Successful management of multiple extrapulmonary complications associated with Rhodococcus equi pneumonia in a foal

This report describes the diagnosis and successful treatment of multiple extrapulmonary sequelae of Rhodococcus equi (R. equi) pneumonia in a 3‐month‐old filly. Bilateral uveitis and hyphaema, haemolytic anaemia and polysynovitis developed in this foal and were likely due to immune‐mediated mechanisms. The challenges associated with diagnosis and treatments of these extrapulmonary disorders are discussed. The filly was treated initially with clarithromycin and rifampin; however, a blood transfusion and immunosuppressive therapy with dexamethasone were required due to progressive haemolysis and for treatment of uveitis and polysynovitis. Bilateral hyphaema was successfully treated with intracameral injections of a recombinant tissue plasminogen activator. The development of antimicrobial resistance in R. equi was an additional challenge encountered in the management of this case and emphasises the importance of culture and in vitro antimicrobial susceptibility testing of isolates from foals with R. equi pneumonia. Extrapulmonary disorders associated with R. equi pneumonia are likely underdiagnosed and associated with a poor prognosis. This case highlights the importance of thorough and ongoing diagnostic assessment of foals with R. equi pneumonia and demonstrates that a successful outcome can be achieved with appropriate and directed treatment.     

Sequelae and complications of Streptococcus equi subspecies equi infections in the horse

Streptococcus equi ssp. equi infection in the horse, or strangles, commonly results in abscessation of the submandibular, submaxillary or retropharyngeal lymph nodes. Although this classical presentation of strangles is associated with a low mortality rate, complications and sequlae may worsen the prognosis and increase mortality rates. This article reviews sequelae and complictions of S. equi ssp. equi infection in the horse, including guttural pouch empyema, bastard strangles and immune mediated diseases such as purpura haemorrhagica, myopathies and myocarditis.     

Haemolytic anaemia and bilateral uveitis associated with leptospirosis in a 6‐year‐old Quarter Horse gelding

A 6‐year‐old Quarter Horse gelding was presented for bilateral uveitis resulting in vision loss as well as icterus. Anaemia with autoagglutination was consistent with a presumptive immune‐mediated haemolytic anaemia. Urinary PCR was positive for Leptospira spp. and microscopic agglutination test (MAT) titres were elevated to multiple serovars supportive of a diagnosis of leptospirosis. Treatments included broad spectrum antibiotics and aggressive anti‐inflammatory medications. While the horse was hospitalised, the development of bilateral corneal ulcers precluded the use of topical ophthalmic anti‐inflammatories for a number of days. The corneal ulceration resolved, vision returned in both eyes and the immune‐mediated haemolytic anaemia resolved. After 9 days of hospitalisation, oral minocycline was administered for 2 weeks at home as well as low dose oral flunixin meglumine and topical ophthalmic diclofenac and atropine. This case represents the first published case of haemolytic anaemia associated with leptospirosis in a horse.     

Presumed immune‐mediated hemolytic anemia in two foals with Rhodococcus equi infection

Objective – To describe the clinical presentation, case management, and outcome in 2 foals with Rhodococcus equi infection associated with presumptive severe immune‐mediated hemolytic anemia. Series Summary – Two foals diagnosed with R. equi pneumonia on the basis of tracheal wash cultures, thoracic radiographs, and thoracic ultrasonography were concurrently diagnosed with hemolytic anemia. Both foals required whole blood transfusions, and were treated with the antimicrobial combination of rifampin and a macrolide (eg, clarithromycin, erythromycin, or azithromycin). Dexamethasone was used to prevent further hemolysis in both foals, and to treat acute lung injury/acute respiratory distress syndrome in 1 of the foals. Both foals survived, and required prolonged antimicrobial therapy. New or Unique Information Provided – Although extra‐pulmonary disorders are commonly diagnosed in foals infected with R. equi, hemolytic anemia is rarely described. Dexamethasone is considered the treatment of choice for immune‐mediated hemolytic anemia, but may be contra‐indicated in foals with severe bacterial infections. In these foals, a relatively low dose and short duration of dexamethasone was utilized in an attempt to minimize immune suppression, although early discontinuation in 1 foal precipitated a second hemolytic crisis.     

