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Characterization of acid phosphatase activities in the equine pathogen Streptococcus equi.

Acid phosphatases hydrolyse phosphomonoesters at acidic pH in a variety of physiological contexts. The recently defined class C family of acid phosphatases includes the 32 kDa LppC lipoprotein of Streptococcus equisimilis. To define further the distribution of acid phosphatases in the genus Streptococcus we have examined the equine pathogens Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Whole cell assays indicated that these organisms possess two acid phosphatases with activity optima at pH 5.0 and pH 6.0-6.5 and that only the former of these was, like LppC, resistant to EDTA. Western blotting with a polyclonal anti-LppC antiserum revealed the presence of a cross-reactive 32 kDa protein in both organisms. The cross-reactive protein in S. equi was shown to be a surface accessible lipoprotein as its processing was inhibited by the antibiotic globomycin and it was released from whole cells by treatment with trypsin. The presence of DNA sequences homologous to the S. equisimilis lppC gene were confirmed by PCR. These data strongly suggest that Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus produce a lipoprotein acid phosphatase homologous to LppC of S. equisimilis.

Acid Phosphatase↗

The molecular basis of Streptococcus equi infection and disease.

Streptococcus equi is the aetiological agent of strangles, one of the most prevalent diseases of the horse. The animal suffering and economic burden associated with this disease necessitate effective treatment. Current antibiotic therapy is often ineffective and thus recent attention has focused on vaccine development. A systematic understanding of S. equi virulence, leading to the identification of targets to which protective immunity can be directed, is a prerequisite of the development of such a vaccine. Here, the virulence factors of S. equi are reviewed.

Animals↗

Localization and characterization of the ligand-binding domain of the fibrinogen-binding protein (FgBP) of Streptococcus equi subsp. equi.

The group C streptococcus Streptococcus equi subsp. equi possesses a 498-residue major cell-wall-associated protein (FgBP) which binds horse fibrinogen (Fg), reacts with convalescent horse serum and protects against lethal S. equi challenge in a small animal model. In the present study, analysis of a panel of 17 purified N- and C-terminal FgBP truncates by ligand affinity blotting and SDS-PAGE revealed that the region required for maximum binding of Fg extended over the first half of the mature protein. The C-terminal two-thirds of this domain is predicted to be alpha-helical coiled-coil and the N-terminal one-third to possess non-coiled-coil single strands. Residues at the extreme N-terminus and within the coiled-coil region are both required for ligand binding. A high incidence of alpha-helical coiled-coil structure also seems to be responsible in part for the aberrant mobility of FgBP on SDS gels. The efficiency with which FgBP binds Fg from different animal species decreases in the order horse > mouse, pig > rat > sheep, dog, bovine, human. Binding to horse Fg is inversely related to temperature over the range 45-4 degrees C and is independent of Ca2+ ions. MS analysis provided corroborative evidence that FgBP is covalently linked to the cell wall peptidoglycan.

Animals↗

Construction of a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain.

Streptococcus equi causes equine strangles, a purulent lymphadenopathy of the head and neck. An avirulent, non-encapsulated strain (Pinnacle) has been used widely in North America as an intranasal vaccine. The aim of the study was to create a specific mutation of the hyaluronate synthase (hasA) gene in Pinnacle to permanently abolish the production of capsule and provide an easily recognisable genetic marker. An internal fragment of hasA was generated by PCR and cloned into pTW100 (Microscience, UK). An encapsulated revertant of Pinnacle was then transformed with the recombinant plasmid by electroporation and cultured under conditions to promote homologous recombination. Among 90 spectinomycin resistant transformants observed, one non-mucoid (non-encapsulated) spectinomycin resistant colony was detected. The presence of plasmid sequence within the hasA gene was confirmed by the PCR. After six passages in antibiotic-free medium, four non-mucoid spectinomycin sensitive colonies were found. Sequence analysis of one of these clones, designated Pinnacle HasNeg, revealed loss of the 3' end of the hasA and the 5' end of the hasB genes. This deletion mutant should serve as a useful candidate to replace Pinnacle since it cannot revert to a mucoid phenotype and can be distinguished genetically from wild type strains.

Animals↗

Characterization of the haemolytic activity of Streptococcus equi.

