Genetic relatedness and virulence gene profiles of Escherichia coli strains isolated from septicaemic and uroseptic patients.

We investigated the relationship between clonality and virulence factors (VFs) of a collection of Escherichia coli strains isolated from septicaemic and uroseptic patients with respect to their origin of translocation. Forty septicaemic and 30 uroseptic strains of E. coli were tested for their phylogenetic groupings, genetic relatedness using randomly amplified polymorphic DNA (RAPD), biochemical fingerprinting method (biochemical phenotypes [BPTs]), adherence to HT-29 cells and the presence of 56 E. coli VF genes. Strains belonging to phylogenetic groups B2 and D constituted 93% of all strains. Fifty-four (77%) strains belonged to two major BPT/RAPD clusters (A and B), with cluster A carrying significantly (P = 0.0099) more uroseptic strains. The degree of adhesion to HT-29 cells of uroseptic strains was significantly (P = 0.0012) greater than that of septicaemic strains. Of the 56 VF genes tested, pap genes was the only group that were found significantly (P < 0.0001) more often among uroseptic isolates. Phylogenetic group B2 contained a significantly higher number of strains carrying pap genes than those in group D. We conclude that uroseptic E. coli are clonally different from septicaemic strains, carry more pap genes and predominantly adhere more to the HT-29 cell model of the gut.

Evaluation of Erysipelothrix rhusiopathiae vaccines in pigs by intradermal challenge and immune responses.

In a vaccine trial, pigs were challenged intradermally with eight E. rhusiopathiae strains of serovars 1a, 1b or 2 given concurrently. The strains were derived from six herds affected with vaccine breakdowns in 1997-1999, one herd without vaccine breakdown and a serovar 2 reference strain. Responses to two commercial bacterins (one implicated in the vaccine breakdowns), and two experimental bacterins (based on field isolates from affected herds) showed distinct differences in protection, particularly in clinical responses measured at 72 h. Less protection was afforded against serovar 1 challenge by the vaccine implicated in the vaccine breakdowns. Antibody and cell-mediated immune (CMI) responses were significantly different between treatments, and highlighted a similar post-vaccinal antibody response was produced against serovar 2 lysate by all vaccines, but only those providing significant protection against serovar 1 [corrected] produced significantly elevated antiserovar I lysate [corrected] antibodies. Vaccination in general significantly reduced CMI responses to the mitogens concanavalin A and phytohaemagglutinin. This experimental pig challenge system was readily able to confirm suboptimal performance of a commercial bacterin that had passed potency tests in mice but was associated with vaccine failure in commercial herds. This vaccine was also the most immunosuppressive to CMI responses associated with E. rhusiopathiae-specific and non-specific stimulation. The best vaccine response was associated with the highest mean serovar 1 antibody response and the highest CMI response (by lymphoproliferation assay) to serovar 2.

Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis.

Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.

Relationship between the immune response of sheep and the population dynamics of bacteria isolated from fleecerot lesions.

In sheep wetted by rain, proliferation of bacteria in the skin-fleece microenvironment invariably discolours the fleece and causes a dermatitic condition known as fleecerot. The changes in population dynamics of fleece bacteria were analysed by carrying out skin washings at randomly selected sites on the back of sheep before, and at 48 h and 96 h after exposure to rain. Gram-positive rods belonging to Bacillus species (10(2)-10(4) cfu/cm2) predominated in dry fleece. Gram-positive cocci (e.g. Micrococcus and Staphylococcus species) as well as Gram-negative rods (pseudomonads) were also present but in lower abundance (less than 10(2) cfu/cm2). Fleece bacterial populations generally increased in numbers during the first 24-48 h of wetting. By 96 h however, skin washings showed a preponderance of Pseudomonas aeruginosa (10(4)-10(6) cfu/cm2) and to a lesser extent, pigmented Micrococcus species. Growth of fleece bacteria was associated with a characteristic green or yellow/orange staining of fleece. Fewer species of bacteria were isolated from sheep showing green staining while those animals with yellow/orange discolourations appeared to have a more mixed microflora composition. The predominance of P. aeruginosa in the wet fleece of sheep displaying either green or yellow/orange bacterial stain, was accompanied by a significant serological response against this species. Since skin bacteria have never been observed to penetrate cutaneously in skin sections biopsied from fleecerot sites, it must be concluded that the sheep skin is sensitized by continuous exposure to antigens that are associated with or released by P. aeruginosa.

Comparison of native and subunit antigens as ELISA reagents for the detection of antibodies against scabby mouth virus.

