Biomedical subjects

J F Timoney

Publications and source records attributed to J F Timoney.

At least 19 recordsLinked to original sources

PubMed1996

Characterisation of murine monoclonal antibodies recognising opsonic, mouse-protective, chaining and mucosally relevant epitopes on the M protein of Streptococcus equi subspecies equi.

Six hybridomas secreting murine monoclonal antibodies (mAbs) specific for the M protein of Streptococcus equi subspecies equi were characterised. The mAbs recognised the major 41 and 46 kDa fragments of M protein in an acid extract of S equi and the 56 and 58 kDa dimer of the native molecule in a mutanolysin extract, but did not react with recombinant M-like protein of S equi subspecies zooepidemicus. One mAb (2A10) showed strong opsonic activity for S equi and protected mice against an experimental challenge with virulent S equi. Two other mAbs were mouse-protective but not opsonic. All the mAbs elicited a strong chaining response from S equi, but had only a weak chaining effect on a strain of S equi (19) that expressed only 4 per cent of the normal amount of M protein. Antibodies in nasopharyngeal mucus of horses recently recovered from strangles were inhibited to different extents by each mAb. These different functional behaviours and the result of inhibition ELISAs suggest that the M protein of S equi carries multiple epitopes.

Animals↗

PubMed1996

The use of gadolinium-BOPTA on magnetic resonance imaging in brain infection.

RATIONALE AND OBJECTIVES: The use of gadolinium (Gd)-BOPTA as a magnetic resonance contrast agent for central nervous system disease was studied in a canine brain abscess model. METHODS: A Streptococcus faecalis brain abscess was evaluated in five dogs at 1.5T. Imaging was performed during the late cerebritis stage, at 5 to 7 days after surgery. Magnetic resonance scans were acquired before and at 1, 5, 15, 30, 45, and 60 minutes after contrast administration, using a dose of 0.1 mmol/kg. Scans also were acquired both before and after contrast injection with the implementation of magnetization transfer. RESULTS: Lesion enhancement, quantified by region-of-interest measurement, peaked at 5 minutes after contrast injection. Both the increase in lesion enhancement from 1 to 5 minutes after injection and the decrease from 5 to 15 minutes after injection, although small, were statistically significant (P < 0.004 and P < 0.03, respectively). The application of magnetization transfer improved lesion enhancement, as measured by signal difference/noise, by 39%. This result also was statistically significant (P < 0.001). CONCLUSIONS: In intraparenchymal brain infection, Gd-BOPTA provides effective lesion enhancement when used at a dose of 0.1 mmol/kg. Further research is needed to compare the magnitude of enhancement achieved with Gd-BOPTA, which has weak protein binding and both hepatobiliary and renal excretion, with that with Gd chelates, which have pure renal excretion.

Animals↗

PubMed1995

Detectability of early brain meningitis with magnetic resonance imaging.

RATIONALE AND OBJECTIVES: The ability of high-field (1.5 T) magnetic resonance imaging (MRI) to detect early brain meningitis was evaluated in a canine model. Contrast dose, timing postinjection, and imaging technique (specifically the use of magnetization transfer) were assessed. METHODS: Imaging of five canines was performed at 1.5 T 24 hours after injection of Cowans staphylococcus into the cisterna magna. Two control animals also were imaged using the same protocol, with one animal receiving a cisternal injection of nutrient broth only and the other no injection. Contrast doses of 0.1, 0.3, and 0.8 mmol/kg gadoteridol (Gd HP-DO3A or Pro-Hance) were compared. Scans were performed at 2, 12, and 22 minutes after an initial injection of 0.1 mmol/kg. At each time point, paired T1-weighted scans with and without magnetization transfer (MT) were acquired. Thirty minutes after the initial injection of contrast, a supplemental dose of 0.2 mmol/kg was given (for a cumulative dose of 0.3 mmol/kg). Scans were then repeated at 2, 12, and 22 minutes after this dose was administered. A second supplemental contrast injection of 0.5 mmol/kg (for a cumulative dose of 0.8 mmol/kg) was given at 70 minutes, and immediate postinjection scans with and without MT were acquired. RESULTS: In the animals receiving a cisternal injection of bacteria, the degree of meningeal enhancement was greatest at 0.8 mmol/kg, intermediate at 0.3 mmol/kg, and least at 0.1 mmol/kg. These conclusions were constant whether imaging was performed with or without MT. Scans in control studies did not demonstrate abnormal meningeal enhancement. High-contrast dose, MT, and acquisition of immediate postcontrast scans all resulted in statistically significant improvement. On masked film review, abnormal meningeal enhancement was noted in only 2 of 5 experimental dogs at a dose of 0.1 mmol/kg (regardless of the use of MT) compared with all animals at a dose of 0.3 mmol/kg. In 18 of 37 dogs (paired scans with and without MT), when abnormal enhancement was noted, the use of MT improved the visualization of abnormal meningeal enhancement. CONCLUSIONS: In early brain meningitis, high-contrast dose (0.3 mmol/kg), MT, and scanning immediately after injection improve detection of abnormal meningeal enhancement, thus facilitating the diagnosis of meningitis. Of these factors, contrast dose is the most important.

