Search

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi , establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

Background: Streptococcus equi subspecies equi infection elicits M protein antibody titers in equids. Interpretation of titers is not generally accepted., Hypothesis: The magnitude of S. equi M protein (SeM) antibody titer after infection (titer ≥1:12 800) will be useful to monitor for the presence of complications or the risk of development of complications., Animals: Forty-eight horses on 1 farm involved in strangles outbreak., Methods: Clinical and observational study. S. equi M protein antibody titers were measured on all horses 8 weeks after infection and select horses 12 and 28 weeks after infection. Horses were categorized: no disease, uncomplicated case, persistent guttural pouch (GP) infection, or complicated cases (metastatic abscesses, purpura hemorrhagica, secondary infections, and dysphagia). Category was compared to titer., Results: Twenty-eight of 48 (58%) developed clinical signs of S. equi infection. Of those, 11 (39%) had uncomplicated strangles, 9 (21%) had persistent GP infection, 5 (18%) were complicated cases, and 3 (11%) had both persistent GP infection and complications. Thirty-three percent of horses (16 of 48) had SeM antibody titers ≥1:12 800 eight weeks after infection. Of horses with titers ≥1:12 800, 6 of 16 had evidence of complications. Of complicated cases, 6 of 8 had titers ≥1:12 800. In this outbreak, the sensitivity (75%; 95% CI [confidence interval] 45-105) for a SeM antibody titer ≥1:12 800 detecting complications was higher than the specificity (43%; 95% CI 23-64)., Conclusions and Clinical Importance: This outbreak demonstrates that SeM antibody titers can be increased after infection (≥1:12 800) in the absence of complications of strangles., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The constitutively expressed hyaluronic acid capsule is an important virulence factor of Streptococcus equi, the cause of equine strangles. Study of the genomic sequence of CF22caps-, a non-encapsulated mutant of S. equi CF22 generated by gamma (Co 60 ) irradiation revealed a non-sense mutation in fasC (SEQ&#95;0302), a sensor kinase gene in FasBCAX an operon with an important regulatory role in expression of streptococcal secreted virulence and matrix binding proteins. The mutation was associated with a significant (p < .05) decrease in transcription of hasA, the synthase gene essential for hyaluronic acid synthesis and, conversely, with small increases in transcription of skc, covR and seM. The early growth phase of CF22caps- was also delayed compared to the CF22caps + parent. In contrast to the human pathogen, S. pyogenes, capsule synthesis in S. equi therefore appears to be controlled by FasBCAX and not by CovRS., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This consensus statement update reflects our current published knowledge and opinion about clinical signs, pathogenesis, epidemiology, treatment, complications, and control of strangles. This updated statement emphasizes varying presentations in the context of existing underlying immunity and carrier states of strangles in the transmission of disease. The statement redefines the "gold standard" for detection of possible infection and reviews the new technologies available in polymerase chain reaction diagnosis and serology and their use in outbreak control and prevention. We reiterate the importance of judicious use of antibiotics in horses with strangles. This updated consensus statement reviews current vaccine technology and the importance of linking vaccination with currently advocated disease control and prevention programs to facilitate the eradication of endemic infections while safely maintaining herd immunity. Differentiation between immune responses to primary and repeated exposure of subclinically infected animals and responses induced by vaccination is also addressed., (Copyright © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation., (Copyright © 2017 American Society for Microbiology.)

Lancefield group G Streptococcus canis is a component of the normal urogenital and pharyngeal flora of the cat. It is also frequently implicated in epizootics of severe disease in closed cat colonies and animal shelters. Given the importance of S canis as a feline pathogen and relative lack of published information on characteristics potentially associated with virulence, the authors have compared isolates from healthy and diseased cats in New York and California using fermentation profiles (biotype) and ScM sequences. With few exceptions, isolates associated with disease were biotype 1. Four alleles of scm were identified of which type 1 dominated in diseased cats. Type 4 allelic variants were found only in healthy cats and all but one were biotype 2. Type 2 and 3 alleles showed extensive N-terminal variation suggesting a plasminogen-binding site as found on the type 1 allele was absent. Cat antisera to ScM were opsonobactericidal, and these potentially protective antibodies increased during convalescence., (British Veterinary Association.)

Immunogenic proteins of Leptospira interrogans serovar Pomona type kennewicki (Lk) including Sph1, LigA, Hsp15 and LipL45 (Qlp42) are up-regulated in infected horses but are undetectable or expressed in trace amounts on cultured organisms. In contrast, LipL32 is abundant on cultured Lk and elicits infection antibody responses. The aim of this study was to develop an ELISA based on LipL32 or Lk sonicate and host-induced proteins to differentiate vaccine from infection serum antibody. IgG specific for recombinant Sph1, LigA, Lk90 (LigA; 379-1225 a.a), Hsp15, LipL45 and LipL32 of Lk were assayed in sera of horses infected naturally with Lk and before and after immunisation with serovar Pomona bacterin. Infection but not vaccine sera reacted strongly with Sph1, LigA and Lk90. LipL45 and Hsp15 reacted moderately with infection sera and weakly with vaccine sera. Lk sonicate and LipL32 reacted strongly with both infection and vaccine sera. As expected, culture-based vaccine failed to stimulate antibody to host-induced proteins. Therefore a dual antigen ELISA based on Lk sonicate or LipL32 combined with host-induced Sph1 and Lk90 will be valuable in differentiating infection from vaccine responses., (British Veterinary Association.)

