Proceeding of the Belgian Equine Practitioners Society

Transcription

1 Proceeding of the Belgian Equine Practitioners Society 15 November 2014 Leuven, Belgium GOLDEN SPONSOR SILVER SPONSOR Next Meeting: Belgian Equine Practitioners Society Study Days 14 Nov Leuven, Belgium Reprinted in the IVIS website with the permission of BEPS

2 NEW INSIGHTS INTO AN OLD DISEASE - STRANGLES MICHAEL HEWETSON, BSC, BVSC, DIPL.ECEIM UNIVERSITY OF HELSINKI ( FINLAND) I. Introduction Strangles is a highly contagious bacterial disease of horses involving the upper respiratory tract and the lymph nodes of the head and neck. Strangles is caused by the obligate β- hemolytic, gram positive cocci Streptococcus equi subspecies equi aka S. equi, and is arguably the most prevalent infectious equine disease worldwide. The disease has been recognized for centuries, and veterinary records describing cases of strangles can be found in the literature as far back as Despite significant advances in diagnosis, treatment and prevention of this disease, it still has a significant morbidity in susceptible populations of horses, ponies and donkeys of all ages, and remains a serious threat to equine populations across the world. Strategies for control rely on early recognition, isolation of suspect or known cases, and careful quarantining of affected premises. Treatment options can be helpful, but the major threat is through subclinical carriers that can harbor the infection in the guttural pouches for many months and sometimes even years. These animals represent an important reservoir of infection in susceptible populations and early identification and effective bacteriological cure of these individuals remains a cornerstone of disease prevention. Attempts to control the disease by vaccination have been largely unsuccessful, possibly because the vaccines are not capable of controlling the disease in the face of a strong epidemiologic challenge. This paper will briefly cover the main presenting features of the disease but will mostly focus on diagnosis, control and management strategies for diseased or susceptible horses. The value of the current vaccination available in Europe will be discussed and the latest information on our understanding of virulence factors will be used to suggest new strategies for the future.

3 II. Epidemiology A. Susceptible animals In strangles outbreaks, the morbidity tends to be very high. All horses (and other equids) are susceptible, although young (1-5 year old) and immunocompromised (e.g. aged) horses are more likely to become infected. Although the morbidity is high, the mortality is low and approximately 75% of horses recovering from the disease will develop solid protective immunity that lasts for up to 5 years 2. Older horses with residual immunity have limited susceptibility and usually develop a milder form of the disease. Colostral antibodies from mares recovered from strangles afford foals protective immunity until weaning 1. The disease does not usually affect other domestic animals; however there is one report of strangles in a dromedary camel! It is important to note that the disease does carry a very low ZOONOTIC potential, with cases reported in elderly immunocompromised patients. B. Transmission S. equi is transmitted (1) via direct contact between infected and susceptible horses (i.e. contact with mucopurulent discharges) or (2) indirectly via contact with fomites (e.g. contaminated stables, fences, equipment, water sources, feed, veterinary equipment etc.). C. Source of infection Infected horses start to shed bacteria 2 to 3 days after developing a fever and should be considered a potential source of infection for at least 4-6 weeks after the clinical signs of strangles have resolved. Be aware that older horses with residual immunity tend to develop a milder atypical form of the disease, but are still able to shed virulent bacteria. These atypical cases can be problematic in a herd outbreak and may act as an ongoing source of infection. Another important source of infection is apparently healthy horses that have recovered from strangles but continue to harbor the organisms in their guttural pouches (or less commonly their sinuses) and infect other horses via normal nasal secretions. These subclinical carriers are very difficult to identify and represent an important reservoir of infection in susceptible populations for many months, and in some cases years.