The haemolytic activity of Streptococcus equi, the cause of equine strangles, was characterized. Production of haemolysin in Todd Hewitt broth was dependent on an equine serum supplement and the logarithmic phase of growth after which activity declined sharply. RNA core also induced haemolysin production from cells harvested at the end of the logarithmic phase of growth. Haemolysis was not affected by cholesterol, was only slightly increased in reducing conditions and was completely inactivated by trypan blue, identifying the haemolytic activity as streptolysin S-like (SLS-like). Purification by hydroxyapatite and Sephacryl column chromatography yielded proteins of molecular weights of approximately 6000 and 17 000-22 000 Da with a 64-fold increase in specific activity. Low molecular weight proteins from the RNA core were still present in the purified toxin. Two non-haemolytic mutants were derived by conjugation with an Enterococcus faecalis-carrying transposon Tn916. Southern blots of HindIII digests of DNA revealed that one of the mutants contained three transposon insertions and the other just one. A lambda phage library of S. equi contained plaques whose haemolytic activity was enhanced by reducing conditions and inhibited by cholesterol, suggesting a streptolysin O-like (SLO-like) activity. However, haemolysin in culture sonicates of host E. coli in which the lambda phage insert was subcloned into plasmid (pUC18), was not affected by these conditions. Seven isolates of S. equi in medium without SLS-like inducers showed no SLO-like activity and no evidence for an SLO-like toxin could be found by immunoblotting with pneumolysin antiserum and monoclonal antibodies or by polymerase chain reaction with primers derived from sequences conserved between the SLO genes of Lancefield group A, C and G streptococci. S. equi does not appear to possess a streptolysin O but does make a streptolysin S-like toxin whose production can be interrupted at just one genetic locus.

Antibodies, Bacterial↗

Studies in atypical Streptococcus equi.

An atypical variety of Streptococcus equi is described. It was shown to be deficient in capsular material, to be very virulent for mice and to possess a cell-wall protein similar to the M-like protein of classical Str equi. Antiserum prepared against classical Str equi effectively opsonised the atypical strains, and induced the formation of long chains by these atypical strains. It is possible that this variant of Str equi can be used to overcome many of the current problems associated with the manufacture and use of strangles vaccines.

Animals↗

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

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

Journal Article↗

Evaluation of a nested PCR test and bacterial culture of swabs from the nasal passages and from abscesses in relation to diagnosis of Streptococcus equi infection (strangles).

REASONS FOR PERFORMING STUDY: Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. OBJECTIVES: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. METHODS: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or from abscesses from horses infected with S. equi (diagnostic sensitivity). RESULTS: All 45 S. equi isolates tested positive in the nested PCR, whereas no amplicon was formed when testing the other 120 Lancefield group C isolates. A total of 43 samples were collected from 11 horses showing clinical signs of strangles during the study period. The diagnostic sensitivity for PCR test was 45% and 80% for samples from the nasal passages and abscesses, respectively; the corresponding diagnostic sensitivity for cultivation was 18% and 20%. The diagnostic sensitivity was significantly higher for PCR than for bacterial cultivation. Furthermore, the shedding of S. equi in 2 infected horse populations was evaluated. An intermittent shedding period of S. equi of up to 15 weeks was recorded in this part of the study. It was also shown that shedding of S. equi occurred both from horses with and without clinical signs. CONCLUSIONS AND POTENTIAL RELEVANCE: The nested PCR test represents a species-specific and -sensitive method for diagnosis of S. equi from clinical samples. It may, however, be desirable in future to develop detection methods with high diagnostic sensitivity and specificity without the potential problems inherent in nested PCR.

Abscess↗

Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.

Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity.

Amino Acid Sequence↗

Immunologically reactive proteins of Streptococcus equi.

Immunologically reactive proteins in acid extracts and culture supernatants of Streptococcus equi were recognized through a combination of chromatographic and immunologic procedures. Both high- and low-molecular-weight components of each of these protein preparations were protective for mice and were, therefore, presumed to contain a variety of hydrolytic products or fragments of the M protein of S. equi. Convalescent horse sera that exhibited strong bactericidal activity for S. equi always reacted with polypeptides in the molecular weight range of 24,000 to 29,000, whereas preinfection sera did not. Rabbit antisera to affinity-purified S. equi protein also reacted with these polypeptides, as well as with a polypeptide of about 36,000 to 37,000 molecular weight. M protein in acid extract and culture supernatant did not cross-react in immunodiffusion, but rabbit antiserum to affinity-purified M protein from an acid extract of S. equi reacted strongly with culture supernatant proteins of approximate molecular weights of 67,000, 58,000, and 43,000. We suggest, therefore, that the M protein in culture supernatant is masked by other sequences that are removed by hot acid during preparation of acid extracts. Polypeptides common to acid extracts of S. equi and Streptococcus zooepidemicus were also identified. These polypeptides had molecular weights of about 55,000 and 31,000.