Reasons for Performing Study: Foals of mares infected with Leptospira interrogans serovar Pomona type kennewicki (Lk) may be aborted/stillborn or delivered as healthy foals. Is fetal survival explained in part by the immune response of the fetus to Leptospira antigens?, Objectives: To describe an outbreak of Leptospira abortion in which infected mares delivered dead/sick or normal foals and determine specificities of antibody in a collection of 54 fetuses from similar outbreaks., Study Design: Outbreak investigation in combination with a case-control study of a larger set of samples from aborted fetuses., Methods: Serology and polymerase chain reaction (PCR) on urine and amniotic fluids were used to diagnose infection during an outbreak of Leptospira abortion. Specificities of immunoglobulin (Ig)M, IgGa and IgGb for recombinant proteins of Lk in archived fluids of fetuses from similar outbreaks were compared by ELISA with those of fluids of fetuses not infected with Leptospira spp., Results: Five fetuses of 11 infected mares in an outbreak survived in utero in the presence of persistent placental infection and were healthy at foaling. Fetuses of 6 mares in the outbreak were aborted or died soon after birth. Significantly greater (P<0.05) IgM reactivity with all recombinant proteins and with Lk sonicate was observed in 54 archived fluids from Leptospira infected fetuses than in fluids of 30 of non-Leptospira infected fetuses. Low levels of IgGa and IgGb specific for LipL32 and Lk sonicate and traces of LigA and Hsp15 specific IgGa were detected in a minority of archived fluids from Leptospira infected fetuses., Conclusion: Although mainly mediated by IgM, a high level of immune competence in aborted fetuses was evidenced by the multiplicity of Leptospira proteins targeted. This is likely to contribute to survival of foals in mares with evidence of placental infection at foaling as detailed in a typical outbreak., (© 2015 EVJ Ltd.)

Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Problem Addressed: Streptococcus zooepidemicus (Sz) and its clonal derivative Streptococcus equi (Se) share greater than 96% DNA identity and elicit immune responses to many shared proteins. Identification of proteins uniquely targeted by the immune response to each infection would have diagnostic value., Objective: The aim of the study was to compare serum antibody responses of horses infected by Se or Sz., Methods and Approach: Antibody levels were measured to panels of recombinant proteins of Sz and Se in sera of horses and ponies before and after experimental and naturally occurring invasive infections by these organisms. Antibody responses to an Se extract vaccine were also measured. Sera diluted 1:200 were assayed in triplicate using optimum concentrations of 9 and 14 immunoreactive proteins of Se and Sz, respectively. Bound IgG was detected using HRP-Protein G conjugate., Results: Antibodies specific for SeM-N2, IdeE2, Se42.0 and Se75.3 (SEQ2190) were elicited by Se but not by Sz infection. Commercial Se extract vaccine did not elicit responses to IdeE2 or Se75.3. Sz infections resulted in significant (p<0.01) responses to Sz115, SzM, ScpC, SzP, MAP and streptokinase an indication these proteins are expressed during opportunistic invasions of the respiratory tract. FSR and HylC specific responses were unique to infections by Sz., Conclusions: The data indicate antibodies to IdeE2, Se75.3 and SeM-N2 may be used to distinguish infection by Se from that caused by the closely related Sz. Se infection, but not vaccination with Se extract elicits antibody to IdeE2 and Se75.3., (Copyright © 2015. Published by Elsevier B.V.)

We describe a case of an infant with recurrent bacteremia caused by Streptococcus equi subsp. zooepidemicus, likely transmitted from mother to infant. Our case highlights the importance of an epidemiological history and molecular diagnostics in ascertaining insights into transmission, pathogenesis, and optimal management., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Reasons for Performing Study: Streptococcus zooepidemicus causes opportunist respiratory and other infections in the horse. Capsule expression is highly variable and known to affect resistance to phagocytosis. Most clinical isolates producing small, dry colonies at 37°C produce mucoid colonies at temperatures below 35°C., Objectives: The aim was to understand the molecular basis of increased capsule expression by equine isolates of S. zooepidemicus at temperatures lower than 35°C., Study Design: Cross-sectional observational study., Methods: Capsule production by groups of equine S. zooepidemicus strains was determined at 23, 30, 35 and 37°C. Hyaluronidase (HylC) at 23 and 37°C was measured by quantitative enzyme-linked immunosorbent assay. Expressions of hasA and hylC at 23 and 37°C were measured by quantitative reverse transcriptase-PCR. The covRS genes in representative S. zooepidemicus were sequenced and checked for mutations., Results: Colonies of randomly selected S. zooepidemicus strains became mucoid or showed marked increase in colony mucoidy following the change in temperature to 23°C. Expression of hasA at 23°C was 45- to 700-fold greater than at 37°C. Transcription of hylC at 23°C was 2.5- to 200-fold greater than at 37°C, yet enzyme concentrations in cultures were significantly higher at 37°C (P<0.05), suggesting that production of HylC is regulated post transcriptionally. The covRS gene in S. zooepidemicus was not mutated as seen in isolates of Streptococcus pyogenes with increased capsule production at 25°C., Conclusions: Sensitivity of capsule expression to temperature above 35°C but not HylC by the general population of equine S. zooepidemicus indicates that capsule is not required for extended colonisation nor for opportunist invasion. Instead, capsule production at lower than body temperature may reflect adaptation to life on skin and mucosal surfaces, where hyaluronic acid contributes to adhesion and resistance to desiccation. Pathogenicity of S. zooepidemicus following opportunist invasion is possibly dependent on factors other than capsule that may be co-regulated with HylC. The Summary is available in Chinese - see Supporting information., (© 2014 EVJ Ltd.)

