A non-competitive chemiluminescence enzyme immunoassay for the equine acute phase protein serum amyloid A (SAA) -- a clinically useful inflammatory marker in the horse.

A non-competitive chemiluminescence enzyme immunoassay for measuring serum amyloid A (SAA) in equine serum was developed. A polyclonal anti-equine-amyloid A antiserum specific for equine SAA was utilized, and the assay was standardized using highly purified equine SAA. An acute phase horse serum was calibrated against the purified SAA and was used as standard when running the assay. Serum SAA concentrations in the range of 3-1210 mg/l could be measured. The reference range of SAA in clinically healthy adult horses was <7 mg/l. The clinical validation of the assay comprised the SAA responses after surgery and experimentally induced aseptic arthritis, and those associated with viral and bacterial infections. The SAA response after surgery (castration) was consistent, with peak concentrations on day 2 and a return to normal SAA concentrations within eight days. The aseptic arthritis produced an SAA response with a pattern similar to that seen after surgery, with peak concentrations of SAA 36-48 h after induction. Seven horses showed a biphasic pattern, with a second rise in SAA concentrations on day 4 and 5. All animals had SAA levels <7 mg/l on day 15. All horses with viral and bacterial infections had SAA concentrations above 7 mg/l. The ranges of SAA concentrations following the different types of inflammation overlap, being consistent with the unspecific nature of the SAA response. This study revealed that SAA is a sensitive and unspecific marker for inflammation, and describes the dynamics of the SAA response after standardized and well defined tissue damage.     

Dynamics in serum of the inflammatory markers serum amyloid A (SAA), haptoglobin,fibrinogen and alpha2-globulins during induced noninfectious arthritis in the horse

Despite the importance of noninfectious joint diseases in equine medicine, little is known about the acute phase response which may be elicited if the local inflammatory process of noninfectious arthritis is sufficiently strong, Therefore the aim of this study was to monitor the systemic inflammatory response during experimentally-induced noninfectious arthritis by studying the dynamics in serum of the acute phase proteins serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins. Twenty-four Standardbred horses, age 3-7 years, found healthy on thorough clinical, radiological, haematological and serum biochemical examination, were injected aseptically into the right midcarpal joint with amphotericin B. Blood samples were drawn before induction of arthritis (0 h), and at 8, 16, 24, 36 and 48 h postinduction and then on Days 3, 4, 5 and 15 postinduction. All horses developed lameness with joint effusion and joint heat as well as increased respiratory rate, heart rate and body temperature. The lameness started to decline after 24-36 h and, in most animals, systemic signs disappeared on Day 2 postinjection. The concentration of the acute phase proteins increased following induction of arthritis. The SAA concentrations were higher than baseline concentrations from 16 h postinduction and were maximal at 36-48 h (227 times baseline concentration). The haptoglobin concentrations were higher than baseline concentrations from 24 h and were maximal at 48-96 h (1.14 times baseline concentration). The maximal concentrations of fibrinogen were seen between 36-72 h postinjection and increased on average 0.87 times from baseline concentrations. The fibrinogen concentrations were higher than baseline concentrations from 24 h postinjection. Alpha2-globulins concentrations showed a minor increase and increased 0.55 times from baseline concentrations. The markers had returned to baseline concentrations by Day 15. Our results demonstrate that amphotericin B-induced arthritis in a single joint gives rise to a systemic acute phase response measurable as increased concentrations in serum SAA, haptoglobin, fibrinogen and alpha2-globulins during the first 2 weeks of the condition and, thereby, that such an increase need not be indicative of infectious arthritis. Further research should be aimed at determining whether chronic noninfectious arthritis in the horse gives rise to increased acute phase protein concentrations in serum.     