4 S. equi is not very persistent in the environment and recent research indicates that the survivability of S. equi on surfaces in the outdoor environment is poor, ranging for 1-3 days 3. Moist shady environments away from direct sunlight may allow S. equi to persist longer however, and current recommendations suggest that contaminated paddocks should be rested for at least 4 weeks to before they can be considered non-infectiuous 1, 4. III. Pathogenesis The incubation period following exposure is 3-14 days and the severity of disease appears to be directly related to the duration of exposure and the challenge load (i.e. number of bacteria) 1. S. equi enters the body via the mouth and nostrils and colonizes the epithelial cells and subepithelial follicles of the pharyngeal and tubal tonsils. Translocation of bacteria then occurs to the local lymph nodes that drain the pharyngeal and tonsillar region. A combination of antiphagocytic factors released by the bacteria prevent neutrophils phagocytosing and killing the bacteria, and accumulation of bacteria and large numbers of degenerating neutrophils within the lymph nodes eventually leads to abscess formation and rupture. Strangles primarily involves the guttural pouch and the lymph nodes of the upper respiratory tract (i.e. submandibular and retropharyngeal lymph nodes); however hematogenous or lymphatic metastasis to distant locations may occur, resulting in abscess formation in other lymph nodes and organs of the body. This is commonly known as bastard strangles. The first clinical sign of disease is fever, and infected horses usually begin to shed bacteria 2-3 days later. In most cases, nasal shedding persists for 2 to 3 weeks, at which time systemic and mucosal IgGb and IgA immune responses facilitate mucosal clearance of the bacteria 5. IV. Clinical signs Horses infected with S. equi will typically develop a fever approximately 2-3 days after exposure and will become lethargic, depressed and inappetant. As the disease progresses, bilateral mucopurulent nasal discharge and swelling of the lymph nodes of the upper respiratory tract will develop (usually the submandibular and retropharyngeal lymph nodes). Obvious lymph node swelling and abscessation is usually evident approximately 1-2 weeks after infection. As the lymph nodes enlarge, affected horses may develop a cough, stridor and dyspnoea due to compression of the larynx and trachea, hence the term Strangles. Pharyngitis and compression of the oesophagus may cause dysphagia in some cases.

5 Retropharyngeal lymph nodes may rupture into and cause empyema of the guttural pouch (Figure 1). Figure 1: Endoscopic view of a ruptured retropharyngeal lymphnode in the medial compartment of the guttural pouch causing guttural pouch empyema (Picture: A. Courouce-Malblanc). Following rupture of the abscesses, most horses will make an uneventful recovery. Complications can occur after an acute episode of strangles however, and you should expect that up to 20% of infected horses may develop some form of complication 6, 7. The most common complications include severe dyspnoea necessitating an emergency tracheotomy, dysphagia, guttural pouch empyema and chondroid formation, metastatic abscessation, purpura haemorrhagica, immune mediated myositis and agalactica 6, 7. Atypical cases may also occur in horses with residual immunity. These horses only demonstrate mild or subclinical signs (usually limited to mild mucopurulent nasal discharge) and may be missed if diagnostic sampling is not performed on all exposed horses. V. Diagnosis A. Detection of recent S. equi infection Samples Diagnosis of a recent strangles infection starts with collecting appropriate samples. Nasopharyngeal swabs or washings are collected most commonly, although aspirates and swabs from draining abscesses are also adequate. Endoscopic examination and lavage is ideal, but probably not necessary for acute cases. NOTE: Nasal washes are more effective than swabs in detection of small numbers of S. equi because a greater surface area within the internal nares is sampled. The technique involves instilling about 50 ml of warm normal saline via a 15-cm length of soft rubber tubing (or a sterile uterine pipette) inserted to the level of the nasal canthus and collecting the washings 1. A practical method of collecting the washings is to place a rectal sleeve over the horses s muzzle following instillation of the saline and collecting the fluid in the fingers of the glove. The volume of one finger of the glove is usually sufficient for bacterial culture and qpcr.