Acute hemorrhagic pneumonia caused by Streptococcus zooepidemicus has emerged as a major disease of shelter dogs and greyhounds. S. zooepidemicus strains differing in multilocus sequence typing (MLST), protective protein (SzP), and M-like protein (SzM) sequences were identified from 9 outbreaks in Texas, Kansas, Florida, Nevada, New Mexico, and Pennsylvania. Clonality based on 2 or more isolates was evident for 7 of these outbreaks. The Pennsylvania and Nevada outbreaks also involved cats. Goat antisera against acutely infected lung tissue as well as convalescent-phase sera reacted with a mucinase (Sz115), hyaluronidase (HylC), InlA domain-containing cell surface-anchored protein (INLA), membrane-anchored protein (MAP), SzP, SzM, and extracellular oligopeptide-binding protein (OppA). The amino acid sequences of SzP and SzM of the isolates varied greatly. The szp and szm alleles of the closely related Kansas clone (sequence type 129 [ST-129]) and United Kingdom isolate BHS5 (ST-123) were different, indicating that MLST was unreliable as a predictor of virulence phenotype. Combinations of conserved HylC and serine protease (ScpC) and variable SzM and SzP proteins of S. zooepidemicus strain NC78 were protectively immunogenic for mice challenged with a virulent canine strain. Thus, although canine pneumonia outbreaks are caused by different strains of S. zooepidemicus, protective immune responses were elicited in mice by combinations of conserved or variable S. zooepidemicus proteins from a single strain., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Streptococcus equi subspecies zooepidemicus (Sz) is a tonsillar and mucosal commensal of healthy horses with the potential to cause opportunistic infections of the distal respiratory tract stressed by virus infection, transportation, training or high temperature. The invasive clone varies from horse to horse with little evidence of lateral transmission in the group. Tonsillar isolates are non-mucoid although primary isolates from opportunist lower respiratory tract infections may initially be mucoid. In this study, a novel stably mucoid Sz (SzNC) from a clonal epizootic of respiratory disease in horses in different parts of New Caledonia is described. SzNC (ST-307) was isolated in pure culture from transtracheal aspirates and as heavy growths from 80% of nasal swabs (n=31). Only 4% of swabs from unaffected horses (n=25) yielded colonies of Sz. A viral etiology was ruled out based on culture and early/late serum antibody screening. Evidence for clonality of SzNC included a mucoid colony phenotype, SzP and SzM sequences, and multilocus sequence typing. SzNC, with the exception of isolates at the end of the outbreak, was hyaluronidase positive. Its SzP protein was composed of an N2 terminal, and HV4 variable region motifs and 18 carboxy terminal PEPK repeats. Biotin labeling of surface proteins revealed DnaK and alanyl-tRNA synthetase (AlaS) on the surface of clonal isolates, but not on non-clonal non-mucoid Sz from horses in the epizootic or unrelated US isolates. Reactivity of these proteins and SzP with convalescent serum indicated expression during infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Streptococcus zooepidemicus (Sz) is a tonsillar commensal of healthy horses but with potential to opportunistically invade the lower respiratory tract. Sz is genetically variable and recombinogenic based on analysis of gene sequences including szp, szm and MLST data. Although a variety of serovars of the protective SzP are commonly harbored in the tonsils of the same horse, lower respiratory infections usually involve a single clone. Nevertheless, isolation of specific clones from epizootics of respiratory disease has been recently reported in horses and dogs in N. America, Europe and Asia. In this report, we provide evidence suggestive of lateral gene exchange and recombination between strains of Sz from cases of respiratory disease secondary to experimental equine herpes 1 virus infection in an isolated group of weanling horses and ponies. Nasal swabs of 13 of 18 weanlings with respiratory disease yielded mucoid colonies of Sz following culture. Comparison of arcC, nrdE, proS, spi, tdk, tpi and yqiL of these Sz revealed 3 Clades. Clade-1 (ST-212) and 2 (ST-24) were composed of 7 and 3 isolates, respectively. ST-24 and 212 differed in all 7 housekeeping as well as szp and szm alleles. Two isolates of Clade-1 were assigned to ST-308, a single locus variant of ST-212 that contained the proS-16 allele sequenced in ST-24. One isolate of ST-308 contained szm-2, the same allele sequenced in Clade 2 isolates; the other was positive for the szp-N2HV2 allele of Clade 2. These observations are consistent with gene transfer between Sz in the natural host and may explain formation of novel clones that invade the lower respiratory tract or cause epizootics of respiratory disease in dogs and horses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Resistance to phagocytosis is a crucial virulence property of Streptococcus equi (Streptococcus equi subsp. equi; Se), the cause of equine strangles. The contribution and interdependence of capsule and SeM to killing in equine blood and neutrophils were investigated in naturally occurring strains of Se. Strains CF32, SF463 were capsule and SeM positive, strains Lex90, Lex93 were capsule negative and SeM positive and strains Se19, Se1-8 were capsule positive and SeM deficient. Phagocytosis and killing of Se19, Se1-8, Lex90 and Lex93 in equine blood and by neutrophils suspended in serum were significantly (P ≤ 0.02) greater compared to CF32 and SF463. The results indicate capsule and SeM are both required for resistance to phagocytosis and killing and that the anti-phagocytic property of SeM is greatly reduced in the absence of capsule.

Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered.

Background: Streptococcus zooepidemicus is an important opportunistic pathogen of the equine respiratory and reproductive tracts. A normal tonsillar and mucosal commensal, it becomes invasive under conditions of stress such as virus infection, weaning, high temperature, prolonged transportation and failure of uterine involution. The aim of this study was to evaluate the vaccine potential of several surface exposed and secreted proteins of a novel mucoid clone of SzNC78 (ST-307) from an epizootic of equine respiratory disease., Methods: An expression gene library of SzNC78 was probed with a pool of convalescent equine sera from a clonal epizootic of respiratory disease. Eleven proteins were selected and purified based on putative function, surface expression or secretion and possible importance as virulence factors. Three additional proteins (AhpC, GAPDH and enolase) were also included based on their putative virulence function. Groups of ICR mice were vaccinated subcutaneously with each recombinant antigen and QuilA and later challenged with SzNC78., Results: SzM protected 100% mice (P<0.01), followed by SzP and HylC which protected 90% mice (P=0.01). MAP, SzMAC and ScpC each protected 63% (P<0.05) mice. No control mouse survived challenge. SzM, MAP and ScpC in combination protected mice against a 10 fold higher dosage of SzNC78 than each antigen given separately. Protection against heterologous challenge (SzW60) was conferred by combinations of HylC+ScpC (60%; P=0.05), followed by HylC+MAP (50%; P=0.06) and ScpC+MAP (40%; P=0.1). Serum antibody responses of horses recently recovered from Sz respiratory infection were highest against ScpC, MAP and SzP. A combination of SKC and Sz115 stimulated no protection against challenge with SzW60., Conclusion: A subset of Sz proteins reactive with convalescent equine antibody have potential as components of experimental vaccines to aid in prevention of opportunistic Sz infections., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Streptococcus zooepidemicus of Lancefield group C is a highly variable tonsillar and mucosal commensal that usually is associated with opportunistic infections of the respiratory tract of vertebrate hosts. More-virulent clones have caused epizootics of severe respiratory disease in dogs and horses. The virulence factors of these strains are poorly understood. The antiphagocytic protein SeM is a major virulence factor and protective antigen of Streptococcus equi, a clonal biovar of an ancestral S. zooepidemicus strain. Although the genome of S. zooepidemicus strain H70, an equine isolate, contains a partial homolog (szm) of sem, expression of the gene has not been documented. We have identified and characterized SzM from an encapsulated S. zooepidemicus strain from an epizootic of equine respiratory disease in New Caledonia. The SzM protein of strain NC78 (SzM(NC78)) has a predicted predominantly alpha-helical fibrillar structure with an LPSTG cell surface anchor motif and resistance to hot acid. A putative binding site for plasminogen is present in the B repeat region, the sequence of which shares homology with repeats of the plasminogen binding proteins of human group C and G streptococci. Equine plasminogen is activated in a dose-dependent manner by recombinant SzM(NC78). Only 23.20 and 25.46% DNA homology is shared with SeM proteins of S. equi strains CF32 and 4047, respectively, and homology ranges from 19.60 to 54.70% for SzM proteins of other S. zooepidemicus strains. As expected, SzM(NC78) reacted with convalescent-phase sera from horses with respiratory disease associated with strains of S. zooepidemicus. SzM(NC78) resembles SeM in binding equine fibrinogen and eliciting strong protective antibody responses in mice. Sera of vaccinated mice opsonized S. zooepidemicus strains NC78 and W60, the SzM protein of which shared partial amino acid homology with SzM(NC78). We conclude that SzM is a protective antigen of NC78; it was strongly reactive with serum antibodies from horses during recovery from S. zooepidemicus-associated respiratory disease.

Reasons for Performing Study: Enterocolitis caused by Clostridium difficile (C. difficile) is a serious, sometimes fatal, disease of neonatal foals and older horses. Toxins A and B (TcdA and B) produced by C. difficile are important virulence factors. Immunisation of mares with receptor binding domains of toxins may prevent or reduce the severity of C. difficile colitis in foals., Objectives: To determine whether antibodies generated in the pregnant mare to the binding regions of TcdA and B will neutralise TcdA and B toxicity., Methods: Sequences encoding the binding domains of each toxin were isolated by PCR amplification from C. difficile JF09, a foal isolate, and cloned and expressed into pET15b. Thirteen mares were immunised twice 2 weeks apart with 200 μg of each recombinant protein with Quil A 2 months prior to foaling. Antibodies were assayed in the sera and colostrum by ELISA and for ability to block the cytopathic activity of each of toxin for equine endothelial cells., Results: All mares produced strong serum antibody responses to the binding domain of each toxin. A high level of toxin-specific antibodies was also detected in colostrum and in most foal sera 2 days after suckling. Diluted sera and colostrum premixed with either TcdA or B had no effect on the morphology of equine endothelial cells. Application of the same concentration of toxins alone or premixed with nonimmune mare/foal serum or colostrum led to an unambiguous cytopathic effect that ranged from complete degradation to varying degrees of cell rounding., Conclusions: Immunisation of pregnant mares with recombinant binding domains of TcdA and B of C. difficile resulted in the production of specific antibodies in serum and colostrum that blocked the cytopathic activity of toxins., Potential Relevance: Results of studies support the feasibility of a prepartum vaccine against C. difficile enterocolitis in foals., (© 2012 EVJ Ltd.)