An Outbreak of Type A2 Influenza Among Horses

The clinical diagnosis of equine influenza was first based on the spectacular contagiousness of the disease, the general clinical resemblances to human influenza and the almost complete absence of complications usually observed in infectious viral arteritis, viral rhinopneumonitis or in other respiratory infections of the horses. The specific viral etiology of the epizootic was ascertained through the isolation of a type A influenza virus and further substantiated by evaluation of the immunological response of the sick horses, as demonstrated by complement fixation and hemagglutination-inhibition tests, using normal and convalescent sera.     

Comparison of Serum Amyloid A in Horses With Infectious and Noninfectious Respiratory Diseases

The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory parameter in the horse, but studies showing SAA responses to specific respiratory disease etiologies are limited. The goal of this study was to evaluate SAA responses in horses with infectious and noninfectious respiratory diseases as well as healthy, control horses. Two hundred seven horses were grouped into the following categories: equine influenza virus (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi), inflammatory airway disease (IAD), and healthy controls. Serum amyloid A concentrations were determined for all horses on serum using a stall-side lateral flow immunoassay test. Serum amyloid A levels were found to be significantly greater for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and horses with IAD when compared to control horses. There was a significant difference between viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi were significantly greater when compared to horses with viral infections (EIV/EHV-4), the wide range of SAA values precluded accurate classification of the infectious cases. In conclusion, SAA is more reliably elevated with infections of the respiratory tract rather than noninfectious airway conditions. This can facilitate early detection of respiratory infections, help track disease progression, and aid practitioners in making recommendations about proper biosecurity and isolation of potentially contagious horses.     

Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations

The aim of the present study was to evaluate whether equine serum amyloid A (SAA) concentrations could be measured reliably with a turbidometric immunoassay (TIA) developed for use with human serum. Intra- and inter-assay imprecision were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by linearity under dilution. The assay was subsequently used for measuring SAA concentrations in clinically healthy horses, horses with inflammatory diseases, horses with non-inflammatory diseases, and in horses before and after castration. In pools with low, intermediate and high SAA concentrations, the intra-assay imprecisions were 24.4%, 1.6% and 2.1%, and the inter-assay imprecisions were 33.2%, 4.6% and 6.5%. Slight signs of inaccuracy were observed, but these inaccuracies were negligible when considering the large dynamic range of the SAA response. The assay was able to detect the expected difference in SAA levels in different groups of horses. It was also able to demonstrate the expected dynamic changes in SAA after castration. In conclusion, equine SAA concentrations can be measured reliably using the TIA designed for human SAA.     

Serum amyloid A in equine health and disease

Serum amyloid A (SAA) is the major acute phase protein in horses. It is produced during the acute phase response (APR), a nonspecific systemic reaction to any type of tissue injury. In the blood of healthy horses, SAA concentration is very low, but it increases dramatically with inflammation. Due to the short half-life of SAA, changes in its concentration in blood closely reflect the onset of inflammation and, therefore, measurement of SAA useful in the diagnosis and monitoring of disease and response to treatment. Increases in SAA concentration have been described in equine digestive, reproductive and respiratory diseases and following surgical procedures. Moreover, SAA has proven useful for detection of some subclinical pathologies that can disturb training and competing in equine athletes. Increasing availability of diagnostic tests for both laboratory and field use adds to SAA's applicability as a reliable indicator of horses’ health status. This review article presents the current information on changes in SAA concentrations in the blood of healthy and diseased horses, focussing on clinical application of this biomarker.     

Equine Respiratory Disease on the Western Canadian Racetracks

The serological results from this study clearly show that both equine influenza and equine rhinopneumonitis viruses were present during spring and autumn epidemics of respiratory disease on Western Canadian racetracks. Approximately 11% of the horses showed significant convalescent titres to influenza while 9% showed significant convalescent titres for equine viral pneumonitis. It was noted in our study a positive vaccination history corresponded with a reduction in the severity of the respiratory infection.     

