J. A. Mumford

Analysis of Equid Herpesvirus 1 Strain Variation Reveals a Point Mutation of the DNA Polymerase Strongly Associated with Neuropathogenic versus Nonneuropathogenic Disease Outbreaks

ABSTRACT Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of EHV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic strain (V592) of EHV-1 and developed PCR/sequencing procedures enabling differentiation of EHV-1 strains circulating in the field. The results indicate the occurrence of several major genetic subgroups of EHV-1 among isolates recovered from outbreaks over the course of 30 years, consistent with the proposal that distinct strains of EHV-1 circulate in the field. Moreover, there is evidence that certain strain groups are geographically restricted, being recovered predominantly from outbreaks occurring in either North America or Europe. Significantly, variation of a single amino acid of the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Strikingly, this variant amino acid occurs at a highly conserved position for herpesvirus DNA polymerases, suggesting an important functional role.

Respiratory disease in thoroughbred horses in training: the relationships between disease and viruses, bacteria and environment

A longitudinal study of respiratory disease in racehorses was carried out to assess its relative associations with different infectious agents and to examine any role that the environmental conditions might play. The relationships between coughing, nasal discharge, pyrexia and lower respiratory tract disease were also examined to provide information for improving clinical diagnosis, particularly of disease of the lower respiratory tract. Lower airway disease was closely associated with infection with Streptococcus zooepidemicus. It was also found that equine herpesvirus seroconversions and S pneumoniae infections were independently associated with the development of nasal discharge. Coughing was a specific, but insensitive measure of lower respiratory tract disease (specificity 84 per cent, sensitivity 38 per cent). However, horses that coughed were very likely to have had lower airway disease for more than one month. Horses housed on straw in loose boxes were twice as likely to suffer from lower airway disease as those kept on shredded paper in American barns. The study was not large enough to assess the significance of rarer infections but it did improve the definition of the problem of respiratory disease in racehorses and revealed some of the trends in the associations between viruses, bacteria and the environment in respiratory disease.

Association between Respiratory Disease and Bacterial and Viral Infections in British Racehorses

ABSTRACT Respiratory disease is important in horses, particularly in young Thoroughbred racehorses, and inflammation that is detected in the trachea and bronchi (termed inflammatory airway disease [IAD]) is more significant in this population in terms of impact and frequency than other presentations of respiratory disease. IAD, which is characterized by neutrophilic inflammation, mild clinical signs, and accumulation of mucus in the trachea, may be multifactorial, possibly involving infections and environmental and immunological factors, and its etiology remains unclear. This 3-year longitudinal study of young Thoroughbred racehorses was undertaken to characterize the associations of IAD and nasal discharge with viral and bacterial infections. IAD was statistically associated with tracheal infection with Streptococcus pneumoniae (capsule type 3), Streptococcus zooepidemicus, Actinobacillus spp., and Mycoplasma equirhinis and equine herpesvirus 1 and 4 infections, after adjustment for variation between training yards, seasons, and age groups. The association with S. pneumoniae and S. zooepidemicus was independent of prior viral infection and, critically, was dependent on the numbers of organisms isolated. S. pneumoniae was significant only in horses that were 2 years old or younger. The prevalence and incidence of IAD, S. zooepidemicus, and S. pneumoniae decreased in parallel with age, consistent with increased disease resistance, perhaps by the acquisition of immunity. The study provided evidence for S. zooepidemicus and S. pneumoniae playing an important etiological role in the pathogenesis of IAD in young horses.

Description of the outbreak of equine influenza (H3N8) in the United Kingdom in 2003, during which recently vaccinated horses in Newmarket developed respiratory disease

Between March and May 2003, equine influenza virus infection was confirmed as the cause of clinical respiratory disease among both vaccinated and unvaccinated horses of different breeds and types in at least 12 locations in the UK. In the largest outbreak, 21 thoroughbred training yards in Newmarket, with more than 1300 racehorses, were affected, with the horses showing signs of coughing and nasal discharge during a period of nine weeks. Many of the infected horses had been vaccinated during the previous three months with a vaccine that contained representatives from both the European (A/eq/Newmarket/2/93) and American (A/eq/Newmarket/1/93) H3N8 influenza virus lineages. Antigenic and genetic characterisation of the viruses from Newmarket and elsewhere indicated that they were all closely related to representatives of a sublineage of American viruses, for example, Kentucky/5/02, the first time that this sublineage had been isolated in the UK. In the recently vaccinated racehorses in Newmarket the single radial haemolysis antibody levels in acute sera appeared to be adequate, and there did not appear to be significant antigenic differences between the infecting virus and A/eq/Newmarket/1/93, the representative of the American lineage virus present in the most widely used vaccine, to explain the vaccine failure. However, there was evidence for significantly fewer infections among two-year-old horses than older animals, despite their having similar high levels of antibody, consistent with a qualitative rather than a quantitative difference in the immunity conveyed by the vaccination.

Foal diarrhoea between 1991 and 1994 in the United Kingdom associated with Clostridium perfringens, rotavirus, Strongyloides westeri and Cryptosporidium spp.