The addition of streptolysin-O (SLO) to the standard antibiotics regimen was shown to be superior to antibiotics alone after experimental infection of foals with Rhodoccocus equi (R. equi). The objective of this study is to investigate this response by determining the site-specific expression of extracellular matrix (ECM) and inflammatory response genes in biopsy samples taken from three distinct lung regions of the infected foals. Twenty-four foals were challenged by intrabronchial instillation of R. equi and assigned to four treatment groups: SLO/antibiotics adjunct therapy, antibiotics-only therapy (7.5 mg/kg clarithromycin and 5 mg/kg rifampin), SLO-only, and saline-only treatments. Treatments were administered twice daily for 16 days unless symptoms progressed to the point where the foals needed to be euthanized. Gene expressions were determined using custom-designed equine real-time qPCR arrays containing forty-eight genes from ECM remodeling and inflammation pathways. A non-parametric Wilcoxon signed-rank test for independent samples was applied to two pairs of time-matched comparison groups, SLO/antibiotics vs. antibiotics-only and SLO-only vs. saline-only, to document the significant differences in gene expressions within these groups. Several genes, MMP9, MMP2, TIMP2, COL1A1, COL12A1, ITGAL, ITGB1, FN1, CCL2, CCL3, CXCL9, TNFα, SMAD7, CD40, IL10, TGFB1, and TLR2, were significantly regulated compared to the unchallenged/untreated control foals. The results of this study demonstrate that enhancement of clinical responses by SLO is consistent with the changes in expression of critical genes in ECM remodeling and inflammatory response pathways.

Successful treatment of leptospirosis is heavily dependent on early diagnosis and prompt initiation of antibiotic therapy. An ELISA test to detect specific IgM antibodies against LipL32 for early diagnosis of leptospirosis is described and evaluated here. One thousand one hundred and eighty sera from clinically suspected leptospirosis cases were enrolled together with 109 healthy volunteers selected from an endemic area between October 2007 and January 2010. Patients were categorized based on their clinical signs and symptoms. Sera were screened for leptospiral antibodies by the microscopic agglutination test (MAT) using a panel of locally circulating serovars followed by enzyme-linked immunosorbent assay (ELISA) based on recombinant LipL32 from Leptospira interrogans serovar Autumnalis strain N2. The sensitivity and specificity of the ELISA test were determined to establish its diagnostic efficiency. The cut-off value was determined to be 0·205. Overall sensitivity and specificity compared to the MAT were found to be 96·4 and 90·4%, respectively. The LipL32-specific IgM ELISA had good sensitivity and acceptable specificity and may be a candidate for the early serodiagnosis of human leptospirosis.

Screening of an expression library of Leptospira interrogans with eye fluids from uveitic horses resulted in identification of a novel protein, LruC. LruC is located in the inner leaflet of the leptospiral outer membrane, and an lruC gene was detected in all tested pathogenic L. interrogans strains. LruC-specific antibody levels were significantly higher in eye fluids and sera of uveitic horses than healthy horses. These findings suggest that LruC may play a role in equine leptospiral uveitis.

Rhodococcus equi is a soil borne bacterium that causes severe morbidity and death in young foals. The economic costs of the disease include loss of life, treatment expenses, veterinary monitoring expenses and, perhaps most importantly, potential reduction in future athletic performance in horses that suffer severe lung abscessations caused by R. equi. Current standard of care for pneumonia caused by R. equi is treatment with a macrolide antimicrobial and rifampicin. However, the hallmark of pneumonia caused by R. equi is severe formation of pyogranulomas and a walling off effect that can prevent systemic antibiotics from reaching antimicrobial concentrations in lung tissues. It is hypothesized that streptolysin O (SLO) used as an adjunct therapy with antibiotics will reduce the duration and severity of disease caused by R. equi pneumonia compared to antibiotic therapy alone. Addition of SLO to the antibiotic enhanced clinical responses compared to the other groups, including the antibiotic alone group. Of particular significance were lower bacterial counts in the lungs and longer survival time in those foals treated with SLO and antibiotics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study has been formulated in order to detect an immunoreactive protein whose identification can play a major role in the early diagnosis of disease. The identified protein will be produced by recombinant methods and used for the recombinant protein based ELISA. A comparison was made between the developed method and the gold standard MAT test to evaluate the serodiagnosis potential of the protein. The protein profile, immunoblot and MALDI-TOF analysis was carried out to identify the immunoreactive protein. The immunoreactive protein identified was used to develop ELISA for the diagnosis of leptospirosis using patients' sera with various clinical manifestations. The immunoreactive protein was identified as Leptospira GroEL chaperonin of molecular weight 60 kDa. The theoretical/experimental molecular weights, pI were found to be 58.5/60 kDa and 5.41/6, respectively. The overall results of the recombinant GroEL-IgM ELISAs showed cumulative sensitivity, specificity, positive predictive value, and negative predictive values of 90.6%, 94.9%, 94.6%, and 91.0%, respectively. The performance of such ELISA appeared better than that of any other serological tests previously evaluated for the diagnosis of leptospirosis in India. Thus, a highly conserved and immunogenic outer exposed GroEL protein during infection clearly merits further use in the serodiagnosis of leptospirosis.