Serological observations on an epidemic of equine influenza in India

During the epidemic of equine influenza which occurred in India in 1987, serum samples were collected at late acute/early convalescent phase (7–9 days), at 5 weeks and at 18–23 weeks after onset of illness, from six affected horses from the Union Territory of Changigarh, and Nawanshahr, Punjab State, India, and were examined for antibodies to A/eq-1 and A/eq-2 influenza viruses by hemagglutination inhibition (HI) tests. It was found that the antibody response to A/eq-1 virus strains, Ludhiana/87 and Prague/56, was stronger and antibodies persisted at high levels in four animals. The fifth animal showed a diagnostic decrease in HI titers while the sixth animal seroconverted. The corresponding HI titers to A/eq-2/Ludhiana virus showed a 4-fold decrease in all six animals.Another nine equine animals in the single convalescent serum samples had detectable or high HI titers against A/eq-1 and A/eq-2 viruses.In serum samples from horses and ponies, taken 5 weeks to 9 months after onset of illness, little or no difference in antibody titers to A/eq-2/Miami/63 and A/eq-2/Fontainebleau/79 strains was found.It seems clear that the antibody titers that ensued were indicative of recent influenza infections. Apparently, two distinct equine influenza viruses, A/eq-1 and A/eq-2, were involved during the epidemic, infecting the equine animals simultaneously in the region.     

Efficacy and duration of immunity of a combined equine influenza and equine herpesvirus vaccine against challenge with an American-like equine influenza virus (A/equi-2/Kentucky/95)

It has been recommended that modern equine influenza vaccines should contain an A/equi-1 strain and A/equi-2 strains of the American and European-like subtype. We describe here the efficacy of a modern updated inactivated equine influenza-herpesvirus combination vaccine against challenge with a recent American-like isolate of equine influenza (A/equine-2/Kentucky/95 (H3N8). The vaccine contains inactivated Influenza strains A-equine-1/Prague'56, A-equine-2/Newmarket-1/'93 (American lineage) and A-equine-2/ Newmarket-2/93 (Eurasian lineage) and inactivated EHV-1 strain RacH and EHV-4 strain V2252. It is adjuvanted with alhydrogel and an immunostim. Horses were vaccinated at the start of the study and 4 weeks later. Four, six and eight weeks after the first vaccination high anti-influenza antibody titres were found in vaccinated horses, whereas at the start of the study all horses were seronegative. After the challenge, carried out at 8 weeks after the first vaccination, nasal swabs were taken, rectal temperatures were measured and clinical signs were monitored for 14 days. In contrast to unvaccinated control horses, vaccinated animals shed hardly any virus after challenge, and the appearance of clinical signs of influenza such as nasal discharge, coughing and fever were reduced in the vaccinated animals. Based on these observations, it was concluded that the vaccine protected against clinical signs of influenza and, more importantly, against virus excretion induced by an American-like challenge virus strain. In a second experiment the duration of the immunity induced by this vaccine was assessed serologically. Horses were vaccinated at the start of the study and 6 and 32 weeks later. Anti-influenza antibody titres were determined in bloodsamples taken at the first vaccination, and 2, 6, 8, 14, 19, 28, 32, 37, 41, 45 and 58 weeks after the first vaccination. Vaccinated horses had high anti-influenza antibody titres, above the level for clinical protection against influenza, against all strains present in the vaccine until 26 weeks after the third vaccination.     

Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: a field study

REASONS FOR PERFORMING THE STUDY: Early recognition of excessive inflammation and infectious complications after surgery, leading to early institution of therapy, reduces post operative discomfort and facilitates recovery. Because serum amyloid A (SAA) is a highly sensitive marker of inflammation, measurements of SAA and other acute phase reactants in the equine surgical patient may be valuable in assisting clinical assessment of post operative inflammation. OBJECTIVES: To investigate changes in inflammatory markers after castration and to correlate levels of acute phase reactants with clinical severity of inflammation after castration. METHODS: Leucocyte numbers and blood levels of iron, SAA and fibrinogen were determined before castration and on Days 3 and 8 post operatively in 2 groups of horses; Group 1 (n = 11) had mild post operative inflammation and an uncomplicated recovery and Group 2 (n = 7) had local clinical signs of moderate to severe inflammation. RESULTS: Both groups had elevated serum SAA levels at Day 3 post operatively. In Group 1 concentrations had returned to preoperative levels by Day 8, whereas in Group 2 concentrations remained elevated. Plasma fibrinogen concentrations in serum increased to equal levels in both groups and stayed elevated throughout the study period. Serum iron concentrations of Group 1 did not change in response to castration, whereas concentrations in Group 2 decreased below preoperative levels on Day 8. Leucocyte numbers remained unchanged during the post operative period in both groups. CONCLUSIONS: Serum SAA and iron profiles reflected the course of inflammation and their levels correlated with the clinical severity of inflammation. In contrast, fever and changes in leucocyte numbers, which are usually considered to be hallmarks of inflammation and infection, were not useful for monitoring post operative recovery. POTENTIAL RELEVANCE: Measurements of SAA and iron may improve post operative monitoring. As sustained inflammation may indicate that the surgical wound has become infected, SAA and iron measurements may facilitate early recognition and hence early treatment of infection.     

Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic

OBJECTIVE: To determine concentrations of 2 acute-phase proteins (serum amyloid A [SAA] and lipopolysaccharide-binding protein [LBP]) in serum samples obtained from horses with colic and identify relationships among these acute-phase proteins and clinical data. ANIMALS: 765 horses with naturally developing gastrointestinal tract diseases characterized by colic (ie, clinical signs indicative of abdominal pain) and 79 healthy control horses; all horses were examined at 2 university teaching hospitals. PROCEDURE: Serum concentrations of SAA and LBP were determined by immunoturbidometric and dot-blot assays, respectively. RESULTS: SAA and LBP concentrations were determined for 718 and 765 horses with colic, respectively. Concentrations of SAA were significantly higher in nonsurvivors than in survivors, and horses with enteritis or colitis and conditions characterized by chronic inflammation (eg, abdominal abscesses, peritonitis, or rectal tears) had SAA concentrations significantly greater than those for horses with other conditions. Serum concentrations of LBP did not correlate with outcome, disease process, or portion of the gastrointestinal tract affected. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating concentrations of SAA were significantly higher at admission in horses with colic attributable to conditions having a primary inflammatory cause (eg, enteritis, colitis, peritonitis, or abdominal abscesses) and were higher in horses that failed to survive the episode of colic, compared with concentrations in horses that survived. Serum concentrations of LBP did not correlate with survival. Analysis of these findings suggests that evaluation of SAA concentrations may be of use in identifying horses with colic attributable to diseases that have inflammation as a primary component of pathogenesis.     

Use of serum amyloid A in equine medicine and surgery

Serum amyloid A (SAA) has become an indispensable part of the management of equine patients in general practice and specialized hospital settings. Although several proteins possess acute phase properties in horses, the usefulness of SAA exceeds that of other acute phase proteins. This is due to the highly desirable kinetics of the equine SAA response. SAA concentrations exhibit a rapid and pronounced increase in response to inflammation and a rapid decline after the resolution of inflammation. This facilitates the detection of inflammatory disease and real-time monitoring of inflammatory activity. SAA may be used in all stages of patient management: (1) before diagnosis (to rule in/rule out inflammatory disease), (2) at the time of diagnosis (to assess the severity of inflammation and assist in prognostication), and (3) after diagnosis (to monitor changes in inflammatory activity in response to therapy, with relapse of disease, or with infectious/inflammatory complications). By assessing other acute phase reactants in addition to SAA, clinicians can succinctly stage inflammation. White blood cell counts and serum iron concentration change within hours of an inflammatory insult, SAA within a day, and fibrinogen within 2–3 days; the interrelationship of these markers thus indicates the duration and activity of the inflammatory condition. Much research on the equine SAA response and clinical use has been conducted in the last decade. This is the prerequisite for the evidence-based use of this analyte. However, still today, most published studies involve a fairly low number of horses. To obtain solid evidence for use of SAA, future studies should be designed with larger sample sizes.     