6 Gram stain Once the samples have been collected, a gram stain should ideally be performed to identify gram positive cocci in chains. The sample should then be submitted for bacterial culture and PCR. Bacterial culture Bacterial culture has traditionally been considered the GOLD STANDARD for diagnosis of strangles and is required for definitive diagnosis. Samples are plated on Columbia CNA or Trypticase soy agar (TSA) with 5% sheep or horse blood and observed for development of mucoid honey coloured colonies which are characteristic for virulent S. equi 1. Remember that S. equi will not be present on the nasopharyngeal mucosa until 24 to 48 hours after the onset of fever, so false negatives are possible in the early stages of the disease. Furthermore, isolation of β-haemolytic colonies may be confounded by the presence of other bacteria, most notably Streptococcus equi subspecies zooepidemicus and Streptococcus dysgalacticae subspecies equisimilis, which may also lead to false negative results 16. For this reason it is important to ensure that the laboratory that you send your samples to is aware of the need to differentiate between the β-haemolytic streptococci, usually by means of selective sugar fermentation tests (S. equi is unable to ferment lactose and sorbitol). Polymerase chain reaction (PCR) PCR should be used as an adjunct to bacterial culture for the diagnosis of strangles. The first quantitative PCR (qpcr) test for diagnosis of S. equi detected the 5 region of the SeM gene [the gene for the antiphagocytic M protein of S. equi] and was reported to be three times more sensitive than bacterial culture 8, 9. It was later discovered however, that this region of the SeM gene was highly variable, and was deleted in some strains of S. equi isolated from persistently infected carriers 18, leading to the possibility of false negative results. To overcome this problem and improve the sensitivity of the test, genome sequencing data for Streptococcus equi subspecies zooepidemicus and subspecies equi was used to develop a triplex qpcr assay targeting two genes specific to S. equi and a unique base pair control DNA sequence inserted into a pseudogene from a S. zoopeidemicus strain (H70) 16. This qpcr assay is able to provide results within 2 hours, has an overall sensitivity of 93.9% and a specificity of 96.6%. Furthermore, it is able to detect S. equi at levels below the threshold for bacterial culture, even in the presence of contaminating bacteria 16. Interpretation of the test should be made with caution however, as the qpcr assay detects BOTH dead and live

7 bacteria. Because of this fact, false positives are possible and horses that are qpcr positive should ideally be followed up with culture to confirm active infection. Serology An SeM-based indirect enzyme-linked immunosorbent assay (ielisa) marketed by IDvet - diagnostic Veterinaire (IDvet) is currently available for the identification of horses recently exposed to S. equi. This ELISA detects antibodies to the SeM protein, but unfortunately there is considerable overlap between normal and exposed animals, and cross reactivity with the S. zooepidemicus M protein (SzM) has led to an increased likelihood of reporting false positive results 19. For these reasons, the SeM-based ielisa test it is currently NOT considered to be a reliable method. The test may however, still have some value for determining the need for vaccination; identifying predisposition to purpura haemorrhagica and aiding in diagnosis of metastatic abscessation and purpura haemorrhagica 1. To circumvent these problems, the animal health trust (AHT) has developed a new serological test for identification of horses exposed to S. equi using data from the S. equi and S. zooepidemicus genome sequencing projects. This ielisa detects IgG antibodies to two novel S. equi-specific protein fragments (antigens A and C) and reveals recent exposure to S. equi with a reported sensitivity of 93.3% and a specificity of 99.3% 20. When considering serological testing, it is important to remember that it is used primarily for screening purposes and should NOT be considered an alternative to conventional diagnostic techniques. Furthermore, remember that seropositive horses do not necessarily pose a risk to other horses (i.e. the test detects exposure, not active infection) and should always be followed up with bacterial culture and PCR to determine the presence of bacteria. B. Detection of subclinical carriers Following an outbreak of strangles, up to 31% of horses may become asymptomatic carriers 9,10. Affected horses do not demonstrate clinical signs of strangles, but continue to harbour bacteria in the guttural pouches for up to 39 months after the initial infection and represent an important reservoir of infection in susceptible populations 9,11. Detection of these asymptomatic carriers is difficult. Bacteria may be shed intermittently and the sensitivity of bacterial culture from nasopharyngeal swabs can be as low as 45% 11. Detection of subclinical