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a protein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component., (Copyright © 2011 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.)

Streptococcus equi (Se), the cause of equine strangles, is highly resistant to phagocytosis by neutrophils and is usually classified as an extracellular pathogen. Large numbers of the organism in tonsillar tissues during the acute phase of the disease are completely eliminated during convalescence by mechanisms not yet understood. In this study we demonstrate in an opsono-bactericidal assay and by cytometry and confocal microscopy that Se is interiorized and killed by equine blood monocytes. This finding supports the hypotheses that adaptive immune clearance is mediated by tonsillar macrophages and that macrophages monocytes could serve as a vehicle for transport from the tonsil to local lymph nodes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Strangles caused by the host adapted Lancefield group C Streptococcus equi subspecies equi (S. equi) is a frequently diagnosed infectious disease of horses worldwide. Critical to the global success of S. equi is its ability to establish persistent infections within the guttural pouches of recovered apparently healthy horses that can result in transmission to in-contact animals. Recent research has identified key events in the S. equi genome, which occurred during its evolution from an ancestral strain of S. equi subspecies zooepidemicus, that may enhance its ability to evade host innate immune responses and rapidly multiply in the tonsillar complex and draining lymph nodes. This review discusses the role of these genetic events on the evolution and emergence of this important host-restricted pathogen.

Streptococcus equi, a clone or biovar of an ancestral Streptococcus zooepidemicus of Lancefield group C causes equine strangles, a highly contagious tonsillitis and lymphadenitis of the head and neck. At least 74 alleles based on N-terminal amino acid sequence of the anti-phagocytic SeM have been observed among isolates of S. equi from N. America, Europe and Japan. A d(N)/d(S) ratio of 5.93 for the 5' region of sem is indicative of positive selective pressure. The aim of this study was to determine whether variations in SeM were accompanied by variations in the surface exposed SzPSe and secreted Se18.9, both of which bind to equine tonsillar epithelium and, along with SeM, elicit strong nasopharyngeal IgA responses during convalescence. Sequences of genes for these proteins from 25 S. equi expressing 19 different SeM alleles isolated over 40 years in different countries were compared. No variation was observed in szpse, except for an Australian isolate with a deletion of a single repeat in the 3' end of the gene. Interestingly, only two SNP loci were detected in se18.9 compared to 93 and 55 in sem and szpse, respectively. The high frequency of nucleotide substitutions in szpse may be related to its mosaic structure since this gene in S. zooepidemicus exists in a variety of combinations of sequence segments and has a central hypervariable region that includes exogenous DNA sequence based on an atypical G-C percentage. In summary, the results of this study document very different responses of streptococcal genes for 3 immunoreactive proteins to selection pressure of the nasopharyngeal mucosal immune response., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although serologic data indicate horses in N. America are exposed to a variety of leptospiral serovars, abortion is almost always associated with Leptospira interrogans serovar Pomona type kennewicki. A variety of wildlife including raccoons, white tailed deer, striped skunks, opossums, and red and grey foxes have been shown to host serovar Pomona and have therefore been suspect as sources of infection for pregnant mares. The aim of the present study was to examine genetic diversity in serovar Pomona type kennewicki in wildlife and in aborting mares. Our approach utilized PCR that targeted tandem repeats at the VNTR - 4 locus and a 1235 bp 5'-sequence of the lk73.5 (sph2) and adjacent upstream sequence unique to serovar Pomona. All isolates/specimens of equine origin in 1992 and 2008 yielded amplicons of 1235 and 595 bp, whereas 14 isolates/specimens from wildlife yielding a 1235 bp amplicon characteristic of serovar Pomona produced amplicons of 1300, 550 bp (3), 1300 bp (10), or 595 bp (6) with the VNTR - 4 primer set. Wildlife therefore hosted at least three different genetic variants of type kennewicki including the genetic variant that predominated in aborting mares. The data are consistent with other studies indicating specific genetic variants of type kennewicki show a strong tendency to be associated with a specific host. Levels of antibody in wildlife sera reactive with rLk73.5, rLig130 and sonicate of type kennewicki were poorly correlated with PCR data, although rLk73.5 was superior to rLig130 in detection of antibody responses. PCR is therefore a more reliable tool for studies of wildlife reservoirs of Leptospira sp. than serologic surveillance that targets host induced proteins or LPS-rich sonicate., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Leptospirosis is a major problem of dairy farms in Tamilnadu, India, resulting in abortions, stillbirths and infertility. Serologic and genetic analyses of samples from cattle, humans and rodents were performed in order to estimate infection prevalence and identify leptospiral species. Five hundred and fifteen sera and 76 urine samples were collected from dairy cattle on 25 farms including a farm that practiced rat control. Sera and kidney samples were also collected from field rats (Rattus norvegicus) in the vicinity of these farms. In addition, sera were collected from farm workers. Serum antibody was measured by the microscopic agglutination test. Leptospires isolated from blood, kidney, and urine were characterized as to serovar. Genomospecies were predicted using random amplified polymorphic DNA (RAPD) profiling. SecY gene sequencing was performed as a tool for tracing of source. Seroprevalence of 87.%, 51.% and 76.5% for cattle, rats and humans, respectively, was observed on endemic farms. Prevalences on a non-endemic farm were lower. Antibodies to Autumnalis, Javanica, Icterohaemorrhagiae and Pomona predominated in both cattle and rats. Thirteen isolates from rat kidneys were identified as serogroup Javanica, serovar Javanica. RAPD comparisons and secY gene sequencing identified these isolates as Leptospira borgpetersenii. These results altogether indicated that L. borgpetersenii was the dominant species in these areas with serovar Javanica apparently derived from rats which provided an important source of infection in cattle resulting a high incidence of infertility, abortion and.still-birth in the Cauvery river valley, Tiruchirappalli, Tamilnadu.

