Elimination of guttural pouch infection and inflammation in asymptomatic carriers of Streptococcus equi

Three protracted outbreaks of strangles were investigated using endoscopic examination and a total of 14 asymptomatic carriers of Streptococcus equi were identified of which 13 showed evidence of carriage in the guttural pouch. Treatment was initiated to eliminate S. equi colonisation since these animals posed an infectious risk to susceptible horses. Two further horses were referred to us with severe guttural pouch pathology and from which S. equi was cultured, and treatment of these cases is also described. Treatment in the first instance was directed towards removal of gross guttural pouch pathology as seen on endoscopic examination. This was done with a combination of irrigation of the pouch with moderate to large amounts of saline, suction of fluid material and endoscopic manipulation of chondroids. Subsequently, antibiotic treatment was used to eliminate S. equi infection. All animals received systemic antibiotics, in some cases combined with topical antimicrobial treatment. Treatment was generally regarded as successful when the guttural pouches appeared normal and S. equi was not detected in nasopharangeal swabs and pouch lavages on 3 consecutive occasions. Successful treatment of one carrier required surgical intervention due to occlusion of both guttural pouch pharyngeal openings. Fourteen of 15 carriers were successfully treated by endoscopic removal of inflammatory material and antibiotic treatment, without surgical intervention. Five carriers originally given potentiated sulphonamide (33%) required further therapy with penicillin or ceftiofur, administered both systemically and topically, before S. equi infection and associated inflammation of the guttural pouches were eliminated.     

Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi

Previous use of repeated nasopharyngeal swabbing and culture of Streptococcus equi showed that healthy carriers developed in more than 50% of 'strangles' outbreaks. The guttural pouches were the only detectable site of S. equi colonisation on endoscopic examination of horses during one of these outbreaks and S. equi was sometimes not detected by culture of nasopharyngeal swabs from carriers for up to 2 or 3 months before nasal shedding resumed sporadically. A more sensitive way of detecting S. equi on swabs from established guttural pouch carriers was therefore required. Conveniently selected 'strangles' outbreaks were investigated in detail using endoscopy, in order to develop and assess a suitable polymerase chain reaction (PCR) test. We report here 3 protracted 'strangles' outbreaks on different kinds of establishments in which between 29 and 52% of sampled horses were infected as detected by culture and/or PCR. Of the infected horses, between 9 and 44% were identified as carrying S. equi after clinical signs had disappeared and the predominant site of carriage was the guttural pouch. Prolonged carriage of S. equi, which lasted up to 8 months, did not cease spontaneously before treatment was initiated to eliminate the infections. The detection and isolation of the carriers, in conjunction with strict hygiene measures, apparently resulted in the control of the outbreaks and allowed the premises to return to normal activity. Comparing PCR and culture, many more swabs were found to be positive using PCR (56 vs. 30% of 61 swabs). Similar results were obtained for guttural pouch samples from 12 established carriers (PCR 76% and culture 59%). These results from repeated samples from relatively few animals need confirming using more long-term carriers. PCR can also detect dead organisms and is, therefore, liable to yield false positive results. Despite this drawback, it is argued that PCR provides a potentially useful adjunct to culture of nasopharyngeal swabs in the detection of asymptomatic carriers of S. equi following outbreaks of 'strangles'.     

Lack of correlation between antibody titers to fibrinogen-binding protein of Streptococcus equi and persistent carriers of strangles.

Previously published studies have neither used nor reported the results of an indirect enzyme-linked immunosorbent assay (iELISA) to measure serologic responses in natural outbreaks of strangles. The concept of using serologic responses to identify persistent carriers of Streptococcus equi has been proposed but not scientifically evaluated. The specific aims of the current study were to determine the duration and level of truncated fibrinogen-binding protein-specific (SeM allele 1) antibody production in ponies involved in a natural outbreak of strangles and to determine if test results from this serologic iELISA could predict persistent carrier status. Serologic samples were obtained before and after an outbreak of naturally occurring strangles infection. Persistent carriers of S. equi were identified via culture and polymerase chain reaction (PCR) testing of lavage fluid collected from the guttural pouches and nasopharynx or swabs of the nasopharynx after recovery from acute disease and at postmortem examination. Logistic regression analysis was used to determine if an association existed between serologic response and persistent carrier state. The ELISA reported in the current study definitively confirmed a recent exposure to S. equi. However, the measured serologic response did not predict carrier status in this strangles outbreak. Therefore, a guttural-pouch endoscopy with subsequent culture or PCR testing to detect S. equi remains the most accurate method available for the identification of persistent carriers.     

