Equine herpesvirus-1 consensus statement.

Equine herpesvirus-1 is a highly prevalent and frequently pathogenic infection of equids. The most serious clinical consequences of infection are abortion and equine herpesvirus myeloencephalopathy (EHM). In recent years, there has been an apparent increase in the incidence of EHM in North America, with serious consequences for horses and the horse industry. This consensus statement draws together current knowledge in the areas of pathogenesis, strain variation, epidemiology, diagnostic testing, vaccination, outbreak prevention and control, and treatment.

Safety and immunogenicity of a live-attenuated auxotrophic candidate vaccine against the intracellular pathogen Rhodococcus equi.

Rhodococcus equi causes serious pneumonia in neonatal foals and is an opportunistic pathogen of people with compromised cellular immunity. No effective vaccine against R. equi disease in foals is available. We tested the safety and immunogenicity of a live, fully attenuated riboflavin auxotrophic candidate vaccine strain of R. equi (R. equi rib-). We demonstrated that R. equi rib- is immunogenic and capable of inducing IFN-gamma responses in immunocompetent BALB/c mice, yet it is safe even in an immunocompromised SCID mouse infection model. Moreover, it protects immunocompetent mice against virulent R. equi challenge. In foals, R. equi rib- was likewise safe and stimulated serum R. equi-specific immune responses. A preliminary immunization strategy did not afford protection against virulent R. equi challenge and therefore, optimization of the vaccine formulation and or vaccination protocol will be necessary.

A case-control study of factors associated with development of clinical disease due to West Nile virus, Saskatchewan 2003.

REASON FOR PERFORMING STUDY: West Nile virus (WNV) was first diagnosed in Saskatchewan equids in 2002. AWNV epidemic was considered highly likely for 2003, which would provide a unique opportunity to study all aspects of WNV subclinical infection and clinical disease development in a relatively naive population. HYPOTHESIS: There are individual equid attributes and management risk factors associated with development of clinical disease. Specifically, this study could address the question of vaccine efficacy for the prevention of development of clinical disease. METHODS: A case-control study was conducted in the summer of 2003 during a province-wide outbreak of WNV. Between 5 and 10 equids were sampled from each of 23 case premises with clinical disease and 23 control premises with no apparent or confirmed clinical disease. Data were analysed to identify risk factors for the development of clinical disease. RESULTS: The proportion of equids serologically positive for natural exposure to West Nile virus was 64% (193/300). Nonvaccinated equids were 23 times (95%CI limits 3.0, 168.5, P = 0.002) more likely to develop clinical disease than those vaccinated. The estimate of vaccine efficacy in this field study was 96% (95%CI limits 67%, 99%). CONCLUSIONS: The study demonstrated that vaccination was strongly associated with the prevention of clinical disease. POTENTIAL RELEVANCE: Vaccination is an effective, practical method of prevention of clinical disease.

Correlation between in vitro secretion of virulence-associated proteins of Campylobacter jejuni and colonization of chickens.

The mechanisms used by Campylobacter jejuni to colonize the (chicken) intestinal tract have not been defined. In this study, we obtained evidence that in the presence of chicken serum and mucus, C. jejuni secreted proteins that may play a role in the colonization of chicken gut (Campylobacter invasion antigen = Cia). C. jejuni strains NCTC11168V1 and 81-176, as well as an NCTC11168V1 flaA mutant, were found to colonize intestinal tract and secrete proteins in the presence of chicken mucus, chicken serum, or fetal bovine serum in cell culture-conditioned medium. C. jejuni strain NCTC11168V26, which was observed to be a poor colonizer compared with the other C. jejuni isolates, did not secrete Cia proteins. Secreted proteins were also recognized by Western immunoblot using sera from birds that had been colonized by C. jejuni. These data suggest that C. jejuni secretes Cia proteins during colonization of chicken gut and that these Cia proteins play an important role in colonization.