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be alpha-crystallin B and vimentin. Similarly, mass spectrometric analyses identified beta-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human alpha-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant beta-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized alpha-crystallin B, beta-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.

Attenuated Salmonella enterica serovar Typhimurium MGN707, expressing the SzP protective protein of the MB9 serovar of Streptococcus equi subspecies zooepidemicus (SzP-MB9) was tested for its safety and efficacy as a nebulised intranasal vaccine against streptococcal uterine infections in mares. In a preliminary study, vaccinated mares (n=5) displayed serum, nasal and uterine responses (P<0.05) to S. Typhimurium lipopolysaccharide (St-LPS). Subsequently, vaccinated mares (expressor group, n=7), but not mares vaccinated with the vector only (control group, n=7), displayed significant increases in SzP-MB9 antibodies in serum, nasal and uterine washes (P<0.05). Assuming the uteri of all nine mares were free of streptococci prior to challenge with 6.3 x 10(9) colony forming units of S. e. zooepidemicus MB9, significantly fewer S. e. zooepidemicus were cultured from the uterine flushings of expressor-vaccinated mares (n=4) compared to control-vaccinated mares (n=5) (P<0.001). The only adverse reaction to vaccination was nasal haemorrhage in one mare., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The clonal Streptococcus equi causes equine strangles, a highly contagious suppurative lymphadenopathy and rhinopharyngitis. An important virulence factor and vaccine component, the antiphagocytic fibrinogen binding SeM of S. equi is a surface anchored fibrillar protein. Two recent studies of N. American, Japanese and European isolates have revealed a high frequency of N-terminal amino acid variation in SeM of S. equi CF32 that suggests this region of the protein is subject to immunologic selection pressure. The aims of the present study were firstly to map regions of SeM reactive with convalescent equine IgG and IgA and stimulatory for lymph node cells and secondly to determine effects of N-terminal variation on the functionality of SeM. Variation did not significantly affect fibrinogen binding or susceptibility of S. equi to an opsonic equine serum. Linear epitopes reactive with convalescent IgG and mucosal IgA were concentrated toward the conserved center of SeM. However, IgA but not IgG from every horse reacted with at least one peptide that contained variable sequence. Lymph node cells (CD4+) from horses immunized with SeM were strongly responsive to a peptide (alphaalpha36-138) encoding the entire variable region. SeM (CF32) specific mouse Mab 04D11 which reacted strongly with this larger peptide but not with shorter peptides within that sequence reacted strongly with whole cells of S. equi CF32 but only weakly with cells of any of 14 isolates of S. equi expressing different variants of SeM. These results in combination suggest that N-terminal variation alters a conformational epitope of significance in mucosal IgA and systemic T cell responses but does not affect antibody mediated phagocytosis and killing., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Streptococcus equi (S. equi subsp. equi) is widely believed to have evolved from an ancestral strain of S. zooepidemicus (S. equi subsp. zooepidemicus) based on high sequence homology. A striking difference is the absence of phage sequences from S. zooepidemicus. In this study we show that the receptor for SeP9, a temperate bacteriophage of S. equi, is the Lancefield group C carbohydrate. However, although SeP9 binds to group C carbohydrate from S. zooepidemicus, it appears not to replicate and produce plaques.

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.

Uveitis can be a serious complication of leptospirosis. Previous studies indicated that the leptospiral lipoproteins LruA and LruB are expressed in the eyes of uveitic horses and that antibodies directed against those proteins show in vitro cross-reactivity with components of equine lens, ciliary body, and/or retina. We now demonstrate that sera from a significant proportion of humans who have leptospiral uveitis also contain antibodies against LruA and LruB. Different categories of nonleptospiral uveitis and autoimmune uveitis were also screened; patients diagnosed with Fuchs uveitis or Behçet's syndrome produced antibodies that cross-reacted with LruA and LruB, suggesting similarities of the autoimmune responses in those diseases with those of leptospiral uveitis.

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.

An outbreak of acute, fatal, hemorrhagic pneumonia was observed in more than 1,000 mixed breed dogs in a single animal shelter. The Department of Anatomic Pathology at the University of California at Davis School of Veterinary Medicine performed necropsies on dogs that were found moribund in acute respiratory distress or found dead with evidence of nasal bleeding. All dogs had hemothorax and an acute, fibrinosuppurative pneumonia. Large numbers of gram-positive cocci were observed within the lungs of all dogs and within septic thromboemboli of remote organs in about 50% of cases. Bacterial cultures from the dogs and their environment revealed widespread beta-hemolytic Streptococus equi subspecies zooepidemicus (Lancefield Group C). Extensive diagnostic testing failed to reveal the consistent presence of copathogens in individual cases. The clinical, epidemiologic, molecular biologic, and pathologic data indicate that a single clone of S. zooepidemicus was the cause of an acutely fatal respiratory infection in these dogs.

