Severe acute rhabdomyolysis associated with Streptococcus equi infection in four horses

Four Quarter Horses (9 months to 7 years of age) with submandibular lymphadenopathy and firm muscles (palpation of which elicited signs of pain) were evaluated; in general, the horses had a stiff gait, and 3 horses became recumbent. Streptococcus equi was cultured from aspirates of lymph nodes or samples of purulent material collected from the auditory tube diverticula. Once the horses were recumbent, their condition deteriorated rapidly despite aggressive antimicrobial and antiinflammatory treatment, necessitating euthanasia within 24 to 48 hours. One horse did not become recumbent and recovered completely. Among the 4 horses, common clinicopathologic findings included neutrophilia, hyperfibrinogenemia, and high serum activities of creatine kinase and aspartate aminotransferase. Necropsies of the 3 euthanatized horses revealed large, pale areas most prominent in the semimembranosus, semitendinosus, sublumbar, and gluteal muscles that were characterized histologically by severe acute myonecrosis and macrophage infiltration of necrotic myofibers. Streptococcus equi was identified in sections of affected muscle by use of immunofluorescent stains for Lancefield group C carbohydrate and S. equi M protein. In the 4 horses of this report, acute severe rhabdomyolysis without clinical evidence of muscle atrophy or infarction was associated with S. equi infection; rhabdomyolysis was attributed to either an inflammatory cascade resembling streptococcal toxic shock or potentially direct toxic effects of S. equi within muscle tissue.     

Epidemiology of Rhodococcus equi infection in horses

Current understanding of the epidemiology of Rhodococcus equi infection on horse farms is reviewed. Infection is widespread in herbivores and their environment, because herbivore manure supplies the simple organic acid substrates on which the organism thrives. There is a progressive development of infection in the soil on horse farms with prolonged use, because: (1) there is a continual supply of nutrients; (2) the organism multiplies progressively as temperatures rise; (3) the bacterium has a robust nature. While this aerobic organism fails to multiply in the largely anaerobic intestine of the adult horse, multiplication to very large numbers may occur in the intestine of a foal in its first 8-12 weeks of life. Farms used for foal breeding over many years may thus become particularly dangerous for foals. Areas for future study include the effectiveness of decontamination, manure-removal programs and dust reduction in reducing challenge to susceptible foals.     

Experimental Babesia equi infection in mature horses

Nine 4-year-old Arabian geldings were experimentally infected with Babesia equi of European origin. All horses developed detectable parasitemia an average of 30 days after they were inoculated, which was accompanied by a decrease in PCV. The infections were generally mild with no animal deaths. All horses became serologically positive by the indirect fluorescent antibody test within an average of 23 days after they were inoculated and by the complement-fixation test 30 days after they were inoculated.     

IdeE reduces the bactericidal activity of equine neutrophils for Streptococcus equi

Streptococcus equi (S. equi) causes equine strangles, a highly contagious and widespread purulent lymphadenitis of the head and neck. Highly resistant to phagocytosis, it produces long extracellular chains in affected lymph nodes. In a screen of clones reactive with convalescent serum from a gene library of S. equi CF32 we identified IdeE, an IgG-endopeptidase and homologue of the leucocyte receptor Mac-1 (CD11b). IdeE is expressed during S. equi infection eliciting both serum and mucosal antibody responses which persisted at significant levels in serum for over 200 days. Release from S. equi into culture medium was detected during the exponential phase of growth. The closely related Streptococcus zooepidemicus appeared to store the protein but not to release it. Antiphagocytic activity for equine neutrophils was dose-dependent and neutralized by IdeE-specific antiserum. Biotinylated IdeE bound weakly to about 77% of purified equine neutrophils and strongly to the remainder.     

Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection

Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ⁺ CD5⁺T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease.     

Rhodococcus equi: equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56 degrees C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly (P less than 0.01) higher with principal sera (2.4 X 10(5) cpm) than with control sera (0.018 X 10(5) cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly (P less than 0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly (P less than 0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.     