A simple ELISA test to detect antibodies against scabby mouth virus (SMV) has been developed. Native whole virions and subunits of SMV generated by boiling the virus in the presence of sodium dodecyl sulphate (SDS) detergent and beta-mercaptoethanol were compared as ELISA assay reagents using naive and hyperimmune sera from sheep and rabbits. Approximately 2 x 10(4) intact virus particles per microtiter well were required to generate a positive to negative signal of 0.8:0.3 ELISA O.D. units when the serum was used at a dilution of 1/100. In contrast, total subunit antigen generated by disrupting and coupling of 250-500 virions per well provided a signal ratio of 1.58:0.3 ELISA O.D. units at a serum dilution of 1/250. Total subunit antigens were therefore 400 times more economical to use than intact virions. In addition, subunit antigens could be readily bound to microtiter plates without the need for removal of the SDS. Secondly, it was not necessary to block non-specific binding sites on the plate with blockers such as gelatin and skim-milk, thereby shortening the time needed to complete the ELISA assay. The total subunit antigen ELISA test was used to detect seroconversion in new born lambs where there was an occurrence of SMV infection in housed sheep. Three bleeds were taken at fortnightly intervals and the ELISA results showed that 9 out of 15 lambs were seropositive for all bleed points. Four of the lambs showed a sequential rise in titer while only one lamb failed to seroconvert.(ABSTRACT TRUNCATED AT 250 WORDS)

Serological assay for swine erysipelas using nitrocellulose particles impregnated with an immunodominant 65 kDa antigen from Erysipelothrix rhusiopathiae.

Pigs (n = 10) that were experimentally challenged with an arthritogenic isolate of Erysipelothrix rhusiopathiae (strain VRS 229; serotype 1a) developed arthritis in at least one of twelve major limb joints. Immunoblots using sera obtained from these pigs at necropsy revealed a major band of immunoreactivity against a subunit polypeptide of apparent molecular mass 65 kDa. The usefulness of the 65 kDa immunodominant subunit as an assay reagent in an ELISA test was examined by presentation of antigen impregnated onto nitrocellulose particles (AINP). This was prepared by electro-transfer of bacterial polypeptides from SDS-PAGE gels to nitrocellulose. Protein bands were visualized by staining with amido black and a strip of nitrocellulose bearing the 65 kDa band was excised and extracted with formic acid. Nitrocellulose particles impregnated with the 65 kDa antigen (65-AINP) were precipitated from solution by neutralization with ammonium hydroxide. 65-AINP was suspended in water and the optimum dilution for ELISA assay was determined by titration to be 0.1 A650 units. Sera from all pigs challenged with VRS 229 reacted against the 65-AINP antigen in the ELISA assay while sera from control, and experimental pigs prior to challenge, failed to do so. The 65-AINP antigen could also be used efficaciously to quantify serological reactivity of pigs experimentally infected with other strains of E. rhusiopathiae representing the three major serotypes (1a, 1b and 2) that are most commonly associated with swine erysipelas infections. Mouse immunizations with 65-AINP also confirmed that nitrocellulose particles bearing the immunodominant subunit antigen will elicit murine antibodies that are monospecific against this determinant.

Comparison of seroreactivity of rams with brucellosis in a complement fixation test, whole cell ELISA and by immunoblotting.

In rams with ovine brucellosis, a high degree of serological correlation exists between the complement fixation (CF) test which utilises antigen extracted from bacteria with hot saline, and the ELISA reactivity using methanol-fixed Brucella ovis as the assay reagent. Since the whole cell ELISA (CELISA) detects mainly antibodies against surface antigens of B. ovis, it was concluded that the similar findings of the two serological tests is due in part to the presence of membrane antigens in the CF test antigen following hot saline extraction of intact bacteria. Immunoblots with pooled sera representing different CF titres confirmed that the major immunoreactive antigens of B. ovis were located in four zones: alpha, beta, gamma 1 and 2 with corresponding apparent molecular masses of 55 and 60 kDa; 27 and 29 kDa; 18.5-20 kDa and 17-18 kDa, respectively. These zones of reactivity were consistently present in immunoblots when assayed against different B. ovis isolates even though Coomassie brilliant blue staining of SDS-PAGE gels revealed some differences in polypeptide banding patterns. However, these intensely-stained CBB bands located at 38 and 40 kDa which distinguished three of the seven B. ovis isolates were considerably less reactive in immunoblots compared to polypeptides that were located at positions equivalent to alpha, beta or gamma reactivities. Intensity of immunoblot reactivity against polypeptides located in the alpha, beta and gamma zones intensified with increasing CF titre. Sera with CF titres greater than 32 also tended to react against bands of higher apparent molecular masses located at 65, 70, 73, 78, 80 and 86 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody response against Pseudomonas aeruginosa membrane proteins in experimentally infected sheep.