Pulmonary ultrasonographic abnormalities associated with naturally occurring equine influenza virus infection in standardbred racehorses

The purpose of this investigation was to determine if naturally occurring acute infectious upper respiratory disease (IRD) caused by equine influenza virus is associated with ultrasonographically detectable pleural and pulmonary abnormalities in horses. Standardbred racehorses were evaluated for signs of IRD, defined as acute coughing or mucopurulent nasal discharge. For every horse with IRD (n = 16), 1 or 2 horses with no signs of IRD and the same owner or trainer (n = 30) were included. Thoracic ultrasonography was performed within 5-10 days of the onset of clinical disease in horses with IRD. Horses without IRD were examined at the same time as the horses with IRD with which they were enrolled. The rank of the ultrasound scores of horses with IRD was compared to that of horses without IRD. Equine influenza virus was identified as the primary etiologic agent associated with IRD in this study. Mild lung consolidation and peripheral pulmonary irregularities were found in 11 (69%) of 16 of the horses with IRD and 11 (37%) of 30 of control horses. Lung consolidation (median score = 1) and peripheral irregularities scores (median score = 1) were greater in horses with IRD compared to horses without IRD (median score = 0; P < .05). Pleural effusion was not observed. Equine influenza virus infection can result in abnormalities of the equine lower respiratory tract. Despite the mild nature of IRD observed in this study, lung consolidation and peripheral pulmonary irregularities were more commonly observed in horses with clinical signs of IRD. Further work is needed to determine the clinical significance of these ultrasonographic abnormalities.     

The effects of equine rhinovirus, influenza virus and herpesvirus infection on tracheal clearance rate in horses.

The response of horses exposed to three common respiratory viruses was studied by measuring tracheal mucociliary clearance rates in the trachea. Tracheal clearance rates (TCR) were determined before, during illness and following recovery in horses exposed to equine rhinovirus (ERhV-2), equine influenza virus (EIV) and equine herpesvirus (EHV-4) by means of lateral scintigraphs made following an injection of technetium-99m sulphide colloid into the tracheal lumen. In six horses exposed to ERhV-2, TCR remained within normal limits. Exposure to EIV resulted in reduced TCR in six of seven horses, with TCR remaining below the 95% confidence limits of normal values for each horse for up to 32 days despite the resolution of clinical signs. Moderate changes were observed in six horses exposed to EHV-4, but significant reductions in TCR were evident in three animals. Measurement of TCR was a useful, minimally-invasive technique which demonstrated that respiratory viruses may cause persistent changes in TCR, even though clinical signs are not evident.     

Surveillance programme for important equine infectious respiratory pathogens in the USA

The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.     

Surveillance of equine respiratory viruses in Ontario

The objective of this project was to develop and implement an active surveillance program for the early and rapid detection of equine influenza viruses in Ontario. For this purpose, from October 2003 to October 2005, nasopharyngeal swabs and acute and convalescent serum samples were collected from 115 client-owned horses in 23 outbreaks of respiratory disease in Ontario. Sera were paired and tested for antibody to equine influenza 1 (AE1-H7N7), equine influenza 2 (AE2-H3N8), equine herpesvirus 1 and 4 (EHV1 and EHV4), and equine rhinitis A and B (ERAV and ERBV). Overall, the cause-specific morbidity rate of equine influenza virus in the respiratory outbreaks was 56.5% as determined by the single radial hemolysis (SRH) test. The AE2-H3N8 was isolated from 15 horses in 5 outbreaks. A 4-fold increase in antibody levels or the presence of a high titer against ERAV or ERBV was observed in 10 out of 13 outbreaks in which AE2-H3N8 was diagnosed as the primary cause of disease. In conclusion, AE2-H3N8 was found to be an important contributor to equine respiratory viral disease. Equine rhinitis A and B (ERAV and ERBV) represented an important component in the equine respiratory disease of performing horses.     

Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis

The aim of the study was to determine the intraarticular serum amyloid A (SAA) response pattern in horses with inflammatory arthritis. Inflammatory arthritis was induced by injection of lipopolysaccharide (LPS) into the radiocarpal joint of four horses. Serum and synovial fluid (SF) samples were collected before and at 4, 8, 12, 24, 48, 72, 96, and 144 h after injection. Concentrations of SAA were measured by immunoturbidometry, and expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. The LPS injection caused systemic and local clinical signs of inflammation. Serum amyloid A appeared in serum and SF within 8 h after LPS injection. Isoelectric focusing showed three major SAA bands with apparent isoelectric points (pI) of 7.9, 8.6, and >9.3 in serum and SF. Synovial fluid contained two additional isoforms with highly alkaline apparent pI values (apparent pI value extrapolated from standard curve = 10.0 and 10.2), which were not present in any of the serum samples. In conclusion, intraarticular injection of LPS induced systemic and local inflammatory responses in the horses. By demonstrating SF-specific SAA isoforms the results of the present study suggest that SAA is synthesized locally in the equine inflamed joint, similar to what has been demonstrated in humans previously. The marked local SAA synthesis suggests an important pathophysiological role in inflammatory arthritis.     

Administration of Perioperative Penicillin Reduces Postoperative Serum Amyloid A Response in Horses Being Castrated Standing

Objectives: To compare postoperative inflammatory responses in horses administered perioperative procaine penicillin and those not administered penicillin using acute phase protein serum amyloid A (SAA) as a marker of inflammation. Study Design: Randomized clinical trial. Animals: Stallions (n=50) castrated under field conditions. Methods: SAA concentrations were determined on days 0, 3, and 8. Six horses were subsequently excluded because of elevated SAA concentrations on day 0. Of the remaining 50 horses, 26 were administered nonsteroidal anti‐inflammatory drug (NSAID) therapy and 24 were administered NSAID and 25,000 U/kg procaine penicillin on day 0, 1, and 2. Results: SAA concentrations increased significantly from preoperative levels in both groups, and on day 8 concentrations were significantly (P<.02) higher in horses administered only NSAID than in those administered procaine penicillin and NSAID. Infectious complications occurred more frequently (P<.01) in horses with preoperatively elevated SAA concentrations (the excluded horses) than in horses with normal preoperative SAA concentrations (the included horses). Conclusions: Perioperative antimicrobial therapy reduced the postoperative SAA response, suggesting that bacteria were present in the surgical wound and contributed to inflammation after castration. Horses with elevated preoperative SAA concentrations developed infectious complications more often than horses with normal preoperative SAA concentrations. Clinical Relevance: Administration of antimicrobials may be important in horses being castrated standing under field conditions. Increased SAA concentrations seem to be an indicator of increased surgical risk in horses and may be useful before elective surgery for planning.     

Ataxia and paresis with equine herpesvirus type 1 infection in a herd of riding school horses

An outbreak of neurologic disease associated with serologic evidence of equine herpesvirus type 1 (EHV-1) infection occurred in a herd of 46 riding school horses. Ataxia and paresis were observed in 14 geldings and 5 barren mares. Eight affected horses had distal limb edema, 1 horse had a head tilt, and 3 others had urinary incontinence. Other clinical signs included fever, depression, and inappetance in 30 horses. Seven horses with neurologic signs were treated with acyclovir. Serum neutralizing antibody titers against EHV-1 increased 4-fold between acute and convalescent samples or exceeded 1: 256 in 19 of 44 horses, confirming recent infection. A significantly greater proportion of horses that seroconverted were mares ( P = .014). Of the 19 horses exhibiting ataxia and paresis, 17 made a complete recovery, 1 made a partial recovery, and 1 was euthanized.     

Longitudinal study describing the clinical signs observed in horses naturally infected with equine influenza

We describe the clinical signs and disease course during an outbreak of equine influenza (EI) in na?ve horses in a police stables in Sydney, New South Wales, Australia.