Sridhar Velineni

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.

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.

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

Abstract 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.

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

ABSTRACT 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 (SzMNC78) 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 SzMNC78. 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, SzMNC78 reacted with convalescent-phase sera from horses with respiratory disease associated with strains of S. zooepidemicus. SzMNC78 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 SzMNC78. 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.

Clones of Streptococcus zooepidemicus from Outbreaks of Hemorrhagic Canine Pneumonia and Associated Immune Responses

ABSTRACT 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.

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.

The Antiphagocytic Activity of SeM of Streptococcus equi Requires Capsule

ABSTRACT 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.

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 Cα 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.

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

ABSTRACT 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.

Recurrent Streptococcus equi subsp. zooepidemicus Bacteremia in an Infant

ABSTRACT 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.