Globetrotting strangles: the unbridled national and international transmission of Streptococcus equi between horses.

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.

Strangles, convalescent Streptococcus equi subspecies equi M antibody titers, and presence of complications.

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.

Evidence for involvement of the Fas BCAX regulon in capsule synthesis by Streptococcus equi.

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 (Co60) irradiation revealed a non-sense mutation in fasC (SEQ_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.

Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions.

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.

In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis.

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.

Comparison of specificities of serum antibody responses of horses to clinical infections caused by Streptococcus equi or zooepidemicus.

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.

Recurrent Streptococcus equi subsp. zooepidemicus Bacteremia in an Infant.

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.

The Antiphagocytic Activity of SeM of Streptococcus equi Requires Capsule.

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.

Clones of Streptococcus zooepidemicus from outbreaks of hemorrhagic canine pneumonia and associated immune responses.

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.

Characterization of a mucoid clone of Streptococcus zooepidemicus from an epizootic of equine respiratory disease in New Caledonia.

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.

Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis.

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.

Jusqu'à 60 % des cas de colite équine n'ont aucune cause connue. Afin d'améliorer la compréhension des causes de colite aigüe chez les chevaux, nous émettons l'hypothèse que les Clostridium perfringens produisant l'entérotoxine (CPE) et/ou la toxine bêta-2 (CPB2) sont des causes courantes et importantes de colites sévères chez les chevaux et/ou qu'une cytotoxine non décrite encore produite par C. perfringens pourrait également causer des colites chez les chevaux. Des échantillons fécaux provenant de 55 chevaux (43 adultes, 12 poulains) avec des évidences cliniques de colite ont été évalués par culture pour la présence de Clostridium difficile, C. perfringens et Salmonella. Les fèces furent également analysées par épreuve immuno-enzymatique (ELISA) pour la présence des toxines A/B de C. difficile et les toxines alpha (CPA), bêta2 (CPB2) et l'entérotoxine (CPE) de C. perfringens. Cinq isolats de C. perfringens par échantillons ont été typés pour les gènes suivants : cpa, cpb, cpb2 consensus, cpb2 atypique, cpe (entérotoxine), etx (toxine epsilon), itx (toxine iota), netB (toxine B de l'entérite nécrotique), et tpeL (cytotoxine large de C. perfringens). Les surnageants de culture de ces isolats ont également été évalués pour leur cytotoxicité envers une lignée cellulaire équine. Tous les échantillons fécaux étaient négatifs pour la présence de Salmonella. Clostridium perfringens et C. difficile furent isolés de 40 % et 5,4 % des échantillons, respectivement. Tous les échantillons de fèces étaient négatifs pour CPE. Les toxines CPA et CPB2 furent détectées à partir de 14,5 % et 7,2 % des échantillons fécaux, respectivement, tous étant positifs pour la présence de C. perfringens en culture. Aucun des isolats n'était positif pour la présence des gènes cpe, etx, netB ou tpeL. Les gènes cpb2 atypiques et cpb2 consensus furent identifiés dans respectivement 15 (13,6 %) et 4 (3,6 %) des 110 isolats. Tous les isolats de C. perfringens équins montraient des effets cytotoxiques nettement plus légers que les témoins positifs produisant CPB, bien que les isolats possédant les gènes cpb2 étaient légèrement mais significativement plus cytotoxiques que les témoins négatifs. En fonction de la population étudiée, il nous est impossible de confirmer notre hypothèse qu'en Ontario des C. perfringens produisant CPE et CPB-2 sont courants chez les chevaux avec colites et nous n'avons pas réussi à identifier une activité cytotoxique in vitro chez les types A isolés.(Traduit par Docteur Serge Messier).

Evidence of lateral gene transfer among strains of Streptococcus zooepidemicus in weanling horses with respiratory disease.

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.

Identification of novel immunoreactive proteins of Streptococcus zooepidemicus with potential as vaccine components.

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.

Characterization and protective immunogenicity of the SzM protein of Streptococcus zooepidemicus NC78 from a clonal outbreak of equine respiratory disease.

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.

Detection of LipL32-specific IgM by ELISA in sera of patients with a clinical diagnosis of leptospirosis.

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.

Streptolysin-O/antibiotics adjunct therapy modulates site-specific expression of extracellular matrix and inflammatory genes in lungs of Rhodococcus equi infected foals.

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.

Antibodies to a novel leptospiral protein, LruC, in the eye fluids and sera of horses with Leptospira-associated uveitis.

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.

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2.

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.

The use of streptolysin O (SLO) as an adjunct therapy for Rhodococcus equi pneumonia in foals.

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.

Association of Streptococcus equi with equine monocytes.

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.