Animals↗

Description of an epizootic and persistence of Streptococcus equi infections in horses.

The age-specific attack rates of Streptococcus equi infections of the upper respiratory tract and lymph nodes (strangles) in horses for the different age groups were 17.6% for broodmares, 47.5% for 1-year-old horses, and 37.5% for foals. Streptococcus equi was isolated from nasal, pharyngeal, or lymph node specimens in 31 (60.8%) of 51 sick horses. A male 1-year-old horse, shipped from Kentucky to farm A, was considered to be the index case. Six (19.4%) of 31 horses with strangles remained as shedders of S equi after clinical signs of the disease had ended. Shedders of S equi were not identified from horses that were exposed to infected horses but never developed strangles.

Animals↗

Identification of carriers of Streptococcus equi in a naturally infected herd.

During an outbreak of strangles in a population of research horses, 4 mares were identified as carriers of Streptococcus equi. Three of the mares had typical signs of strangles (severe regional lymphadenitis with or without rupture of abscessed lymph nodes). The 4th mare experienced episodes of serous to mucopurulent nasal discharge, but never had more than a mild degree of lymph node enlargement. Streptococcus equi was isolated from the abscessed lymph nodes and from nasopharyngeal swab specimens from the first 3 mares from 6 to 19 weeks after rupture of involved nodes. Streptococcus equi was isolated from the nasopharynx of the 4th mare on introduction into the herd and intermittently over the ensuing 6 months. During the 7th month, mare 4 was placed in isolation, where she continued to shed S equi for 4 more months. A complete physical examination during the 10th month, including radiography of the head and thorax, did not reveal any relevant abnormalities, but a pharyngeal swab specimen was culture-positive for S equi. This isolate was used to inoculate 2 yearling colts, which developed strangles and from which S equi was reisolated. Shedding of S equi by mare 4 ceased in the 11th month, and at necropsy 2 months later, S equi was not recovered from any organ or tissue. Corticosteroid administration 3 weeks prior to necropsy had induced neither shedding of the organism nor clinical signs of strangles. The study provided clinical, epidemiologic, and bacteriologic evidence to support the existence of a carrier state following natural infection with S equi.

Abscess↗

Characterization of the antiphagocytic activity of equine fibrinogen for Streptococcus equi subsp. equi.

The antiphagocytic property of equine fibrinogen for Streptococcus equi subsp. equi strain CF32 was examined in vitro. The results of bactericidal assays demonstrated that the presence of fibrinogen enhanced the ability of overnight and early log-phase cultures of strain CF32 to resist killing by equine neutrophils by 12-fold and seven-fold, respectively (p > 0.01). In addition, fibrinogen-coated bacteria treated with fibrinogen specific F(ab')2 fragments were 32% more susceptible to killing by equine neutrophils after opsonization in serum (p > 0.05), indicating that specific epitopes on fibrinogen may be important for its antiphagocytic effect. Since complement deposition is inhibited on subsp. equi (Boschwitz JS, Timoney JF, Infect Immun 1994; 42, 3515-20, we examined the effect of fibrinogen on complement deposition by using colloidal gold labeling of surface-bound C3. No significant differences were detected in the quantity of C3 deposited on the cell surface after opsonization with serum, serum plus fibrinogen, or plasma. These results suggest that the antiphagocytic property of fibrinogen is not related to the inhibition of complement deposition on the bacterial surface. Pretreatment of CF32 with M protein specific antibody inhibited fibrinogen binding by 72%, and a strain of subsp. equi expressing low levels of M protein bound 64% less fibrinogen than CF32, suggesting that the some of the fibrinogen deposited on the surface of subsp. equi is bound to M protein.

Animals↗

Purification and antigenicity of an M-like protein of Streptococcus equi.