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine.

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty naïve horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.

Immunization with recombinant modified vaccinia Ankara (rMVA) constructs encoding the HA or NP gene protects ponies from equine influenza virus challenge.

Two novel recombinant strains of modified vaccinia Ankara (rMVA) for the vaccination of horses against equine influenza virus were developed, and preliminary evidence of their immunogenicity in ponies was demonstrated [Breathnach CC, Rudersdorf R, Lunn DP. Use of recombinant modified vaccinia Ankara vectors for equine influenza vaccination. Vet Immunol Immunopathol 2004:98;127-36]. The present study assessed the protective efficacy of these rMVA strains in ponies, examined the advantage of combining rMVA vaccination with a DNA priming dose, and investigated the protection resulting from equine influenza nucleoprotein (NP) versus haemagglutinin (HA) vaccination. Twenty yearling ponies, seronegative for equine influenza virus, were divided into four groups of five. Group 1 and Group 2 ponies were vaccinated using a DNA prime-rMVA boost vaccination regimen, with HA- or NP-expressing vectors, respectively. Group 3 ponies were vaccinated with rMVA-HA only. Group 4 ponies served as unvaccinated controls. Vaccines were administered on days 0, 42 and 70, and all ponies were challenge infected with influenza virus on day 100. Antigen-specific antibody and cellular immune responses to each vaccination regimen were monitored throughout the experiment. Both groups of HA-vaccinated ponies were significantly protected from clinical disease following challenge infection, demonstrating the efficacy of rMVA vaccination with or without a DNA prime. NP-vaccination provided more limited protection from clinical disease. The protective post-vaccinal immune responses were characterized by antigen-specific IgGa, IgGb and IgA antibodies which were induced both in serum and in nasal secretions. Virus-specific lymphoproliferative and IFN-gamma mRNA responses were also elicited by each vaccination regimen. These data demonstrate that vaccination of horses with rMVA alone, or as part of a prime-boost regimen, is an effective means of inducing protective immunity to influenza virus infection, and also indicate that NP-specific immune responses can contribute to protection of horses.

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep.

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.

Meniscal tears in horses: an evaluation of clinical signs and arthroscopic treatment of 80 cases.

REASONS FOR PERFORMING STUDY: There is little published information available describing clinical signs, arthroscopic findings and prognosis of meniscal injuries in horses. OBJECTIVES: To evaluate the effect on the outcome not only of the arthroscopic findings and treatment, but also of the clinical and radiographic signs in these horses. METHODS: The following were recorded for each case: the meniscal injury, graded according to severity; clinical and radiographic findings prior to surgery; any concurrent injury in the joint seen at arthroscopy. The effect of these factors and the grade of injury on the outcome were analysed using Fisher's exact test or Chi-square analysis. Only horses whose meniscal injury was judged to be the primary cause of lameness were included in the series. RESULTS: A series of 80 meniscal injuries were diagnosed and treated arthroscopically by the authors at the Liphook Equine Hospital and 47% of horses returned to full use. Statistically, poor prognosis was associated with increasing severity of the meniscal injury, the presence of concurrent articular cartilage lesions and radiographic abnormalities in the joint. Arthroscopic treatment of many lesions was limited by the inaccessibility of parts of the femorotibial joint. POTENTIAL RELEVANCE: Further work is required to improve and evaluate arthroscopic techniques for the treatment of these injuries.

Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere.