Standing surgical removal of inspissated guttural pouch exudate (chondroids) in ten horses

OBJECTIVE: To report use of a modified Whitehouse approach in standing horses for management of inspissated guttural pouch empyema. STUDY DESIGN: Retrospective study. ANIMALS: Adult horses (n=10) with guttural pouch empyema. METHODS: Inspissated exudate in 1 or both guttural pouches was removed surgically through a modified Whitehouse approach, with the horses standing and sedated. Medical records of affected horses were reviewed to determine history; physical, endoscopic, and radiological examination findings; surgical technique; complications, and outcome. RESULTS: All horses had purulent nasal discharge; 3 horses had dysphagia, 2 had recurrent laryngeal neuropathy on the side affected by guttural pouch empyema, and 1 had persistent soft palate displacement. Inspissated exudate was removed safely without causing apparent discomfort. Eight horses returned to their previous level of athletic activity after surgery; 1 horse dysphagic before surgery, was euthanatized because of persistent dysphagia after surgery, and 1 horse died 1 week after surgery for unknown reasons. Streptococcus equi subsp equi was isolated from the affected guttural pouch of 3 horses. CONCLUSIONS: Inspissated exudate can be removed surgically from the guttural pouch in standing horses through a modified Whitehouse approach. CLINICAL RELEVANCE: To eliminate risks associated with general anesthesia and avoid surgical suite contamination, removal of chondroids can be performed in standing sedated horses through a modified Whitehouse approach.     

Clinical and epidemiological investigation of chronic upper respiratory diseases caused by beta-haemolytic Streptococci in horses

An outbreak of strangle-like disease involving 26 horses farmed in central Italy was investigated by clinic examination, endoscopy, cytology, bacteriology and polymerase chain reaction (PCR). At weekly interval, a total of three nasal swabs and one guttural pouches lavage fluid (GPLF) were collected, and no Streptococcus equi subsp. equi carrier was found. Some horses showed upper airways disease and endoscopic signs of pharyngeal lymphoid hyperplasia of different grade and/or abnormal endoscopic appearance of guttural pouches. Streptococcus dysgalactiae subsp. equisimilis was isolated from 14 horses while S. equi subsp. zooepidemicus was isolated from six horses. PCR confirmed the biochemical and serological identification of all isolates and was positive in 10 bacteriological negative samples. The absence of S. equi and the frequent detection of S. equisimilis and S. zooepidemicus suggest that beta-haemolytic streptococci other than S. equi could be the causative agent of strangle-like disease.     

Cystic structures in the guttural pouch (auditory tube diverticulum) of two horses.

Two horses were examined for compression of the pharynx from the dorsal pharyngeal wall. Neither horse had a patent opening of 1 of the guttural pouches. Radiography of the guttural pouch region revealed a retropharyngeal opacity that occluded 1 guttural pouch. Organisms were not isolated on bacteriologic culture of fluid obtained from the affected guttural pouch. Surgical exploration of the guttural pouch revealed the lining to be easily removeable by blunt dissection in 1 horse; however, the lining was more firmly attached and removal was not attempted in the second horse. A fenestration between the normal and affected guttural pouch was created in both horses to allow for drainage. The etiopathogenesis of the cyst in the guttural pouches is unknown.     

Molecular epidemiology of strangles outbreaks in the UK during 2010

The sequence of the Streptococcus equi subspecies equi (S equi) M-like protein (SeM) gene was determined for 105 isolates of S equi from strangles outbreaks in the UK during 2010 and compared with previous data from 2007 to 2008. Twenty-three distinct alleles were identified, including 11 novel alleles. One allele giving rise to a putative truncated M protein was identified from the guttural pouch of an asymptomatic carrier. Allele 9 was the most prevalent, comprising 57.7 per cent of isolates, followed by allele 6 (10.3 per cent). Significant changes in allele prevalence were found between 2007, 2008 and 2010, with an increasing prevalence in SeM-9-related alleles and a corresponding decreasing prevalence in SeM-6-related alleles observed over the period (P<0.001). Geographical proximity of outbreaks caused by some uncommon alleles was apparent between 2007, 2008 and 2010.     

In vivo pathogenicity and resistance to phagocytosis of Streptococcus equi strains with different levels of capsule expression.

The glossy non-encapsulated strain of Steptococcus equi, NCTC 9682, was compared with the matt strain Hidaka/95/2 which expresses a medium sized capsule and with the mucoid CF32 which expresses a large sized capsule in phagocytosis assays and for virulence in inoculated horses. The three strains, NCTC 9682, Hidaka /95/2 and CF32 produced 2.0, 3.1, and 5.3 mg/g wet cells respectively after 3 h incubation, but similar amounts of M-like proteins, cytotoxin and mitogen. NCTC 9682 showed no resistance to phagocytosis by equine neutrophils regardless of the presence of opsonin while strains Hidaka /95/2 and CF32 showed almost complete resistance to phagocytosis. Furthermore, NCTC 9682 produced no clinical disease although it infected the guttural pouch and caused seroconversion. Typical strangles with guttural pouch invasion was observed in all horses infected with encapsulated strains.     