8 carriers by bacterial culture and qpcr of guttural pouch washings obtained via endoscopy examination and lavage is significantly more sensitive and is currently the diagnostic test of choice to identify subclinical carriers of the disease 1,9. Because of its improved sensitivity, qpcr is a very useful adjunct to bacterial culture for detecting asymptomatic carriers and can also be used to determine the success of elimination of S. equi from the guttural pouch following treatment. Serological methods are not currently considered useful for detecting strangles carriers 21. VI. Control of an outbreak A. Prevent spread of disease In an outbreak situation, it is essential to STOP MOVEMENT of horses to and from the affected premises until it has been determined to be non-infectious. ISOLATE all infected horses on the property immediately and QUARANTINE healthy horses. This is best achieved by establishing distinct dirty and clean geographical areas on the property. Horses are then divided into three groups: 1) infected horses; 2) exposed healthy horses; and 3) non-exposed healthy horses. Infected horses are isolated in the dirty area, while susceptible exposed and non-exposed healthy horses are quarantined separately in the clean area. When setting up the quarantine and isolation facilities, make every effort to ensure that the potential for fomite transmission is not overlooked. Staff should also be designated into dirty and clean groups and it needs to be made clear that staff working in the dirty area must not deal simultaneously with susceptible animals. If this is unavoidable, infected horses should be dealt with after susceptible animals. Everyone handling infected or exposed horses should use barrier precautions (i.e. gowns, gloves, and plastic boots) and disinfection facilities should be provided. Equipment used for an infected horse should ONLY be used on that horse and the stable facilities, vehicles and equipment that has come into contact with infected horses must be cleaned and disinfected (e.g. Virkon, sodium hypochlorite, phenolics etc.). Remember that contaminated paddocks should be rested for at least 4-6 weeks before they can be considered non-infectious and water sources should be cleaned and thoroughly disinfected. Once isolation facilities have been established, test all symptomatic horses to confirm the diagnosis (i.e. culture and qpcr on nasopharyngeal washes). Monitor the rectal temperatures

9 of all healthy susceptible horses twice daily and move any febrile animals into the dirty area immediately. Shedding does not begin until a day or two after onset of pyrexia, so monitoring rectal temperatures is a very effective way of identifying new cases and isolating them before they transmit infection. It is also essential to interview all horse owners on the property to try and establish the source of infection (i.e. identify new or recent introductions, establish movement patterns of infected horses and determine recent contacts with potential carriers or infected horses). B. Establish if convalescing horses are infectious after clinical recovery and identify carriers Isolation and quarantine should be continued until no new cases have occurred for at least 30 days and all isolated horses have tested negative on three nasopharyngeal swabs or washes taken at weekly intervals and submitted for qpcr and bacterial culture 9. Furthermore, all nonaffected horses in the clean area should ideally be screened by ielisa to identify any horses that were inadvertently exposed to S. equi and have not developed clinical signs (i.e. atypical cases) or may be unknown persistently infected asymptomatic carriers. Any seropositive horses should be isolated immediately and tested. Horses that are consistently negative on three consecutive occasions can be removed from isolation. Endoscopy of the URT and guttural pouches should be performed on any horse that continues to test positive, as these may be potential asymptomatic carriers and may require further treatment to prevent spread of disease 9,11. VII. Treatment A. Treatment of horses with acute disease Treatment strategy in cases of acute strangles depends upon the stage and severity of disease 1. Horses with early clinical signs In most cases, it is advisable to let the disease run its course. Mortality is low in uncomplicated cases and most horses will recover over a period of 2 to 3 weeks with supportive therapy. ISOLATE affected horses and provide them with adequate rest in a warm