Characterization of a Streptococcus equi ssp. equi Isolate From a Strangles Outbreak in Thailand

Strangles, which is caused by Streptococcus equi ssp. equi, is one of the major infectious respiratory diseases in horses. Knowledge of isolates from different areas of the world is important for investigating the different strains of the disease. In contrast to many other countries, currently little is known about S. equi ssp. equi isolates in Thailand. In 2014, a farm in Thailand imported 20 horses from Europe. Approximately 1 month after arrival, 50% of the horses had developed pyrexia, mucopurulent nasal discharge, and abscesses of the mandibular lymph nodes. Nasal swabs of mucopurulent discharge were sent to a diagnostic laboratory, and two isolates of S. equi ssp. equi were identified. One of the isolates was further characterized using seM gene polymerase chain reaction and sequence analysis. The seM sequence was then compared to the database of PubMLST-seM. It was found to contain SeM allele 48, an allele isolated from horses in the United Kingdom in 2006 and 2010. This result demonstrates the usefulness of SeM allele identification as a tool for investigating the source of related strains and for the epidemiologic study of strangles. To the best of the authors' knowledge, this is the first report of the identification of an SeM allele of the S. equi ssp. equi isolate in Thailand.     

Rhodococcus equi pneumonia in an adult horse

A 20‐year‐old, Thoroughbred mare in the fifth month of gestation was examined for weight loss, pyrexia and lethargy. Physical examination, ultrasonography and radiography revealed a severe abscessing pneumonia and a dead fetus. The mare did not respond to symptomatic treatment and died suddenly. Necropsy revealed multifocal pulmonary abscessation. Rhodococcus equi was isolated from the lungs, liver and kidneys. Specific immune function of the mare and presence of the virulence associated protein A (VapA) of the R. equi isolated was not determined. It is likely that immunosuppression is required for systemic R. equi infections in adult horses; however, it is unknown if VapA is necessary to produce disease in adult horses.     

Cutaneous Alternaria infectoria infection in a dog in association with therapeutic immunosuppression for the management of immune‐mediated haemolytic anaemia

A 4‐year‐old, ovariohysterectomized, English springer spaniel on immunosuppressive therapy was re‐examined for the review of its immune‐mediated haemolytic anaemia and the recent development of skin lesions. For the 3 months since hospital discharge, the dog had been receiving 1.3 mg/kg prednisolone and 2.6 mg/kg ciclosporin, both administered orally twice daily. Physical examination revealed hepatomegaly and multiple, purulent, crusting, erosive to ulcerative lesions over different body areas. Onychorrhexis had occurred on one digit and the underlying corium had blackened. There were two proliferative and one plaque‐like lesions in the mouth. Thick walled fungal hyphae were detected in impression smears from all skin lesions and staining with periodic acid–Schiff’s stain confirmed the presence of multiple fungal hyphae and spores in all biopsies examined. Fungal culture isolated a heavy, pure growth of an Alternaria sp. which was identified as A. infectoria by sequencing the internal transcribed spacer 1 region of the rRNA gene. The animal’s condition prevented detailed investigation of the oral lesions. Withdrawal of the ciclosporin and reduction of the prednisolone dosage resulted in spontaneous resolution of the skin lesions within 40 days. Further gradual decrements in the prednisolone dosage to zero were carried out without recurrence of the immune‐mediated haemolytic anaemia. After 12 months, there has been no recurrence of either the skin lesions or the anaemia. To the authors’ knowledge, this is the first reported case of A. infectoria infection in a dog.     

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.     

Clinical Evaluation of a Peptide‐ELISA based upon N‐terminal B‐cell Epitope of the VapA Protein for Diagnosis of Rhodococcus equi Pneumonia in Foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide‐based enzyme‐linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence‐associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11‐14. The peptide corresponds to the N‐terminal B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut‐off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA‐specific IgGb antibodies against N‐terminal B‐cell epitope of the VapA protein rather than IgG antibodies. The VapA‐IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6‐week‐old foals showed that the VapA‐IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA‐IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA‐specific IgGb antibodies may be a better predictor of R. equi disease in foals.     