We reported previously that equine-2 influenza A virus (H3N8) had evolved into two genetically and antigenically distinct "Eurasian" and "American" lineages. Phylogenetic analysis, using the HA1 gene of more recent American isolates, indicated a further divergence of these viruses into three evolution lineages: A South American lineage, a Kentucky lineage, and a Florida lineage. These multiple evolution pathways were not due to geographic barriers, as viruses from different lineages co-circulated. For the Kentucky lineage, the evolution rate was estimated to be 0.89 amino acid substitutions per year, which agreed with the previously estimated rate of 0.8. For the South American lineage, the evolution rate was estimated to be only 0.27 amino acid substitutions per year. This low evolution rate was probably due to a unique alternating Ser138 to Ala138 substitutions at antigenic site A. For the Kentucky lineage, there was a preference for sequential nonsynonymous substitutions at antigenic site B, which was also a "hot spot" for amino acid substitutions. Convalescent sera had minimal cross-reactivity to viruses of different lineages, indicating antigenic distinctions among these viruses. In contrast to human H3N2 viruses, our results suggested that the evolution of equine-2 influenza virus resembled the multiple evolution pathways of influenza B virus.

Effect of vaccination on experimental infection with Bordetella bronchiseptica in dogs.

OBJECTIVE: To determine comparative efficacy of vaccines administered IM and intranasally, used alone or sequentially, to protect puppies from infection with Bordetella bronchiseptica and determine whether systemic or mucosal antibody response correlated with protection. DESIGN: Randomized controlled trial. ANIMALS: 50 specific-pathogen-free Beagle puppies. PROCEDURE: In 2 replicates of 25 dogs each, 14-week-old puppies that were vaccinated against canine distemper virus and parvovirus were vaccinated against B bronchiseptica via intranasal, IM, intranasal-IM, or IM-intranasal administration or were unvaccinated controls. Puppies were challenge exposed via aerosol administration of B bronchiseptica 2 weeks after final vaccination. Clinical variables and systemic and mucosal antibody responses were monitored for 10 days after challenge exposure. Puppies in replicate 1 were necropsied for histologic and immunohistochemical studies. RESULTS: Control puppies that were seronegative before challenge exposure developed paroxysmal coughing, signs of depression, anorexia, and fever. Vaccinated puppies (either vaccine) that were seronegative before challenge exposure had fewer clinical signs. Puppies that received both vaccines had the least severe clinical signs and fewest lesions in the respiratory tract. Vaccinated dogs had significantly higher concentrations of B bronchiseptica-reactive antibodies in serum saliva before and after challenge. Antibody concentrations were negatively correlated with bacterial growth in nasal cavity and pharyngeal samples after challenge exposure. CONCLUSIONS AND CLINICAL RELEVANCE: Parenterally and intranasally administered vaccines containing B bronchiseptica may provide substantial protection from clinical signs of respiratory tract disease associated with infection by this bacterium. Administration of both types of vaccines in sequence afforded the greatest degree of protection against disease.

A new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge.

Flu Avert IN vaccine is a new, live attenuated virus vaccine for equine influenza. We tested this vaccine in vivo to ascertain 1) its safety and stability when subjected to serial horse to horse passage, 2) whether it spread spontaneously from horse to horse and 3) its ability to protect against heterologous equine influenza challenge viruses of epidemiological relevance. For the stability study, the vaccine was administered to 5 ponies. Nasal swabs were collected and pooled fluids administered directly to 4 successive groups of naïve ponies by intranasal inoculation. Viruses isolated from the last group retained the vaccine's full attenuation phenotype, with no reversion to the wild-type virus phenotype or production of clinical influenza disease. The vaccine virus spread spontaneously to only 1 of 13 nonvaccinated horses/ponies when these were comingled with 39 vaccinates in the same field. For the heterologous protection study, a challenge model system was utilised in which vaccinated or naïve control horses and ponies were exposed to the challenge virus by inhalation of virus-containing aerosols. Challenge viruses included influenza A/equine-2/Kentucky/98, a recent representative of the 'American' lineage of equine-2 influenza viruses; and A/equine-2/Saskatoon/90, representative of the 'Eurasian' lineage. Clinical signs among challenged animals were recorded daily using a standardised scoring protocol. With both challenge viruses, control animals reliably contracted clinical signs of influenza, whereas vaccinated animals were reliably protected from clinical disease. These results demonstrate that Flu Avert IN vaccine is safe and phenotypically stable, has low spontaneous transmissibility and is effective in protecting horses against challenge viruses representative of those in circulation worldwide.