10 stable, soft palatable food and easy access to food and water. Administration of non-steroidal anti-inflammatories will ameliorate the clinical signs and may hasten recovery. CONTROVERSY exists as to antimicrobial treatment of these horses and there is no data in the literature to support or refute the use of antibiotics in acute cases. Horses that are treated with antibiotics in the acute stages of the disease do not usually develop abscess, BUT may have an increased risk of bacteriemia, septicaemia and metastatic abscessation (no data). The primary concern regarding the use of antibiotics is that they inhibit the development of a protective immunity, and thus these horses remain highly susceptible to re-infection 12. Remember that the majority of horse (75%), that are not given antibiotics will recover uneventfully and will develop a strong immunity that lasts for up to 5 years. Thus, in the author s opinion, antibiotic treatment in uncomplicated cases is NOT necessary and ill advised. Horses with lymph node abscessation In addition to the measures already discussed, horses with visible external lymphadenopathy may benefit from hot packs to enhance abscess maturation (no data) and surgical drainage (Figure 2). If surgical drainage is attempted, it is CRITICAL to wait until the abscess has matured and thinned out ventrally. Once opened, twice daily flushing with 3-5% iodine is recommended. Figure 2: Purulent exudate draining from a submandibular abscess following surgical drainage (Picture: J.M. Betsch). Antibiotic administration at this stage of the disease is contraindicated as it prolongs abscess maturation and rupture 1. If however, the horse remains systemically sick (i.e. persistent fever, anorexia and depression) or retropharyngeal abscesses cause secondary problems (e.g. dyspnoea, dysphagia), prescribe antibiotics. The best first choice antimicrobial is PENICILLIN and most horsed will require a 3 to 5 days course of treatment.

11 B. Treatment of subclinical carriers Treatment of subclinical carriers is dependent upon the consistency and volume of material in pouches (i.e. empyema vs. chondroids). Guttural pouch empyema Repeated LAVAGE of the guttural pouches via rigid or indwelling catheters, administration of benzylpenicillin, and topical administration of 20% acetylcysteine solution is the treatment of choice for guttural pouch empyema. The lavage fluid most commonly used is isotonic saline or polyionic fluid, and it helps to sedate the horse during the procedure so that it lowers its head, facilitating drainage of purulent material through the nostrils. Some clinicians prefer to lavage the guttural pouches under endoscopic guidance and use a suction pump attached to the endoscope to aid the elimination of empyema. Administration of benzylpenicillin [both systemically and directly into the guttural pouches] appears to improve treatment success rates 1. Topical administration of benzylpenicillin directly into guttural pouch using a gelatin-penicillin mix is more effective than a straight aqueous solution as it remain in the pouches for a longer period of time 13. Installation is easiest through a catheter inserted up the nose and endoscopically guided into the pouch opening. The catheter works best with the last 2cm bent at an angle to aid entry under the pouch flap. It is recommended to elevate the horse s head after infusion to prolong contact time in the guttural pouch. Topical installation of 20% acetylcysteine solution is a useful adjunct therapy as it reduces mucous viscosity and facilitates natural drainage of purulent material through the nostrils, however mild irritation of the guttural pouch may occur. NOTE: recipe for making a gelatin/penicillin mix for admnistration into the guttural pouch 13. Weigh out 2 grams of gelatin and add 40 ml sterile water Heat or microwave to dissolve the gelatin Meanwhile add 10 ml sterile water to 10,000,000 iu sodium benzylpenicillin G Cool gelatin and then mix with penicillin solution to make a total volume of 50 ml Dispense into syringes and leave overnight at 48 o C to set