Animals↗

PubMed1995

Cloning and sequence analysis of a protective M-like protein gene from Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus, a Lancefield group C streptococcus, is a frequently isolated opportunist pathogen from a variety of animal hosts, including the horse. Previous studies have indicated that equine strains carry antigens with characteristics of the antiphagocytic M proteins on the Lancefield groups A and G streptococci. We have cloned a protective M-like protein gene (SzPW60) of an equine strain of S. equi subsp. zooepidemicus W60 and determined its sequence. This gene encodes a protein with a molecular weight of 40,123 which protects mice against subsp. zooepidemicus but not subsp. equi, stimulates antibodies which opsonize subsp. zooepidemicus but not equi, and reacts with antiserum to the protein of the parent strain. The predicted amino acid structure shows significant homology with the carboxy termini of groups A and G M proteins but no other homology. The M-like protein, although showing an extensive region of alpha helix, lacks the A, B, and C repeats found in group A M proteins and has a shorter signal sequence. A proline-rich region upstream from the LPSTGE motif contains 20 repeats of the tetrapeptide PEPK. The presence of this repeat region may account for the slow migration of the M-like protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗

PubMed1994

Epidemiologic and immunologic characteristics of Streptococcus equi infection in foals.

A 2-phase study was performed to characterize the effects of Streptococcus equi infection in unexposed and previously exposed foals. In phase I, 22 weanling foals involved in a naturally occurring S equi epizootic were studied, along with a comparison group of 11 unexposed foals, matched for age, sex, and breed. Six months later (phase II), an epizootic was experimentally induced in previously exposed and unexposed foals from phase I. The prevalence and duration of clinical signs, the relative risk of developing disease, bacteriologic culture results, hematologic responses, and mucosal and serum immunologic responses were determined. Disease protection in phase-I and -II foals was associated with high values for serum S equi M protein-specific IgG at the onset of the epizootic (P < 0.02 for phase 1 and P < 0.01 for phase II), and with a rapid (within 2 weeks of exposure) mucosal S equi M protein-specific IgG response (P < 0.05 for phase I and P = 0.01 for phase II).

Analysis of Variance↗

PubMed1994

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↗

PubMed1994

Biochemical differences among human and animal streptococci of Lancefield group C or group G.

Pyogenic streptococci of Lancefield group C or group G from human or animal sources were examined with a view to increasing the number of diagnostic tests useful for their differentiation. Human strains of group G produced L-prolyl-L-arginine aminopeptidase but isolates of Streptococcus equisimilis (group C) did not. Tests for alpha-L-glutamate aminopeptidase together with fermentation of glycogen or sorbitol distinguished S. dysgalactiae from strains of S. equisimilis isolated from animals. It was confirmed that fermentation tests were helpful in the study of S. equi and S. zooepidemicus and that enzyme reactions helped distinguish between S. canis and the human strains of group G.

Aminopeptidases↗

PubMed1994

Inhibition of C3 deposition on Streptococcus equi subsp. equi by M protein: a mechanism for survival in equine blood.

The effect of the M protein of Streptococcus equi subsp. equi on complement deposition, complement degradation, and bacterial survival in equine whole blood was examined in vitro. Preincubation of bacteria with rabbit M protein-specific immunoglobulin G (IgG) inhibited the survival of the M+ strain in whole blood by 20-fold (P < 0.01). In addition, preincubation of bacteria with M protein-specific F(ab')2 fragments inhibited the survival of M+ cells in whole blood by 3.8-fold (P < 0.01). In the absence of specific antibody, an M+ strain (CF32) of S. equi subsp. equi survived 100-fold better in whole blood than an M- isolate (strain 19) (P < 0.01). Complement inactivation by cobra venom factor significantly enhanced the ability of the M- and M+ strains of S. equi subsp. equi to survive in whole blood, the latter in the presence or absence of M protein-specific IgG. The major opsonic forms of C3, C3b and iC3b, were present on both M- and M+ cells after opsonization in nonimmune plasma. However, colloidal gold staining indicated that the M- strain bound four times as much C3 as the M+ strain (P < 0.02) and that preincubation of the M+ strain with M protein-specific IgG or F(ab')2 fragments also enhanced the amount of C3 deposited by a factor of 4 (P < 0.02). Therefore, at least part of the M protein's ability to enhance bacterial survival in equine whole blood may be related to its ability to interfere with the deposition of equine complement on the bacterial surface.