Early pathogenesis of equine Streptococcus equi infection (strangles)

Reasons for performing study: Little is known about entry and subsequent multiplication of Streptococcus equi following exposure of a susceptible horse. This information would have value in design of intranasal vaccines and understanding of shedding and protective immune responses. Objectives: To determine entry points and sites of subsequent replication and dispersion of S. equi at different times after intranasal infection or commingling exposure. Methods: Previously unexposed horses and ponies were subjected to euthanasia 1, 3, 20 or 48 h following intranasal inoculation with biotin labelled or unlabelled S. equi CF32. Some ponies were inoculated with suspensions of equal numbers of CF32 and its mutants lacking capsule, S. equi M‐like protein or streptolysin S. Others were infected by commingling exposure and subjected to euthanasia after onset of fever. Tonsils and lymph nodes were cultured for S. equi and tissues sectioned for histopathological examination and fluorescent microscopy. Results: Tonsillar tissues of both the oro‐ and nasopharynx served as portals of entry. Entry was unexpectedly rapid but involved few bacteria. Small numbers of organisms were detected in tonsillar crypts, in adjacent subepithelial follicular tissue and draining lymph nodes 3 h after inoculation. By 48 h, clumps of S. equi were visible in the lamina propria. At onset of fever, tonsillar tissues and one or more mandibular and retropharyngeal lymph nodes were heavily infiltrated by neutrophils and long chains of extracellular S. equi. Mutant S. equi lacking virulence factors were not seen in draining lymph nodes. Conclusions: Although very small numbers of S. equi entered the lingual and nasopharyngeal tonsils, carriage to regional lymph nodes occurred within hours of inoculation. This observation, together with visual evidence of intracellular and extracellular multiplication of S. equi in tonsillar lymphoid tissue and lymph nodes over the following days, indicates involvement of potent antiphagocytic activity and failure of innate immune defences. Relevance: Future research should logically address the tonsillar immune mechanisms involved including identification of effector cell(s) and antigens.     

Septic tendonitis of the deep digital flexor tendon in a Thoroughbred horse referred for weight loss and intermittent fever

This case report describes the history, clinical and diagnostic findings, treatment and outcome of a 3‐year‐old Thoroughbred gelding. The horse was examined for chronic weight loss, intermittent fever of 4 months' duration and acute lameness of 2 weeks' duration, and was finally diagnosed with septic tendonitis of the deep digital flexor tendon and digital flexor tendon sheath caused by Streptococcus equi ssp. zooepidemicus. Investigation of the lower respiratory tract also allowed isolation of S. equi ssp. zooepidemicus. Although surgical treatment was recommended, because of the severity of the lesion and the guarded prognosis for a return to full athletic function, the owner declined surgery. The horse was therefore treated aggressively with antimicrobials using systemic, local and regional approaches, and with anti‐inflammatory drugs. At 20 months after discharge, the horse was able to race, and he is now in full work with 20 races finished successfully.     

Subscapular lymph node abscessation as a result of metastatic Streptococcus equi subspecies equi infection: An atypical presentation of bastard strangles in a mare

The case reported here represents an atypical presentation of bastard strangles in an 18‐year‐old Arab mare. The horse initially presented for progressive neck pain characterised by reluctance to lateral and ventroflexion of the neck. Subsequent diagnostics revealed a subscapular abscess and aspirates of the mass cultured positive for Streptococcus equi ssp. equi. Surgical drainage and debridement of the abscess was performed under general anaesthesia. Six months post surgery, the mare had made a complete recovery.     

Recognition of a B‐cell Epitope of the VapA Protein of Rhodococcus equi in Newborn and Experimentally Infected Foals

The aim of this study was to evaluate the usefulness of the previously identified B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein of Rhodococcus equi and its association with R. equi pneumonia. A modified peptide designated PN11‐14 corresponding to the epitope was recognized by all sera from experimentally infected foals with virulent R. equi ATCC103⁺ containing the virulence plasmid but not by its plasmid‐cured derivative ATCC103^? strain. Marked levels of VapA‐specific immunoglobulin (Ig)G were detected in all sera from the ATCC103⁺ infected foals at 2 weeks after the infection. One control animal had high titres as determined by the peptide enzyme‐linked immunosorbent assay (ELISA), indicating the ELISA may not absolutely differentiate between foals with R. equi pneumonia and healthy exposed foals in farms where the prevalence of disease is high. However, numbers of animals used were small. Further evaluation of the peptide ELISA with field samples is necessary to determine whether the assay is diagnostically useful. This study showed that levels of passive transfer of maternal IgG antibodies to the epitope in newborn foals could be measured. Interestingly, the maternally derived antibodies were found to significantly (P < 0.05 by Student's t‐test) decline 2 weeks after birth. Seroconversion against naturally occurring VapA expressing R. equi could be detected in some foals at 4 weeks of age. Antibodies to the epitope peaked and were significantly (P < 0.05) greater in foals aged between 6 and 8 weeks. These results indicated that the peptide ELISA could be used to monitor anti‐VapA antibodies in foals, particularly those at the age of 4–6 weeks. It is possible that the ELISA may be of some use as a diagnostic test on farms where R. equi is non‐endemic. Further studies using large number of field samples are needed to verify this assumption.     

Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development

For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular‐based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.     

Molecular characterisation of 'strangles' outbreaks in the UK: the use of M-protein typing of Streptococcus equi ssp. equi

Reasons for performing study: Strangles is the most commonly diagnosed and important infectious disease of horses worldwide. Very little is known about the temporo‐spatial and molecular epidemiology of strangles. The disease is not notifiable in the UK and there are few published data on the geographical locations of outbreaks. Objective: To investigate whether typing of a surface protein (SeM) of Streptococcus equi ssp. equi (S. equi), the causative agent of strangles, is a useful epidemiological tool. Methods: The variable region of the SeM gene was amplified from 145 isolates of S. equi by PCR and sequenced. Different SeM gene alleles were assigned based on the SeM database, grouped into phylogenetic clusters using split decomposition analysis and plotted against the submitting veterinary practices. Results: In this study 21 S. equi SeM alleles were found, including 9 previously unidentified alleles and representing 4 phylogenetic groups. S. equi containing SeM alleles 9 and 7 were the most commonly isolated and there was a high number of low frequency alleles. The occurrence of an outbreak cluster in the north‐west of the UK is also reported. Conclusions: Strangles outbreaks can be differentiated on the basis of their SeM allele sequences. The data provide further evidence of SeM mutation leading to the emergence of novel, but related SeM alleles that are geographically linked. Sequencing of the SeM gene is a useful tool for the elucidation of strangles epidemiology at a regional and a national level. Potential relevance: This technique may allow differentiation or linkage of strangles outbreaks and as such may be an effective tool for local as well as national and international disease surveillance.     

Molecular Typing and Anti‐microbial Susceptibility of Clinical Isolates of Streptococcus equi ssp. zooepidemicus from Equine Bacterial Endometritis

The anti‐microbial susceptibility and genetic diversity of 65 strains of Streptococcus equi ssp. zooepidemicus (Sez) isolated from mares presenting clinical signs of endometritis was determined by disk agar diffusion and pulsed field gel electrophoresis (PFGE) methods, respectively. Overall, Sez isolates were susceptible to β‐lactams, enrofloxacin, trimethoprim‐sulfamethoxazole and gentamicin. These anti‐microbials could be recommended as empiric anti‐microbial therapy in cases of endometritis caused by Sez. Pulsed field gel electrophoresis typing revealed a great genetic diversity (56 different PFGE macrorestriction profiles) and a low level of genetic relatedness amongst the isolates.     

Production of an anticanine globulin (polyspecific) reagent for laboratory investigation

Objectives : Canine allo‐ or autoantibodies are clinically important, but attachment of these immunoglobulin G (IgG) antibodies does not produce observable haemagglutination. Antibody to canine globulins is required to demonstrate sensitisation of red blood cells. Commercial reagents are available, but these often differ in sensitivity and specificity. Rabbit anticanine globulins (polyspecific) were produced for use in canine blood compatibility testing and in the investigation of immune‐mediated haemolytic anaemia. Methods : Canine sera was pooled, IgG was purified and subsequently used to immunise rabbits. A rising titre of anticanine IgG was demonstrated by indirect enzyme‐linked immunosorbent assay. Rabbit anticanine complement was isolated and investigated by agglutination of complement‐coated canine red blood cells. Both antibodies were purified and checked for crossreactivity before being combined to polyspecific anticanine globulins. The obtained reagent was used to indicate sensitised canine red blood cells and free antibodies in three different types of clinical samples, including blood for compatibility testing and that for investigation of immune‐mediated haemolytic anaemia and screening for post‐transfusion alloantibodies and was also compared to commercial Coombs’ reagent. Results : The product provided results in accordance with those from commercial Coombs’ reagent. The sensitivity for canine crossmatching was 100% and specificity for diagnosing immune‐mediated haemolytic anaemia was 87%. Clinical Significance : This product is helpful for canine crossmatching purposes and in the investigation of immune‐mediated haemolytic anaemia.     