12 Chondroids Chondroids form in the guttural pouch following long term inspissation of purulent material (Figure 3). These chondroids contain large numbers of bacteria and need to be removed. Endoscopic removal using retrieval basket is effective in most cases, however surgical removal using a modified Whitehouse approach or hyovertebrotomy may ne necessary in some cases. Figure 3: Endoscopic view of the guttural pouch. A single large chondroid is visible (Picture: J.M. Betsch). VIII. Prevention A. Quarantine and bacteriological screening Quarantine and bacteriological screening remains a cornerstone of disease prevention. All new introductions to a property housing susceptible horses should be QUARANTINED for 3-4 weeks and monitored for clinical signs. Bacteriological screening using three consecutive negative PCR tests on nasopharyngeal washes or swabs taken at weekly intervals will increase the likelihood of detecting asymptomatic carriers 9. Any horses that are PCR positive must undergo endoscopic examination and should test negative for a guttural pouch PCR wash and bacterial culture before being considered non-infectious. Serological methods are also available to identify horses that have been exposed to strangles and may provide an affordable alternative to bacteriological screening. The animal health trust (AHT) has developed an indirect enzyme-linked immunosorbent assay (ielisa) test that can detect recent exposure to S equi with a reported sensitivity of 93.3% and a specificity of 99.3% 20. Because of the increased likelihood of reporting false positive results due to cross reactivity with the S. zooepidemicus M protein (SzM), the SeM-based ielisa marketed by

13 IDvet is not considered to be a reliable method 19. New introductions to a property should be screened using the AHT ielisa, and any horses that have a positive test must undergo endoscopic examination and should test negative for a guttural pouch PCR wash and bacterial culture before being considered non-infectious. Unfortunately prevention through quarantining and bacterial screening is particularly difficult where there is frequent moving and mixing of horses and remains one of the biggest challenges for prevention of strangles outbreaks. B. Vaccination There is currently one licensed S. equi vaccine in Europe. This is a live deletion mutant vaccine strain TW 928 which is marketed by Intervet (MSD animal health) as Equilis StrepE 14. During initial trials, administration of the vaccine intramuscularly conferred 100% protection from subsequent challenge, however severe injection site reactions precluded the use of this route. Subsequent testing determined that submucosal vaccination in the upper lip was safe and conferred 50% protection from development of lymph node abscesses following an intranasal S. equi challenge; and a further 25% of vaccinates had reduced clinical signs of disease. Unfortunately the vaccine will only provide short term immunity (approximately 3 months), and thus it is only of value in situations where healthy susceptible horses are at a high risk of contracting the disease and require a short duration of immunity (e.g. co-mingling at shows and competitions, outbreak situations). The vaccine should only be administered to healthy horses and is safe to use from 4 months of age. NOTE: The basic vaccination schedule for Equilis StrepE is two submucosal vaccinations four weeks apart. Horses in high-risk situations should be re-vaccinated with a single dose every three months. For horses in medium-risk situations re-vaccination every six months rather than every three months can be considered. If horses are re-vaccinated every six months, a prompt booster with a single dose should be given if an outbreak of strangles occurs more than three months after the last booster. Horses that have been vaccinated within the last 3 months do not require another booster in an outbreak situation. Vaccination of horses during an outbreak is controversial. In the author s opinion, vaccination of healthy horses that are at risk of strangles during an outbreak may be of benefit, but remember that an effective immune response to the vaccine requires 7-10 days and NO

14 vaccine has any guarantee. Furthermore, it is NOT recommended to vaccinate horses recovering from infections for 1-2 years due to the risk of purpura haemorrhagica. Future developments The results of recent studies using multilocus enzyme electrophoresis suggest that S. equi evolved from an archetype of S. zoopeidmicus 15. Despite this close relationship, S. zooepidemicus does not stimulate immunity that is cross-protective, and so researchers are focusing on identifying immunogens that are expressed by S. equi but are absent from S. zooepidemicus. This research has been greatly facilitated by the ongoing S. equi and S. zooepidemicus genome sequencing projects at the Welcome Trust Sanger Institute ( and Once S. equi specific immunogens are identified, future vaccine development can be focused on multi-component subunit vaccines that make use of several immunogenic S. equi surface proteins. Theses vaccine would offer an alternative to live attenuated vaccines with greater efficacy and fewer side effects. NOTE: Authorities believe we can eliminate S. equi as it has all the characteristics of an infection that can be eradicated. These characteristics include infection limited to horses, poor survivability in the environment, little to no antigenic variability, low carrier rates and the availability of both a PCR for detection, and moderately effective vaccines that could be used in combination with an eradication program. IX. Selected References 1. Sweeney, C.R., Timoney, J.F., Newton, J.R., Hines, M.T. Streptococcus equi infections in horses: guidelines for treatment, control, and prevention of strangles. J Vet Int Med. 2005; 19: Hamlen H.J., Timoney J.F., Bell R.J. Epidemiologic and immunologic characteristics of Streptococcus equi infection in foals. J Am Vet Med Assoc. 1994;204: Weese J.S., Jarlot C., Morely P.S. Survival of Streptococcus equi on surfaces in an outdoor environement. Canadian Vet Med Assoc. 2009;50 (9):