The mystery of fungal infection in the guttural pouches

Advances in the understanding of guttural pouch physiology and novel therapeutic approaches to mycotic infections in the horse are reviewed. It is suggested that the guttural pouches may contribute to the regulation of arterial blood temperature, cooling the circulation to the brain to below body temperature. Aspergillus spp. is the major organism found in a guttural pouch affected with mycosis but it is unclear why this agent becomes aggressive. Conventional therapy aims to prevent fatal haemorrhage and to treat any neurological lesions but it is desirable to try to prevent the disease. A technique consisting of inserting a transarterial coil into the internal carotid, external carotid and maxillary arteries in normal and affected horses has been reported to be rapid, safe and effective in occluding the arteries and in inducing regression of the mycotic lesions without adjunctive medical treatment. When faced with acute and uncontrollable epistaxis in the field, the most effective means to reduce haemorrhage is probably the occlusion of both common carotid arteries. However, how such arterial occlusions can result in the successful management of guttural pouch mycosis without antifungal medication remains a mystery.     

Empyema of the guttural pouch (auditory tube diverticulum) in horses: 91 cases (1977-1997)

OBJECTIVE: To identify features of guttural pouch (auditory tube diverticulum) empyema in horses and compare findings of uncomplicated guttural pouch empyema with guttural pouch empyema complicated by chondroids. DESIGN: Retrospective study. ANIMALS: 91 horses with guttural pouch empyema. PROCEDURE: Medical records of horses with guttural pouch empyema were reviewed. RESULTS: The most common owner complaint and abnormal finding was persistent nasal discharge. Chondroids were detected in 21% (19/91) of affected horses. Streptococcus equi was isolated from the guttural pouch in 14 of 44 horses; for Streptococcus spp, in vitro resistance to sulfadimethoxine and trimethoprim-sulfamethoxazole was detected. Retropharyngeal swelling and pharyngeal narrowing were significantly more prevalent in horses with chondroids, compared with horses with uncomplicated empyema. Ninety-three percent of affected horses were discharged from the hospital; at time of discharge, 66% had complete resolution of disease, 19% had improvement without resolution, and 15% did not have improvement. CONCLUSIONS AND CLINICAL RELEVANCE: Horses with persistent nasal discharge should be examined endoscopically for guttural pouch empyema. Treatment with lavage offers a good prognosis for resolution of uncomplicated guttural pouch empyema. Aggressive treatment with lavage and endoscopic snare removal of chondroids offers a good prognosis and may make surgical intervention unnecessary.     

Lessons learned from a strangles outbreak on a large Standardbred farm

Streptococcus equi subspecies (ssp.) equi infection (strangles) remains one of the most frequently diagnosed and costly infectious diseases of horses. Large breeding herds, where a disease outbreak competes for personnel and financial resources needed for foaling management, present a special challenge for equine practitioners. A 15‐month outbreak involving 62 clinical cases of strangles occurred on a large Standardbred breeding farm (average population of 1400 horses). Sixteen asymptomatic horses were found to be PCR (polymerase chain reaction)‐positive for S. equi ssp. equi. During the outbreak, serological samples from 48 clinically normal horses were found to be seropositive for S. equi ssp. equi, confirming herd‐wide exposure. After several clinical cases of strangles had been diagnosed, an intranasal S. equi ssp. equi vaccine was administered to clinically normal horses (n = 558) considered to be at risk of exposure. Strangles complications included 7 fatalities (none in vaccinated horses) and 6 cases of purpura haemorrhagica (4 in vaccinated horses). Midway through the outbreak, injectable, sustained release ceftiofur crystalline free acid (CCFA), given as an initial dose followed by a second dose 4 days later, was used exclusively for systemic antimicrobial treatment of clinically affected and PCR‐positive horses. This antimicrobial regimen coincided with a reduction in disease incidence and eventual resolution of the outbreak. Two horses with persistent guttural pouch infection were endoscopically confirmed as carrier horses. The herd history demonstrated that a strangles outbreak will often result in asymptomatic carrier horses and that identification and treatment of these horses are necessary to eliminate long‐term sources of infection. Ceftiofur crystalline free acid was found to be a suitable antimicrobial due to its activity against S. equi ssp. equi and the efficiencies associated with twice parenteral dosing during a 10‐day treatment period. Occurrence of purpura in 4 vaccinated horses suggests that vaccination should be reserved for healthy seronegative horses and avoided during an active outbreak.     