A cell wall component of Streptococcus equi analogous to the M protein of group A streptococci has been identified and purified. A highly purified product has been obtained from cells by hot acid extraction, followed by acid precipitation, ammonium sulfate fractionation, and column chromatography. This product reacts with S. equi antiserum. The existence of this fraction in S. equi has been confirmed by the failure of trypsin-treated cells and their extracts to remove the long-chaining capacity of S. equi antiserum. The antigenicity of this M-like protein when incorporated in adjuvant has been assessed in rabbits and horses. In the rabbit, multiple doses of as low as 10 mug resulted in the production of serum capable of inducing long-chain formation and opsonizing and mouse protective antibody. Two doses of 100 mug elicited similar responses. The bactericidal capacity of this serum could be eliminated by absorption with whole S. equi cells or their extracts, but not by absorption with trypsin-treated cells or extracts therefrom. Horses vaccinated 15 months previously with a whole-cell killed vaccine and given 200 mug of the purified protein intramuscularly showed evidence of an anamnestic response to the antigen.

Absorption↗

Detection of DNA restriction fragment polymorphisms in Streptococcus equi.

Large-restriction-fragment (LRF) polymorphisms in Streptococcus equi (S equi subspecies equi) were studied by pulsed-field gel electrophoresis. Five or six chromosomal fragments of between 194 and 915 kb were separated by digestion with the restriction endonuclease Notl. All 20 isolates of S equi, including 12 from independent Japanese outbreaks, four from independent American outbreaks, two from a single Irish outbreak, us vaccine strain F43, and type strain NCTC 9682 were successfully typed. Seven distinctive, reproducible and stable types were identified. The 12 Japanese isolates collected between 1992 and 1998 were of LRF type II suggesting that they were derived from the same source. The remaining eight isolates were of six types. The results indicate that LRF typing should be a useful technique for investigating the source and transmission of S equi.

Animals↗

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

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

Animals↗

Microencapsulation of Streptococcus equi antigens in biodegradable microspheres and preliminary immunisation studies.

Streptococcus equi subspecies equi is the causative agent of strangles, a bacterial infection of the respiratory tract of equidae. Current strategies to prevent strangles rely on antimicrobial therapy or immunisation with inactivated bacteria, S. equi bacterin, or M-like protein (SeM) extract. The aim of this work was to investigate whether immunisation with whole killed S. equi or a bacterial lysate entrapped in poly(lactide-co-glycolide) (PLGA) microspheres might induce protective immunity to mice. Animals were treated with a dose of antigen equivalent to 25 microg of SeM. For intranasal route animals were primed on days 1, 2 and 3 and were boosted on day 29. For intramuscular route, primary immunisation was carried out with a single injection on day 1 and animals were boosted on day 29. On day 43 animals were submitted to a challenge with a virulent strain of S. equi. Vaccination with antigen-containing microspheres induced higher serum antibody levels in mice treated by the intranasal route, whereas intramuscular immunisation did not reveal any difference between control and treatment groups. Microencapsulated antigens achieved to fully protect mice against experimental infection irrespective of the route of administration used. Following intranasal or intramuscular administration soluble antigen failed to protect mice against challenge. These studies indicate that PLGA microspheres are a potential carrier system for the delivery of S. equi antigens.

Administration, Intranasal↗

Inhibition of hyaluronan synthesis in Streptococcus equi FM100 by 4-methylumbelliferone.

As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production was also observed in group C Streptococcus equi FM100 cells treated with 4-methylumbelliferone (MU), although there was no effect on their growth. In this study, the inhibition mechanism of hyaluronan synthesis by MU was examined using Streptococcus equi FM100, as a model. When MU was added to a reaction mixture containing the two sugar nucleotide donors and a membrane-rich fraction as an enzyme source in a cell-free hyaluronan synthesis experiment, there was no change in the production of hyaluronan. On the contrary, when MU was added to the culture medium of FM100 cells, hyaluronan production in the isolated membranes was decreased in a dose-dependent manner. However, when the effect of MU on the expression level of hyaluronan synthase was examined, MU did not decrease either the mRNA level of the has operon containing the hyaluronan synthase gene or the protein level of hyaluronan synthase. Solubilization of the enzyme from membranes of MU-treated cells and addition of the exogenous phospholipid, cardiolipin, rescued hyaluronan synthase activity. In the mass spectrometric analysis of the membrane phospholipids from FM100 cells treated with MU, changes were observed in the distribution of only cardiolipin species but not of the other major phospholipid, PtdGro. These results suggest that MU treatment may cause a decrease in hyaluronan synthase activity by altering the lipid environment of membranes, especially the distribution of different cardiolipin species, surrounding hyaluronan synthase.

Cell-Free System↗