Animals↗

PubMed1993

An R-like protein of Streptococcus uberis stimulates opsonising antibodies.

A pepsin-sensitive R-like antigen with a molecular mass of 65 kilodaltons was extracted from Streptococcus uberis strain ATCC 19436 by trypsinisation and purified by diethyl-aminoethanol anionic exchange chromatography. The antigen reacted with sera from infected cows and stimulated opsonic antibody in the guinea pig. The amino acid composition of the antigen was generally similar to that previously reported for the R antigen of group C streptococci.

Animals↗

PubMed1993

Purification and characterization of equine complement factor C3.

A rapid method for purifying equine C3 which yields milligram quantities of pure C3 is described. Protein from equine plasma was selectively precipitated with polyethylene glycol, and the C3 was purified by anionic and cationic exchange HPLC. The yield from this procedure was 12%. The purified C3 was composed of an alpha chain (118 kD) and a beta chain (68 kD) linked by at least one disulfide bond, and it had an isoelectric point of 4.7. Amino acid analysis indicated a strong conservation of amino acid usage between equine and human C3. The N-terminal sequences of the alpha and beta chains were homologous to human, mouse, and rat C3, and activation of C3 produced breakdown products similar in molecular weight to C3b and iC3b of other species. Equine C3 appeared to be functionally dependent upon a reactive thiolester as treatment of fresh equine serum with methylamine abrogated its hemolytic activity.

Amino Acid Sequence↗

PubMed1993

Properties of a protective protein antigen of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae is a widely distributed mucosal commensal of the alimentary tracts of vertebrates. Antibodies to a 66-64 kDa protein released from the cell surface have been shown to be involved in protective immunity. Mice immunized with the purified 66-64 kDa protein from strain T28, serotype 2b were protected against challenge by the United States challenge strain E1-6P (serotype 1a) and by the official German challenge strain Frankfurt 1 (serotype N). Thus, protection is not serotype specific, a result consistent with previous observations that polysaccharide, non-proteinaceous antigens are the type specific antigens useful in serotyping. The 66-64 kDa protein appears to be most immunogenic when complexed to glycolipid. This may be due to an adjuvant effect of polysaccharide antigens. Further studies on the correlation between antibody titers to the 66-64 kDa protein and protection in pigs, turkeys and mice in vivo should be helpful in developing a basis for an in vitro assay to replace the mouse protection test in vaccine testing.

Animals↗

PubMed1993

The protective M proteins of the equine group C streptococci.

The group C streptococci are the most commonly isolated bacteria from disease states in the horse. Important virulence factors of S. equi and S. zooepidemicus are the hyaluronic acid capsule and the antiphagocytic fibrillar M protein located on the surface of the cell wall and extending into and through the capsule. The hyaluronic acid capsule is non-antigenic and so is not involved in protective immunity. The M protein, a superantigen, elicits very strong B and T cell responses that may result in protective immunity mediated by opsonic antibodies in plasma and by locally synthesized IgG and IgA on the pharyngeal mucosa. However, vaccines based on acid or mutanolysin extracted M protein do not confer a high level of protection against field exposure. Protective antibodies to S. equi or S. zooepidemicus can in part be assayed by the bactericidal test that measures opsonization for equine neutrophils. A mouse-challenge model has also been used to test immunizing potency of streptococcal extracts and in a passive protection test for protective antibody. There is as yet no means of distinguishing protective opsonic or mucosal antibodies from other antibodies produced against the many epitopes on the M molecule.

Animals↗

PubMed1993

Strangles.

The etiology, epizootiology, pathogenesis, and clinical presentation of strangles are described. Streptococcus equi, the causative organism, is highly host-adapted to Equidae and shows no antigenic variation. Protective immunity apparently is mediated by a combination of serum opsonic and nasopharyngeal mucosal humoral responses. Vaccines based on M protein or inactivated bacterial suspensions may reduce the clinical attack rate by 50%, a level of protection much lower than that produced during recovery from strangles.