A case control study of foal diarrhoea in the United Kingdom was carried out over a 3-year period. Clostridium perfringens was significantly associated with foal diarrhoea (Odds Ratio (OR) = 3.0), being isolated from 57% of 421 animals with diarrhoea but from only 27% of 223 healthy foals. Also, C. perfringens was significantly associated with fatal diarrhoea (OR = 4.5). About half of diarrhoea with a fatal outcome was attributable to this organism. The other pathogens significantly associated with diarrhoea were rotavirus (OR = 5.6), Cryptosporidium spp. (OR = 3.2) and the nematode Strongyloides westeri, which was significant only when present in large numbers (> 2000 eggs/g of faeces: OR = 6.1). Salmonella spp. (OR = 14.2) and Cryptosporidium spp. (OR = 3.0) were the only other pathogens associated with fatal illness. Overall, C. perfringens, rotavirus, and large numbers of Cryptosporidium spp. or S. westeri were isolated from 80% of foals with diarrhoea. Thermophilic Campylobacter spp., Yersinia enterocolitica, Escherichia coli and other parasites were not associated with diarrhoea. Carriage of C. perfringens, rotavirus and Cryptosporidium spp. was significantly greater in healthy foals in contact with cases of diarrhoea than in foals that were not in contact with diarrhoea (P < 0.05). There were no statistical interactions between any of the pathogens associated with diarrhoea although separate cases from one location often involved more than one pathogen.

Antigenicity and immunogenicity of equine influenza vaccines containing a Carbomer adjuvant.

Equine influenza vaccines containing inactivated whole virus and Carbomer adjuvant stimulated higher levels and longer lasting antibody to haemagglutinin in ponies than vaccines of equivalent antigenic content containing aluminium phosphate adjuvants. Five months after the third dose of vaccine containing Carbomer adjuvant, ponies were protected against clinical disease induced by an aerosol of virulent influenza virus (A/equine/Newmarket/79, H3N8). In contrast ponies which received vaccine containing aluminium phosphate adjuvant were susceptible to infection and disease. There was an inverse correlation between prechallenge levels of antibody detected by single radial haemolysis (SRH) and duration of virus excretion, pyrexia and coughing. All ponies with antibody levels equivalent to SRH zones of > or = 154 mm2 were protected against infection and all those with levels > or = 85 mm2 were protected from disease.

Immunity to equine influenza: relationship of vaccine-induced antibody in young Thoroughbred racehorses to protection against field infection with influenza A/equine-2 viruses (H3N8)

Field outbreaks of influenza that occurred in vaccinated Thoroughbred racehorses in Newmarket in 1995 and 1996 were investigated by nucleoprotein ELISA and serology. Investigations showed that serum levels of vaccine-induced single radial haemolysis (SRH) antibody correlated closely with protective immunity against equine influenza and were consistent with observations made in previous experimental studies using nebulised aerosol challenge. In the second part of this study, antibody levels stimulated by vaccination were investigated to examine probable protection in high risk groups, such as yearlings and horses in training. Results for yearlings correlated closely with experimentally derived antibody profiles described for several equine influenza vaccines. The horses in training had levels of antibody immediately prior to revaccination, which were higher than those measured in the yearlings. In conclusion, SRH antibody, used in the investigation of outbreaks and surveillance of post vaccination responses, was shown to correlate with and validate experimental vaccination and challenge models currently used in ponies in the licensing of modern vaccines. There may be benefit from serological monitoring of horses following vaccination through identification of susceptible periods to infection and demonstration of poor vaccine responders. This would allow appropriate and timely amendment of vaccination strategies to maximise protective immunity against influenza.

Inflammatory airway disease, nasal discharge and respiratory infections in young British racehorses

REASONS FOR PERFORMING STUDY Respiratory disease is important in young Thoroughbred racehorses, but the variation in the rates of occurrence between different ages and training groups has not been characterised. OBJECTIVES To determine the rates of respiratory disease, particularly inflammatory airway disease (IAD), as well as evidence of infection, and their variation between age and group. METHODS Horses were examined monthly in 7 British flat training yards over a 3 year period. IAD was defined as increased mucus in the trachea with increased proportions of neutrophils in tracheal wash samples. Frequencies of disease outcomes were estimated from the data. RESULTS The prevalence of IAD was 13.8% and the incidence was 8.9 cases/100 horses/month. Rates varied with training and age groups, decreasing in older animals. The prevalence of nasal discharge (ND) was 4.1%. Rates of bacterial isolation were more common than viral infections. The incidence and prevalence of several bacterial species decreased with age. CONCLUSIONS IAD and ND were common in young racehorses, varying significantly between training groups and decreasing with age, consistent with infection playing a role in aetiology. POTENTIAL RELEVANCE The high prevalence of IAD in 2-year-old horses in Britain suggests that routine endoscopic examination may be helpful in providing early diagnosis and appropriate therapy. The transmission of bacteria and viruses within and between groups of young animals and the role of infection, stable environment and factors inherent to each horse, including their genetic make-up, in the multifactorial aetiology of the disease all merit further study.

Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL

Complementary DNAs encoding ORFs 2 to 7 equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-specific synthetic peptide (residues 75 through 97) induced EAV-neutralizing antibody in vaccinated horses. The defined antigenic region of GL is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

Antigenicity and immunogenicity of experimental equine influenza ISCOM vaccines

A comparison of the antigenicity and immunogenicity of ISCOM vaccines prepared from equine influenza viruses H3N8 and H7N7 was made with inactivated whole-virus vaccines containing equivalent amounts of virus haemagglutinin. ISCOMs stimulated superior antibody responses in terms of both amount and duration. As with conventional whole-virus vaccines, the levels of antibody to virus haemagglutinin induced by ISCOMs correlated with protection.