccines. Surveillance programmes are essential for this process to operate effectively and to decrease the risk of disease spread through the international movement of subclinically infected vaccinated horses. Not only is surveillance necessary to inform vaccine companies which strains are in circulation, but it serves as an early warning system for horse owners, trainers and veterinary clinicians, facilitating the implementation of appropriate prophylactic and control measures. OBJECTIVE: To summarise the genetic analysis of EI viruses detected in Ireland from June 2007 to January 2010. METHODS: The HA1 gene of 18 viruses was sequenced and phylogenetic analysis undertaken. RESULTS: All viruses belonged to the Florida sublineage of the American lineage. Clade 2 viruses predominated up to 2009. The viruses identified on 4 premises in 2007 displayed 100% nucleotide identity to A/eq/Richmond/1/07, the current clade 2 prototype. The first clade 1 virus was identified in November 2009 and, thereafter, clade 1 viruses were responsible for all the outbreaks identified. The Irish clade 1 viruses differ from the clade 1 virus responsible for the EI outbreaks in Japan and Australia in 2007. No virus of the Eurasian lineage was isolated during this surveillance period. CONCLUSIONS: In 2010 the ESP recommended that the vaccines should not include a H7N7 virus or a H3N8 virus of the Eurasian lineage but that they should contain both a clade 1 and clade 2 virus of the Florida sublineage. The surveillance data presented here support these recommendations and indicate that they are epidemiologically relevant. POTENTIAL RELEVANCE: These data also serve as a scientific basis for investigating the source of epizootics and outbreaks both nationally and internationally.

Management and environmental factors involved in equine influenza outbreaks in Ireland 2007-2010.

REASONS FOR PERFORMING STUDY: Outbreaks of equine influenza (EI) in endemic populations continue to cause economic loss despite widespread vaccination. HYPOTHESIS: To identify the key management and environmental factors that determine the risk of horses contracting EI in an endemic country and to identify control strategies. METHODS: Real time-polymerase chain reaction (RT-PCR), virus isolation and haemagglutination inhibition were carried out on nasopharyngeal swabs and clotted blood samples collected from horses and ponies showing signs of respiratory disease. On premises where a diagnosis of EI was confirmed, the attending veterinary surgeon was asked to participate in an epidemiological investigation. RESULTS: Between June 2007 and January 2010, EI outbreaks were diagnosed on 28 premises located in 13 of the 32 counties of Ireland. Veterinary advice was sought on average more than 5 days after the first clinical signs were observed. The majority of diagnoses were made by RT-PCR. Data from 404 horses on 16 premises were used in the epidemiological analysis. In 15 premises, EI was identified following movement of horses. Housing type, teaser stallions or fomites/personnel contributed to virus spread. Vaccination status, number of years vaccination, time since last vaccination and age influenced disease expression. Isolation and vaccination were effective control measures on the premises where they were implemented. CONCLUSIONS: Preventative measures include: isolation, clinical monitoring, serological testing and vaccination of new arrivals, booster vaccination of horses at 6 monthly intervals, maintenance of effective boundaries between equine premises and avoidance of stabling in single air spaces. Control measures include: prompt isolation of suspected cases, rapid diagnosis by RT-PCR, booster vaccination of cohorts and implementation of biosecurity measures to avoid transmission by fomites and personnel. POTENTIAL RELEVANCE: Implementation of these preventative and control measures should reduce the economic losses associated with outbreaks of EI.

Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses.

REASONS FOR PERFORMING STUDY: Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. OBJECTIVES: To develop a sensitive, rapid, real-time RT-PCR (rRT-PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. METHODS: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5' UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT-PCR were verified by virus isolation and ERBV positive samples were verified by rRT-PCR using a different set of primers. RESULTS: The detection limit of the rRT-PCR for both viruses was 10-100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co-circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT-PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). CONCLUSIONS: The rRT-PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. POTENTIAL RELEVANCE: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.

Influenza A viruses with truncated NS1 as modified live virus vaccines: pilot studies of safety and efficacy in horses.