Streptococcus equi, a clonal descendent of an ancestral S. zooepidemicus, causes equine strangles, a highly contagious purulent lymphadenitis of the head and neck. The aim of this study was to evaluate as vaccine components novel surface exposed or secreted S. equi proteins identified in an expression gene library with sera from resistant horses. Six proteins expressed by S. equi CF32 but not by S. zooepidemicus 631 were used to vaccinate one group of eight ponies. A second pony group was immunized with five adhesin and other proteins encoded by genes of Linkage Gr 1. All ponies made strong serum antibody responses to each protein as measured by ELISA but none were resistant to subsequent comingling challenge with S. equi CF32. These results in combination with evidence that recovered horses rapidly clear intranasally inoculated S. equi and do not make detectable serum antibody responses to its surface proteins suggest that acquired immune-mediated tonsillar clearance and not serum antibody must be stimulated by an effective strangles vaccine.

Evasion of phagocytosis is an important virulence determinant of Streptococcus equi (S. equi subsp. equi), the cause of equine strangles and distinguishes it from the closely related but much less virulent S. zooepidemicus (S. equi subsp. zooepidemicus). We describe Se18.9, a novel H factor binding protein secreted by S. equi but not by S. zooepidemicus that reduces deposition of C3 on the bacterial surface and significantly reduces the bactericidal activity of equine neutrophils suspended in normal serum for both S. equi and S. zooepidemicus. Se18.9 is secreted abundantly by actively dividing cells and is also bound to the bacterial surface. Strong serum and mucosal antibody responses are elicited in S. equi infected horses. Although a gene identical to se18.9 was not detected in S. zooepidemicus, sequences encoding proteins of similar size with similar signal peptide sequences were found in 3 of 12 randomly selected strains. Since Se18.9 is unique to S. equi, and immunoreactive with convalescent sera and mucosal IgA, it has potential for immunodiagnosis and for study of mucosal antibody response to S. equi.

Immunological protection of horses from equine herpesvirus-1 (EHV-1) infection and disease depends on the cooperation of virus-specific humoral and cellular immune responses. EHV-specific mucosal immunity may be an important component of such immune responses. This study demonstrates the induction of anti-EHV cytotoxic cellular immune responses in various mucosal and systemic lymphoid tissues associated with the upper respiratory tract (URT) of the horse. Four young horses (1-2 years of age) were inoculated intranasally with the Army 183 strain of EHV-1 and euthanized 1 week later. One untreated foal served as a non-infected control. Mucosa-associated tonsillar tissues, draining lymph nodes and PBMC were harvested. Virus-specific memory and effector cytolytic activity were individually assessed using 4 h chromium release assays, with and without in vitro restimulation with EHV-1, respectively. EHV-specific cytotoxic activity was detected ex vivo in several URT-associated mucosal lymphoid tissues of horses, particularly within the lining of the nasopharynx, a principal site of EHV-1 replication. This activity was also detected in the circulation of some horses 1 week post-challenge. Virus-specific memory cytotoxic activity was elevated in the circulation, and detectable in the draining lymph nodes of all horses following challenge infection.

The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

The tonsil of the soft palate was an oval, flat structure located centro-rostrally on the oral surface of the soft palate. Its stratified squamous non-keratinized epithelium was perforated by holes or small crypts the deeper parts of which were loosely spongiform inter-digitated with lymphoid tissue. These unusual features have not previously been reported in tonsils of any species. Crypts and reticulated epithelium as found in the lingual and palatine tonsils were not observed. Lectins showed varying affinities for specific layers of the epithelium. M cells were not observed. A few Langerhans cells were distributed among surface epithelial cells. Lymphoid tissue was arranged loosely and in isolated lymphoid follicles in the subepithelial lamina propria mucosae. Although IgA+ cells and macrophages were proportionately more numerous the amount of lymphoid tissue was much less than in the lingual and palatine tonsils. Most of the follicular germinal centres lacked a darkly stained corona. CD4 positive were more numerous than CD8+ lymphocytes and were distributed in the parafollicular and inter-follicular areas. Large clusters of mucus acini positive for glycogen, acidic and neutral mucopolysaccharides separated lymphoid tissue from deeply placed striated muscle. Only a few high endothelial venules were observed in the parafollicular and inter-follicular areas. These had relatively few vesiculo vacuolar or other organelles in their high endothelial cells and few lymphocytes attaching to their walls.

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.

Objective: To develop a method for typing Streptococcus equi on the basis of the DNA sequence of the genes that produce an M-like protein and to compare isolates among the United States, Japan, and other countries., Sample Population: S equi strains CF32, Hidaka/95/2, and NCTC9682 as well as 82 other isolates from the United States, Japan, and other countries obtained during 1975 to 2001., Procedure: DNA sequences of the structural genes ( SeM and SzPSe) that produce M-like proteins were determined for 3 representative strains to find a variable region. Variability in this region of SeM was then determined for the other isolates. Amino acid sequences were deduced and analyzed phylogenetically by use of the neighbor-joining method., Results: Sequence diversity was detected in the N-terminal region of SeM but not in SzPSe of the 3 representative strains. Base substitutions in the variable region of SeM varied in a nonsynonymous manner, resulting in variation in the amino acid sequence. Eighty-five isolates were categorized as 32 types of SeM on the basis of differences in the deduced amino acid sequences., Conclusions and Clinical Relevance: This study documented a region in the N-terminal portion of SeM that varies in a nonsynonymous manner. This information should be useful in molecular epidemiologic studies of S equi.