Efficacy of a cold-adapted, intranasal, equine influenza vaccine: challenge trials.

A randomised, controlled, double-blind, influenza virus, aerosol challenge of horses was undertaken to determine the efficacy of a cold-adapted, temperature sensitive, modified-live virus, intranasal, equine influenza vaccine. Ninety 11-month-old influenza-naïve foals were assigned randomly to 3 groups (20 vaccinates and 10 controls per group) and challenged 5 weeks, 6 and 12 months after a single vaccination. Challenges were performed on Day 0 in a plastic-lined chamber. Between Days 1 and 10, animals were examined daily for evidence of clinical signs of influenza. Nasal swabs for virus isolation were obtained on Day 1 and Days 1 to 8 and blood samples for serology were collected on Days 1, 7 and 14. There was no adverse response to vaccination in any animal. Following challenge at 5 weeks and 6 months, vaccinates had significantly lower clinical scores (P = 0.0001 and 0.005, respectively), experienced smaller increases in rectal temperature (P = 0.0008 and 0.0007, respectively) and shed less virus (P<0.0001 and P = 0.03, respectively) over fewer days (P<0.0001 and P = 0.002, respectively) than did the controls. After the 12 month challenge, rectal temperatures (P = 0.006) as well as the duration (P = 0.03) and concentration of virus shed (P = 0.04) were significantly reduced among vaccinated animals. The results of this study showed that 6 months after a single dose of vaccine the duration and severity of clinical signs were markedly reduced amongst vaccinated animals exposed to a severe live-virus challenge. Appropriate use of this vaccine should lead to a marked reduction in the frequency, severity and duration of outbreaks of equine influenza in North America.

Equine vaccination.

Equine infectious disease remains a constant and important threat to the health of domesticated horses. Vaccination plays a critical role in protecting against such disease, but at the present time the efficacy of some equine vaccination strategies is in doubt. The best strategy for resolving these concerns is an improved knowledge of the immunologic basis of successful vaccination, combined with the appropriate integration of effective vaccines into well-designed disease control policies.

Clinical and pathologic findings in donkeys with hypothermia: 10 cases (1988-1998).

OBJECTIVE: To describe clinical signs and clinicopathologic findings in donkeys with hypothermia. DESIGN: Retrospective study. ANIMALS: 10 hypothermic donkeys. PROCEDURE: Information on signalment, history, physical examination findings, results of diagnostic tests, treatments, and necropsy findings was extracted from medical records of all donkeys with hypothermia between 1988 and 1998 and compared with information from medical records of all normothermic donkeys and hypothermic horses admitted to the hospital during the same period. RESULTS: Donkeys were more likely to be hypothermic than horses. The mean age of hypothermic donkeys was 6 years (range, 7 months to 11 years), compared with 4.2 years (range, < 1 month to 15 years) for normothermic donkeys; this difference was not significant. Ten of 12 horses with hypothermia were neonates; there were no hypothermic neonatal donkeys. At admission, 7 of 8 hypothermic donkeys were in good body condition and all hypothermic donkeys were weak. Six hypothermic donkeys were able to maintain sternal recumbency, 1 remained in lateral recumbency, and 3 were able to stand. Of the 10 hypothermic donkeys, 2 survived, 1 died, and 7 were euthanatized. Histologically, the thyroid glands from 4 of 5 hypothermic donkeys appeared abnormal and were similar to those of foals with hypothyroidism. During the months that hypothermic donkeys were admitted, there was not a significant difference in environmental temperatures on days of admission between hypothermic and normothermic donkeys. CONCLUSIONS AND CLINICAL RELEVANCE: Hypothermia is a problem in donkeys during cold winter months, and may not be secondary to other disease or related to diet or management.