Ulcerative keratitis caused by beta-hemolytic Streptococcus equi in 11 horses

Purpose To describe 11 clinical cases of ulcerative keratitis in horses associated with beta-hemolytic Streptococcus equi in Florida, USA. METHODS: Retrospective clinical study (1996-99). RESULTS: Beta-hemolytic Streptococcus equi was cultured from 11 horses with deep ulcers, descemetoceles or iris prolapse (n = 8), a suture abscess found with a penetrating keratoplasty for a stromal abscess (n = 1), and ulceration that developed following keratectomy/irradiation for corneal squamous cell carcinoma (n = 2). Beta-hemolytic Streptococcus equi subspecies zooepidemicus was found in 10 eyes and subspecies equi in one. Marked signs of uveitis including miosis and hypopyon were present in 8/11 (72.7%) eyes. Keratomalacia was severe in all eyes. The mean diameter of the ulcers associated with beta-hemolytic Streptococcus was 10.2 +/- 6.1 mm. Eight of the eyes required conjunctival flap surgery (four grafts dehisced) and one eye corneal transplantation. Two eyes were treated with medication only. Isolate sensitivity to antibiotics included ampicillin (6/11), bacitracin (11/11), cephalothin (11/11), chloramphenicol (11/11), gentamicin (5/11), polymyxin B (2/11), and tobramycin (1/11). All isolates were resistant to neomycin. The average healing time was 44.7 +/- 26.7 days. The visual outcome was positive in 8/11 eyes, and the globe retained in 9/11 eyes. CONCLUSIONS: Although Gram-positive bacteria predominate in the normal conjunctival microflora of horses throughout the world, Gram-negative bacteria and fungi are more often isolated from equine ulcers. Beta-hemolytic Streptococcus spp. are associated with a very aggressive ulcerative keratitis with the capability to digest conjunctival graft tissue. Clinical signs are pronounced. Aggressive surgical and intensive medical therapy with topical antibiotics and protease inhibitors is indicated.     

Diagnostic testing patterns for Streptococcus equi subsp. equi in Ontario horses during the years 2008 to 2018

This retrospective study describes testing patterns and the incidence of Streptococcus equi subsp. equi in Ontario to assess the utility of laboratory data for surveillance purposes. Laboratory records for equine infectious disease test submissions were extracted from the Animal Health Laboratory (AHL) at the University of Guelph for the years 2008 to 2018. Yearly and seasonal trends in S. equi testing and the proportion of tests that returned positive results were assessed. The number of samples submitted for S. equi testing decreased over the 11-year period (odds ratio = 0.96, 95% confidence interval: 0.92 to 0.999; P = 0.04). A generalized linear model identified a significant seasonal effect for animals recognized as clinically ill, with the highest test positivity noted in the winter. Although this study identified important trends in the incidence of S. equi in Ontario, the variability in information accompanying test submissions made the data challenging to interpret, highlighting the need for more complete diagnostic submission data for S. equi.     

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.     

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.     

Effects of dexamethasone on development of immunoglobulin G subclass responses following vaccination of horses

OBJECTIVE: To determine the effects of dexamethasone on development of IgG subclass responses following vaccination of healthy horses. ANIMALS: 11 mature Thoroughbreds. PROCEDURE: Horses received 2 IM injections at 2-week intervals of a vaccine containing inactivated infectious bovine rhinotracheitis, bovine viral diarrhea, and parainfluenza-3 viral antigens and were then randomly assigned to 2 groups. Six horses received dexamethasone (0.2 mg/kg of body weight, IM) twice weekly for 8 weeks starting the day of the first vaccination. Five control horses received an equivalent volume of saline (0.9% NaCl) solution. Antigen-specific serum IgG subclass titers were determined weekly after vaccination by use of an ELISA. RESULTS: Vaccination resulted in similar antigen-specific serum IgG(T) titers in dexamethasone-treated and control horses. In contrast, although control horses developed IgGa and IgGb responses after vaccination, corticosteroid administration completely inhibited these responses in treated horses. CONCLUSIONS AND CLINICAL RELEVANCE: Cortico steroids can have profound effects on primary immune responses in horses and can significantly affect IgG responses to inactivated vaccines. Corticosteroid treatment regimens commonly used to treat diseases in horses may result induction of a nonprotective IgG subclass response, leaving treated horses susceptible to disease. Additionally, mechanisms regulating IgGa and IgGb responses appear to differ from those regulating IgG(T) responses. Further defining these mechanisms is a critical step in designing effective vaccines, and corticosteroid-induced immunomodulation may be a valuable tool for studying immune responses in horses.     

Rhodococcus (Corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses

The capacity of hematogenous polymorphonuclear neutrophilic leukocytes (PMNL) to kill Rhodococcus equi was compared in horses of various ages. A radioisotope bactericidal assay was used to determine the capacity of PMNL to kill R equi. Assays were conducted on PMNL from horses in 3 groups: group I, 13 foals with a mean age of 3.3 days; group II, 10 group-I foals at a mean age of 35.7 days; and group III, adult dams of group-I foals. Bacteria were obtained from the lungs of a foal with R equi pneumonia and opsonized with fresh adult equine serum that contained R equi specific antibody. The mean peak percentage of R equi killed by PMNL was 78.9 for group I, 90.1 for group II, and 87.9 for group III. There was no significant difference (P greater than 0.05) among groups; however, 15% of foals in group I (2 foals) had a mean peak percentage of 30.5 killed, which was significantly (P less than 0.05) lower than the percentage for other foals in group I. The results of our investigation indicated that the capacity of PMNL to kill opsonized R equi is similar in neonatal, young, and adult horses. However, some neonatal foals have a substantially lower capacity to kill R equi, which may be an important factor in the pathogenesis of R equi infections.     