Rhodococcus equi foal pneumonia: Update on epidemiology,immunity, treatment and prevention

Pneumonia in foals caused by the bacterium Rhodococcus equi has a worldwide distribution and is a common cause of disease and death for foals. The purpose of this narrative review was to summarise recent developments pertaining to the epidemiology, immune responses, treatment, and prevention of rhodococcal pneumonia of foals. Screening tests have been used to implement earlier detection and treatment of foals with presumed subclinical R. equi pneumonia to reduce mortality and severity of disease. Unfortunately, this practice has been linked to the emergence of antimicrobial-resistant R. equi in North America. Correlates of protective immunity for R. equi infections of foals remain elusive, but recent evidence indicates that innate immune responses are important both for mediating killing and orchestrating adaptive immune responses. A macrolide antimicrobial in combination with rifampin remains the recommended treatment for foals with R. equi pneumonia. Great need exists to identify which antimicrobial combination is most effective for treating foals with R. equi pneumonia and to limit emergence of antimicrobial-resistant strains. In the absence of an effective vaccine against R. equi, passive immunisation remains the only commercially available method for effectively reducing the incidence of R. equi pneumonia. Because passive immunisation is expensive, labour-intensive and carries risks for foals, great need exists to develop alternative approaches for passive and active immunisation.     

Genetic Susceptibility to Rhodococcus equi

Rhodococcus equi pneumonia is a major cause of morbidity and mortality in neonatal foals. Much effort has been made to identify preventative measures and new treatments for R. equi with limited success. With a growing focus in the medical community on understanding the genetic basis of disease susceptibility, investigators have begun to evaluate the interaction of the genetics of the foal with R. equi. This review describes past efforts to understand the genetic basis underlying R. equi susceptibility and tolerance. It also highlights the genetic technology available to study horses and describes the use of this technology in investigating R. equi. This review provides readers with a foundational understanding of candidate gene approaches, single nucleotide polymorphism‐based, and copy number variant‐based genome‐wide association studies, and next generation sequencing (both DNA and RNA).     

Lineages of Streptococcus equi ssp. equi in the Irish equine industry

Background

Streptococcus equi ssp. equi is the causative agent of ‘Strangles’ in horses. This is a debilitating condition leading to economic loss, yard closures and cancellation of equestrian events. There are multiple genotypes of S. equi ssp. equi which can cause disease, but to date there has been no systematic study of strains which are prevalent in Ireland. This study identified and classified Streptococcus equi ssp. equi strains isolated from within the Irish equine industry.

Results

Two hundred veterinary isolates were subjected to SLST (single locus sequence typing) based on an internal sequence from the seM gene of Streptococcus equi ssp equi. Of the 171 samples which successfully gave an amplicon, 162 samples (137 Irish and 24 UK strains) gave robust DNA sequence information. Analysis of the sequences allowed division of the isolates into 19 groups, 13 of which contain at least 2 isolates and 6 groups containing single isolates. There were 19 positions where a DNA SNP (single nucleotide polymorphism) occurs, and one 3 bp insertion. All groups had multiple (2–8) SNPs. Of the SNPs 17 would result in an amino acid change in the encoded protein. Interestingly, the single isolate EI8, which has 6 SNPs, has the three base pair insertion which is not seen in any other isolate, this would result in the insertion of an Ile residue at position 62 in that protein sequence. Comparison of the relevant region in the determined sequences with the UK Streptococcus equi seM MLST database showed that Group B (15 isolates) and Group I (2 isolates), as well as the individual isolates EI3 and EI8, are unique to Ireland, and some groups are most likely of UK origin (Groups F and M), but many more probably passed back and forth between the two countries.

Conclusions

The strains occurring in Ireland are not clonal and there is a considerable degree of sequence variation seen in the seM gene. There are two major clades causing infection in Ireland and these strains are also common in the UK.