Descriptive epidemiologic study of disease associated with influenza virus infections during three epidemics in horses.

OBJECTIVE: To describe 3 epidemics of respiratory tract disease caused by influenza virus infections in a large population of horses. DESIGN: Cross-sectional and prospective longitudinal observational studies. ANIMALS: All horses stabled at a Thoroughbred racetrack. PROCEDURES: During a 3-year period, descriptive information was collected as horses arrived at the racetrack and throughout race meetings. Routine observations and physical examinations were used to classify horses' disease status. Cause of epidemics was established by use of serologic testing and identification of influenza virus in nasal secretions. RESULTS: An epidemic of respiratory tract disease caused by influenza virus infections was identified during each year of the study. Attack rates of infectious upper respiratory tract disease (IURD) ranged from 16 to 28%. Incidence of disease caused by influenza virus infections during racing seasons in the second and third years was 27 and 37 cases/1,000 horses/mo, respectively. Physical distributions of stall locations revealed that affected horses were stabled throughout the population; horses affected later in epidemics were often clustered around horses affected earlier. Mucopurulent nasal discharge and coughing were observed in 83 and 62% of horses with IURD, respectively. Median duration of clinical disease was 11 days. Serologic testing was the most sensitive method used to detect influenza virus infections; 76% of affected horses seroconverted to influenza virus. CONCLUSIONS AND CLINICAL RELEVANCE: Epidemics of IURD were observed annually in association with influenza virus infections. Few precautions were taken to limit spread of infection. Preventing or decreasing the likelihood of exposure and improving immunity in the population could substantially decrease risk of disease in similar populations.

Risk factors for disease associated with influenza virus infections during three epidemics in horses.

OBJECTIVE: To identify risk factors associated with respiratory tract disease in horses during 3 epidemics caused by influenza virus infections. DESIGN: Cross-sectional and prospective longitudinal observational studies. ANIMALS: 1,163 horses stabled at a Thoroughbred racetrack. PROCEDURES: Investigations were conducted during a 3-year period. An epidemic of respiratory tract disease caused by influenza virus infections was identified in each year. Routine observations and physical examinations were used to classify horses' disease status. Data were analyzed to identify factors associated with development of disease. RESULTS: Results were quite similar among the epidemics. Concentrations of serum antibodies against influenza virus and age were strongly associated with risk of disease; young horses and those with low antibody concentrations had the highest risk of disease. Calculation of population attributable fractions suggested that respiratory tract disease would have been prevented in 25% of affected horses of all horses had high serum antibody concentrations prior to exposure. However, recent history of vaccination was not associated with reduction in disease risk. Exercise ponies had greater risk of disease than racehorses, which was likely attributable to frequent horse-to-horse contact. CONCLUSIONS AND CLINICAL RELEVANCE: Particular attention should be paid to young horses, those with low serum antibody concentrations, and horses that have frequent contact with other horses when designing and implementing control programs for respiratory tract disease caused by influenza virus infections. It appears that control programs should not rely on the efficacy of commercial vaccines to substantially reduce the risk of disease caused by influenza virus infections.

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.

Validity of saddle pressure measurements using force-sensing array technology--preliminary studies.

Back pain is a common and poorly understood clinical problem. An important factor in this regard is the induction or exacerbation of back pain from badly designed or poorly fitting saddles. This study used a pressure-sensing mat to investigate saddle fit. The aims of the study were to confirm the accuracy and reliability of the force-sensing array technology when used to measure pressure beneath the saddles of horses, and to collect some initial data from normal healthy horses with well-fitting saddles. Experiments were undertaken to establish that a linear relationship existed between the total force (weight) exerted and the pressure measured beneath the saddle, using both a wooden horse and a live horse in the standing position. Further studies were performed to demonstrate that characteristic changes of the centre of pressure occur while horses move at the walk, sitting trot, rising trot, and canter.