Immunity to Rhodococcus equi: antigen-specific recall responses in the lungs of adult horses

Rhodococcal pneumonia is an important disease of young horses that is not seen in immunocompetent adults. Since all foals are normally exposed to Rhodococcus equi in their environment, we hypothesized that most develop protective immune responses. Furthermore, these antigen-specific responses were hypothesized to operate throughout adult life to prevent rhodococcal pneumonia. A better understanding of the mechanisms of immune clearance in adult horses would help define the requirements for an effective vaccine in foals. Adult horses were challenged with virulent R. equi by intrabronchial inoculation into the right lung, and pulmonary immune responses were followed for 2 weeks by bronchoalveolar lavage. Local responses in the inoculated right lung were compared to the uninfected left lung and peripheral blood. Challenged horses rapidly cleared R. equi infection without significant clinical signs. Clearance of bacteria was associated with increased mononuclear cells in bronchoalveolar lavage fluid (primarily lymphocytes) and inversion of the normal macrophage:lymphocyte ratio. There was no significant increase in neutrophils at 7 days post-challenge. Flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, both CD4+ and CD8+. Prior to challenge, most adult horses demonstrated low proliferative responses when pulmonary lymphocytes were stimulated with soluble R. equi ex vivo. However, clearance was associated with marked increases in lymphoproliferative responses to soluble R. equi antigen and recombinant VapA, a virulence associated protein of R. equi and candidate immunogen. These results are compatible with previous work in mice which showed that both CD4+ and CD8+ T-cells play a role in immune clearance of R. equi. Recognition of VapA in association with clearance lends further support to its testing as an immunogen. Importantly, the cellular responses to R. equi challenge were relatively compartmentalized. Responses were more marked and the sensitivity to antigen dose was increased at the site of challenge. The blood, including peripheral blood mononuclear cells, was an insensitive indicator of local pulmonary responses.     

Pneumonia in horses induced by intrapulmonary inoculation of Streptococcus equi subsp. zooepidemicus

To evaluate the possibility that Streptococcus equi subsp. zooepidemicus (S.z) the causative bacterial agent of equine shipping fever pneumonia (ESFP), as well as to investigate its pathogenesis, 10 horses (seven Thoroughbreds and three Anglo-Arab species, ranging from 2-4 years in age) were experimentally inoculated, via an endoscope, into bronchus of the lung lobe with a dose of 30 ml of 1-7 x 10(8) CFU/ml of S.z. After inoculation, autopsy and pathological examinations were sequentially conducted 30 min, 1, 2, 3, 4, 17, 20 hr and 2 weeks later. Pneumonia induced by the intrapulmonary inoculation of S.z was characterized by small purulent pneumonic foci in the inoculated areas. With the lapse of time, these foci developed into serous hemorrhagic pneumonia, hemorrhagic purulent pneumonia, and then purulent, coagulation necrotic pneumonia. These pathomorphological characteristics of experimental pneumonia closely resemble those naturally occurring ESFP. There is strong evidence that S.z. is implicated as a causal factor in ESFP. S.z. grew in the mucus, exudate, and pulmonary effusions. Further, the bacteria showed resistance against phagocytosis by pulmonary alveolar macrophages (PAM) and neutrophils. Inhibition of PAM and neutrophil function is considered to be important in the development of pneumonia. With the progression of the disease, the neutrophils often adhered to the endothelial surface of the alveolar capillary lumen and played a role in generating coagulation necrosis of lung tissues.     

The equine immune response to Streptococcus equi subspecies zooepidemicus during uterine infection

The purpose of this study was to describe strain-specific immune responses to Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) during uterine infection in horses. Five isolates of S. zooepidemicus were differentiated into four strains antigenically by bactericidal testing in blood of 12 horses, and genetically by pulsed-field gel electrophoresis. Eight healthy mares were then divided into two groups, each inoculated with one strain intrauterinely on three successive oestrous cycles followed by a second strain for three successive cycles, first and second strains being reversed for each group. Immune responses to both strains were assessed by bactericidal testing and immunoblotting over eight cycles. Both techniques indicated that immune responses to each strain arose at different times. Immunoblots showed greater binding to the first inoculated strain than to the second (P < 0.05). These data confirm that immune responses to S. zooepidemicus during uterine infection are partly strain-specific.