Shedded sheep inoculated epicutaneously with P. aeruginosa and then wetted experimentally by a sprinkler system, rapidly develop a green bacterial stain. This was associated with an outpouring of serious exudates onto the skin surface in the fleecerot lesion site. Histopathological analysis of dermatitic lesions revealed an infiltration of polymorphonuclear leucocytes into the dermis and the formation of a mosaic of microabscesses beneath the sloughed sheets of cornified epithelium. P. aeruginosa if present, was always localized as aggregates at the leading front of the seropurulent exudate and was never observed to invade the dermis. Animals that had been inoculated with P. aeruginosa but kept dry, showed no signs of dermatitis or serological reactivity against the inoculated bacterium. In contrast, sheep that had been inoculated and wetted, reacted serologically against P. aeruginosa whole cells in an enzyme-linked immunosorbent assay (ELISA). Eleven of 18 sheep were considered to be high-antibody responders and registered an ELISA ratio > 2.5 at one or more time points over the duration of the experiment (14 weeks). Analysis of ELISA reactivity of fleecerot sheep against fractionated cell envelope proteins of P. aeruginosa showed a preferential antibody response to outer (OMP) rather than inner (IMP) membrane proteins. Immunoblots revealed strong antibody activity against 2 major OMPs-Opr F and Opr H with apparent molecular masses of 39 and 21 kDa respectively. OMPs prepared from sarkosyl-resistant outer membrane vesicles were electrophoretically identical to OMPs prepared by a more rapid and efficient organic phase partitioning procedure (Chin and Dai, 1990). Although two other OMPs-Opr E (44 kDa) and Opr G (25 kDa) were seen in Coomassie blue-stained SDS-PAGE gels of P. aeruginosa OMPs, they were not reactive with sera from fleecerot affected sheep. It is likely that sheep with high levels of circulating serum antibody against major outer membrane proteins of P. aeruginosa may, in the event of a fleecerot episode, exude such antibodies onto the skin surface. This could provide a strategy for the control of ovine fleecerot by vaccination if highly conserved outer membrane proteins of P. aeruginosa were found to be protective.

Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.

Identification of immunoreactive membrane antigens of Brucella ovis by ELISA profiling.

Enzyme-linked immunosorbent assay (ELISA) profiling was used to identify the immunoreactive membrane antigens of Brucella ovis. Immunoreactive membrane antigens obtained after detergent extraction of the bacterial membrane complex (inner and outer membranes) were resolved into five peaks (A, B, B1, C and D) by gel permeation chromatography. Aliquots from each of the chromatographed fractions were coupled to 96-well microtitre plates and immunoreactive fractions identified with sera from two rams. Serum from ram 1 which had been vaccinated with a single injection of formalin-killed B ovis emulsified in incomplete Freund's adjuvant identified A and B as the major immunoreactive peaks. Serum from ram 2, which had been successfully infected with B ovis, reacted mainly against peaks A, B1, C and D. This observation facilitated the use of A, B, B1, C and D peak antigens as test reagents to examine the serological response of 12 other rams exposed to B ovis by vaccination or intraconjunctival or intravenous inoculation. Sera from rams which developed productive infections reacted strongly against peaks A, B1, C and D while vaccinated rams had preferential antibody activity against peaks A and B.

Temporal ELISA response of rams to Brucella ovis following experimental infection or vaccination.

Sera from rams vaccinated with antigens extracted chaotropically from Brucella ovis by potassium thiocyanate treatment were used to optimise a whole-cell, enzyme-linked immunosorbent assay (CELISA) and to monitor the temporal serological response of rams which had been challenged with infected semen by the intranasal or intrapreputial route. Three patterns of CELISA response were detected. Thirteen of 15 rams intranasally challenged did not respond serologically (pattern 1 or nil response). Only one of 15 rams in the intranasal group exhibited a rise and fall response with CELISA (pattern 2), while another showed a rise and surge response (pattern 3). The numbers of rams in the intrapreputial group which displayed a pattern 1 or 2 or 3 response were four, nine and two, respectively. No ram with a pattern 2 response excreted B ovis in the semen or showed any other evidence of infection, whereas rams with a pattern 3 response excreted B ovis in the semen and developed palpable lesions. Intrapreputially challenged rams that were CELISA-positive consistently mounted an antibody response against B ovis about two to four weeks earlier than intranasally challenged rams.

Monoclonal antibodies against ovine IgA purified from lung lavage fluid.