Animals↗

PubMed1992

Equine glomerulonephritis and renal failure associated with complexes of group-C streptococcal antigen and IgG antibody.

A 12-year-old thoroughbred gelding died from diffuse global glomerulonephritis, 3 months after a lower respiratory infection from which Streptococcus zooepidemicus was isolated. Immunopathological studies (immunofluorescence, immunodiffusion, immunoperoxidase testing and immunoblotting) indicated the presence of an immune reactant renal disease associated with IgG antibody and streptococcal antigens.

Animals↗

PubMed1991

A convenient gel holder for preparative electrophoretic separation of aggregated bacterial proteins.

A simple custom-made gel holder for preparative SDS-PAGE to separate aggregated bacterial antigens is described. The gel holder fits easily into commercially available gel electrophoresis apparatus and proteins or peptides are collected in a stream of distilled water through a channel in the gel. The performance of the device was illustrated by the successful separation and purification of fragments of aggregated Streptococcus equi M protein and of Erysipelothrix rhusiopathiae protective antigens. Yields of up to 1.2 mg per run were obtained. The purified proteins retained immunological reactivity and were of sufficient purity for amino acid compositional and sequence analysis.

Antigens, Bacterial↗

PubMed1991

Antigenic and genetic homogeneity of Streptococcus uberis strains from the bovine udder.

DNA- fingerprints (Hind III) of Streptococcus uberis field isolates from New York State and Europe showed substantial homogeneity, but were different to those of the type strain of the newly proposed psychrophilic species S. parauberis. S. uberis strains had major SDS-heat extracted antigens of molecular masses (Mr) less than 14, 40-41, 42-43, 59-61, 80-86 and 118-122 kDa following immunoblotting with rabbit hyperimmune sera. Bovine sera and milk reacted with the 40-41 and 118-122 kDa antigens. Variations in the Mr of particular bands were too unevenly distributed to permit formation of subgroups. Although cross reactive, the sizes of the antigens of S. parauberis strain NCDO 2020 were substantially different to those of S. uberis, the most prominent antigen having a Mr of 50 kDa. The antigenic and genetic data therefore strongly support the introduction of S. parauberis as a distinct species. S. uberis strains reacted with antiserum to Lancefield groups B, E, G and P, their grouping reactions showing no correlation with DNA and immunoblot fingerprints. Lancefield grouping of S. uberis therefore appears to have little value in identification.

Animals↗

PubMed1991

Characterization of a protective protein antigen of Erysipelothrix rhusiopathiae.

Although vaccination is widely practiced against infection by Erysipelothrix rhusiopathiae in pigs and turkeys, the protective antigen(s) involved have not been fully characterized or purified to homogeneity. Antigens of E. rhusiopathiae strain T28, serotype 2b, and of FRANKFURT XI, serotype N, in culture supernatant and in extracts made with hot acid, 10 mM NaOH, ultrasound or EDTA were compared by SDS-PAGE and immunoblotting and in a mouse protection test. EDTA and 10 mM NaOH yielded highly protective extracts; culture supernatant was less protective and ultrasonic or hot acid extracts stimulated little or no protection in mice. Protective antisera from swine, horses and mice recognized prominent bands of molecular mass (m.m.) of 66-64 and 40-39 kDa in EDTA and 10 mM NaOH extracts. Mice immunized with preparations of the 66-64 kDa band purified by preparative electrophoresis were protected. Both antigens were trypsin sensitive, contained no detectable polysaccharide, and showed a marked tendency to aggregate in the absence of SDS.

Animals↗

PubMed1991

A comparison of different methods of extraction of the M-protein from Streptococcus equi.

The molecular weights of the proteins produced in different extracts of Streptococcus equi were compared on immunoblots with antisera against acid extracted and mutanolysin extracted M-protein. Acid and alkaline extracts of S. equi contained some peptides of similar molecular weight that reacted with antiserum against an acid extracted 41,000 m.w. fragment suggesting that these fragments contained common epitopes. Comparison of the amino acid compositions of the 35,000 m.w. fragment of the alkaline extract and the 41,000 m.w. fragment of the acid extract suggest that these immunologically reactive fragments were probably derived from the same protein. Little cross-reactivity was observed between antisera against S. equi acid extracted protein and the native 58,000 m.w. M-protein. This suggests that conformational epitopes on the native M molecule are not present after acid treatment.

Amino Acids↗