Description of an epizootic and persistence of Streptococcus equi infections in horses

The age-specific attack rates of Streptococcus equi infections of the upper respiratory tract and lymph nodes (strangles) in horses for the different age groups were 17.6% for broodmares, 47.5% for 1-year-old horses, and 37.5% for foals. Streptococcus equi was isolated from nasal, pharyngeal, or lymph node specimens in 31 (60.8%) of 51 sick horses. A male 1-year-old horse, shipped from Kentucky to farm A, was considered to be the index case. Six (19.4%) of 31 horses with strangles remained as shedders of S equi after clinical signs of the disease had ended. Shedders of S equi were not identified from horses that were exposed to infected horses but never developed strangles.     

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.     

Serum bactericidal responses to Streptococcus equi of horses following infection or vaccination

An indirect test based on horse blood was used to study bactericidal responses of the horse to Streptococcus equi following infection or vaccination. Bactericidal antibody appeared in convalescent sera between two and four weeks and high titres were usually attained by eight weeks. Infection without clinical evidence of abscessation was also effective in eliciting strong bactericidal responses. Serum bactericidal activity of horses either recovered from strangles or immunised with commercial bacterin had declined eight months after vaccination. However, horses that developed strangles eight to 10 months after vaccination exhibited rapid and substantial increases in serum bactericidal activity. Groups of yearlings immunised with commercial S equi vaccines consisting either of M protein or bacterin developed clinical strangles within six months of vaccination although the majority of the animals had exhibited strong serum bactericidal activity a few weeks before occurrence of the disease. Similarly, a group of seven yearling ponies hyperimmunised with experimental vaccine, rich in M protein, were found to be highly susceptible to an intranasal challenge of 5 X 10(8) colony forming units of S equi, although their sera exhibited strong bactericidal activity at the time of challenge. These observations suggest that the role of serum bactericidal antibody in protection of the horse against strangles has been overrated.     

Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles

The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.     

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi.     

Complications associated with Streptococcus equi infection on a horse farm

Complications associated with Streptococcus equi infection developed in 15 (20.3%) of 74 horses on one farm included death, guttural pouch empyema, purpura hemorrhagica, upper respiratory tract obstruction, pneumonia, pleuropneumonia, agalactia, mesenteric lymph node abscessation, and periorbital abscessation. Death was attributed to pneumonia in 3 horses and to upper respiratory tract obstruction in 2 horses. One horse was euthanatized because of severe purpura hemorrhagica.     

Equine rhabdomyolysis

A 1.5-year-old Quarter Horse gelding with a history of chronic nasal discharge and leukocytosis presented with signs of increased lethargy and muscular pain. The horse quickly became recumbent and unable to rise and was euthanized due to a poor prognosis. At necropsy, severe bilateral guttural pouch empyema was observed, as well as numerous well-demarcated areas of pallor within the skeletal muscles of all major muscle groups. Polymerase chain reaction testing of the guttural pouch exudate confirmed an infection with Streptococcus equi subsp. equi, and an S. equi-associated immune-mediated rhabdomyolysis was initially considered to be the most likely diagnosis. This report briefly discusses the various etiologies that should be considered in cases of equine myopathy, and it demonstrates the complexity of these poorly understood muscular disorders.     

Evaluation of a nested PCR test and bacterial culture of swabs from the nasal passages and from abscesses in relation to diagnosis of Streptococcus equi infection (strangles)

REASONS FOR PERFORMING STUDY: Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. OBJECTIVES: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. METHODS: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or from abscesses from horses infected with S. equi (diagnostic sensitivity). RESULTS: All 45 S. equi isolates tested positive in the nested PCR, whereas no amplicon was formed when testing the other 120 Lancefield group C isolates. A total of 43 samples were collected from 11 horses showing clinical signs of strangles during the study period. The diagnostic sensitivity for PCR test was 45% and 80% for samples from the nasal passages and abscesses, respectively; the corresponding diagnostic sensitivity for cultivation was 18% and 20%. The diagnostic sensitivity was significantly higher for PCR than for bacterial cultivation. Furthermore, the shedding of S. equi in 2 infected horse populations was evaluated. An intermittent shedding period of S. equi of up to 15 weeks was recorded in this part of the study. It was also shown that shedding of S. equi occurred both from horses with and without clinical signs. CONCLUSIONS AND POTENTIAL RELEVANCE: The nested PCR test represents a species-specific and -sensitive method for diagnosis of S. equi from clinical samples. It may, however, be desirable in future to develop detection methods with high diagnostic sensitivity and specificity without the potential problems inherent in nested PCR.     

Nonsurgical removal of chondroid masses from the guttural pouches of two horses

Chondroid masses were successfully removed from the guttural pouches of 2 horses, using an endoscope, a snare, and a vacuum pump. This technique is an alternative to surgical removal of chondroid masses in patients when basic lavage of the affected guttural pouch is ineffective. Complications were not encountered. Advantages of this snare technique include avoidance of surgery and its potential complications, minimal recovery time, and minimal expense.