15 4. Jorm LR. Laboratory studies on the survival of Streptococcus equi subspecies equi on surfaces. In: Plowright W, Rossdale PD, Wade JF, eds. Proceedings of Equine Infectious Diseases VI. Newmarket, UK: R & W Publications Ltd; 1992: Galan JE, Timoney JF. Mucosal nasopharyngeal immune response of the horse to protein antigens of Streptococcus equi. Infect Immun. 1985;47: Sweeney CR, Whitlock RH, Meirs DA, et al. Complications associated with Streptococcus equi infection on a horse farm. J Am Vet Med Assoc 1987;191: Ford J, Lokai MD. Complications of Streptococcus equi infection. Equine Pract. 1980;4: Timoney JF, Artiushin SC. Detection of Streptococcus equi in equine nasal swabs and washes by DNA amplification. Vet Rec. 1997; 141: Newton JR, Verheyen K, Talbot NC, et al. Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi. Equine Vet J. 2000;32: Knowles EJ, Mair TS, Butcher AS, Waller AS, Wood JLN. Use of a novel serological test for exposure to Streptococcus equi subspecies equi in hospitalixed horses. The Vet Rec. 2010;166: Newton, J.R., Wood, J.L.N., Dunn, K.A., DeBrauwere, M.N. and Chanter, N. Naturally occurring persistent and asymptomatic infection of the guttural pouches of horses with Streptococcus equi. Vet Rec. 1997;140: Piche, C.A. Clinical observations on an outbreak of strangles. Can. vet. J. 1984;25: Verheyen K, Newton JR, Talbot NC, et al. Elimination of guttural pouch infection and inflammation in asymptomatic carriers of Streptococcus equi. Equine Vet. J. 2000; 32: Hartford, O.M., Foster, T.J., Jacobs, A.A.C., Streptococcus equi vaccine. United States Patent 5,895,654. Application Number: Filed: January 27, Jorm, L.R., Love, D.N., Bailey, G.D., McKay, G.M., Briscoe, D.A. Genetic structure of populations of beta-haemolytic Lancefield group C streptococci from horses and their association with disease. Res Vet Sci. 1994; 57: Webb K, Barker C, Harrison T, Heather Z, Steward KF, Robinson C, et al. Detection of Streptococcus equi subspecies equi using a triplex qpcr assay. Vet J. 2013; 195(3):

16 17. Chanter N, Talbot NC, Newton JR, Hewson D, Verheyen K. Streptococcus equi with truncated M-proteins isolated from outwardly healthy horses. Microbiology 2000; 146(6): Kelly C, Bugg M, Robinson C, Mitchell Z, Davis-Poynter N, Newton JR, et al. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. J Clin Microbiol. 2006; 44(2): Robinson C, Steward KF, Potts N, Barker C, Hammond TA, Pierce K, et al. Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure. Vet J. 2013; pii: S (13) Davidson A, Traub-Dargatz JL, Magnuson R, Hill A, Irwin V, Newton R, et al. Lack of correlation between antibody titers to fibrinogen-binding protein of Streptococcus equi and persistent carriers of strangles. J Vet Diag Invest. 2008; 20(4):