REASONS FOR PERFORMING STUDY: Three previously described NS1 mutant equine influenza viruses encoding carboxy-terminally truncated NS1 proteins are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, and thus are candidates for use as a modified live influenza virus vaccine in the horse. HYPOTHESIS: One or more of these mutant viruses is safe when administered to horses, and recipient horses when challenged with wild-type influenza have reduced physiological and virological correlates of disease. METHODS: Vaccination and challenge studies were done in horses, with measurement of pyrexia, clinical signs, virus shedding and systemic proinflammatory cytokines. RESULTS: Aerosol or intranasal inoculation of horses with the viruses produced no adverse effects. Seronegative horses inoculated with the NS1-73 and NS1-126 viruses, but not the NS1-99 virus, shed detectable virus and generated significant levels of antibodies. Following challenge with wild-type influenza, horses vaccinated with NS1-126 virus did not develop fever (>38.5 degrees C), had significantly fewer clinical signs of illness and significantly reduced quantities of virus excreted for a shorter duration post challenge compared to unvaccinated controls. Mean levels of proinflammatory cytokines IL-1beta and IL-6 were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post challenge. CONCLUSION AND CLINICAL RELEVANCE: These data suggest that the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza. This type of reverse genetics-based vaccine can be easily updated by exchanging viral surface antigens to combat the problem of antigenic drift in influenza viruses.

Molecular characterization of equine rotavirus in Ireland.

Group A rotaviruses are important causative agents of severe, acute dehydrating diarrhea in foals. A total of 86 rotavirus-positive fecal samples, collected from diarrheic foals from 11 counties in three of the four provinces of Ireland, were obtained from the Irish Equine Centre in Kildare during a 7-year (1999 to 2005) passive surveillance study and were characterized molecularly to establish the VP7 (G type) and VP4 (P type) antigenic specificities. Fifty-eight samples (67.5%) were found to contain G3 viruses, while in 26 samples (30.2%) the rotaviruses were typed as G14 and in 2 samples (2.3%) there was a mixed infection, G3 plus G14. All samples except for two, which were untypeable, were characterized as P[12]. Fifty-eight percent of the samples were obtained from County Kildare, the center of the Irish horse industry, where an apparent shift from G3P[12] to G14P[12] was observed in 2003. By sequence analysis of the VP7 protein, the G3 Irish strains were shown to resemble viruses of the G3A subtype (H2-like) (97.1 to 100% amino acid [aa] identity), while the G14 Irish strains displayed 93.9 to 97.1% aa identity to other G14 viruses. In the VP8* fragment of the VP4 protein, the P[12] Irish viruses displayed high conservation (92.3 to 100% aa) with other equine P[12] viruses. Worldwide, G3P[12] and G14P[12] are the most prevalent equine rotavirus strains, and G3P[12] vaccines have been developed for prevention of rotavirus-associated diarrhea in foals. Investigations of the VP7/VP4 diversity of the circulating equine viruses and the dynamics of strain replacement are important for better assessing the efficacies of the vaccines.

Diagnosis of equine infectious anaemia during the 2006 outbreak in Ireland.

In 2006 there was an outbreak of equine infectious anaemia (EIA) in Ireland. This paper describes the use of the diagnosis of clinical and subclinical cases of the disease. In acute cases the ELISAs and the immunoblot were more sensitive than the AGID. In one mare, fluctuating antibody levels were observed in all the serological assays before it seroconverted by AGID. Viral RNA and DNA were detected by RT-PCR and PCR in all the tissues from the infected animals examined postmortem. The PCR detected viral DNA in plasma regardless of the stage of the disease. In contrast, the RT-PCR detected RNA in only 52 per cent of the seropositive animals tested and appeared to be most sensitive for the detection of virus early in infection. Both PCR and RT-PCR demonstrated potential to detect acutely infected horses earlier than some of the official tests. The serological data suggest that the usual incubation/seroconversion period for this strain of the virus was approximately 37 days but may be more than 60 days in a few cases.

Effective priming of foals born to immune dams against influenza by a canarypox-vectored recombinant influenza H3N8 vaccine.

A classical limitation of early life immunization is the interference by maternally derived antibodies, which are known to inhibit the immune response to modified-live and killed vaccines. Several studies have convincingly shown that even minute amounts of maternally derived antibodies against equine influenza can strongly interfere with successful vaccination of foals born to immune mares. In this study we evaluated the response of foals born to vaccinated mares to immunization with a canarypox-vectored recombinant vaccine against equine influenza virus H3N8. The recombinant vaccine was able to efficiently prime foals in the presence of maternally derived immunity against influenza as was evidenced by a clear anamnestic antibody response when a secondary vaccination with the same vaccine was performed. The canarypox-vectored recombinant influenza vaccine therefore offers a unique opportunity to overcome the limitations of early life vaccination in the face of maternally derived immunity in foals.