Ovine secretory IgA (sIgA) has been purified to relative homogeneity by ammonium sulphate precipitation (to 40 per cent w/v) of lung lavage fluid from 3- or 12-month-old lambs, followed by molecular sieve chromatography on a Sephacryl S300 matrix. Three peaks A, B and C with molecular sizes corresponding to 550,000, 400,000 and 165,000 respectively were eluted from the column. Immunoelectrophoresis, radial immunodiffusion and enzyme-linked immunosorbent assays (ELISAs) with class specific antiserum confirmed that peak B contained only IgA. Polyacrylamide gel electrophoresis of peak B under reducing conditions resolved three subunits corresponding to secretory component, heavy and light chains. Hybridomas generated by fusing spleen cells from IgA-primed Balb/C mice with murine myeloma (Sp2/0) were screened for IgA-specific monoclonal antibodies against a panel of ovine IgG2, IgG1, IgA and IgM. One particular clone, F3-4B4, identified as murine IgG1, was monospecific against ovine IgA with no cross reactivity against bovine immunoglobulins. This hybridoma was successfully tested as a serological probe by ELISA profiling to locate IgA containing fractions in the course of immunoglobulin purification from biological fluids.

Comparison of different antigenic preparations for the detection of ovine serum antibodies against Brucella ovis by ELISA.

An enzyme-linked immunosorbent assay has been developed for the detection of antibodies against Brucella ovis using serum from control rams (Con-S), naturally infected rams (Inf-S), rams inoculated intravenously with B. ovis (IV-S) and rams vaccinated intramuscularly (IM-S). The serum was titrated by serial double dilutions from 1/25 to 1/25,600 against whole bacteria, B. ovis lipopolysaccharide and a detergent-extracted component of the outer membrane complex of B. ovis as antigens immobilised on microtitre plates. Sheep antibodies bound to antigen were assayed with rabbit anti-sheep gammaglobulin and alkaline phosphatase conjugated protein A. A high level of antibody activity against intact B. ovis cells was detected in Inf-S and IM-S. When lipopolysaccharide was the immobilised antigen, only IM-S yielded significant antibody activity. The component from detergent extracts of the outer membrane complex of B. ovis reacted best with serum (up to 1/6,400) from field-infected rams, while serum from vaccinated and intravenously inoculated rams registered significant titres up to a serum dilution of 1/800 and 1/200 respectively. These results indicate that ELISA is a very sensitive test but its value as a serodiagnostic procedure is dependent upon the choice of antigen used in the assay.

Immunoreactivity of fractionated antigens obtained from autoclaved extracts of an arthritogenic isolate of Erysipelothrix rhusiopathiae.

The immunoreactive antigens in heat-extracted (autoclaved) preparations of an arthritogenic strain of Erysipelothrix rhusiopathiae (isolate VRS 229, serotype 1a) have been identified by gel diffusion precipitin (GDP) tests and a novel application of the enzyme linked immunosorbent assay (ELISA) procedure. Antigens precipitated by ethanol treatment of autoclaved extracts of this strain were resolved into 4 major peaks (A,B,C and D) after gel permeation chromatography on Sephacryl S200. Peak A was confirmed as a protein peak (Lowry positive) which was excluded from the gel. This peak was identified to be ELISA-reactive when assayed with serum from pigs infected with other isolates corresponding to serotypes 1a, 1b and 2. However, it did not form precipitin lines in GDP tests. Peak B was Lowry-positive and also contained carbohydrates. It was not as reactive in ELISA tests but rapidly formed precipitin lines with serum from pigs infected with the homologous isolate, but only erratically with serums from pigs infected with other serotype 1a and 1b isolates, and not with serotype 2 isolates. Peaks C and D were high in carbohydrate and phosphate content respectively but were both non-reactive in GDP tests and only slightly so by ELISA. Since serotypes 1 and 2 are the most predominant among isolates from infected pigs it is likely that the commonly recognised A antigen is a useful ELISA reagent for the diagnosis of E. rhusiopathiae infection; B antigen on the other hand, would probably be of limited diagnostic value.

Dermal and serological response against Pseudomonas aeruginosa in sheep bred for resistance and susceptibility to fleece-rot.

Genetically select lines of Merino sheep have been bred at Trangie (NSW Agriculture and Fisheries) for resistance (R) or susceptibility (S) to fleece-rot and flystrike. It is believed that fleece characters are primarily responsible for the R or S phenotype. When transferred to the wetter coastal environment of Sydney, R and S sheep with no more than 6 weeks wool cover, continued to show significant differences in the incidence and severity of fleece-rot dermatitis. To test the hypothesis that these sheep might also exhibit differences in their local skin reactions and immune responsiveness, 3 intradermal injections of killed Pseudomonas aeruginosa were administered at monthly intervals. After primary intradermal challenge, R sheep had a higher incidence of skin induration and a stronger inflammatory response (increased induration diameter) than S sheep. Compared to S sheep, R sheep also developed higher levels of circulating antibodies against whole cell antigen and both inner and outer membrane proteins of P. aeruginosa. These responses were maintained in R sheep with each consecutive challenge while S sheep showed a decline in their immune responsiveness. Differences in antibody response against outer membrane proteins were also detected when antigenically naive sheep from each genetic line were sensitised by epicutaneous challenge with P. aeruginosa under experimental wetting conditions. Intradermal challenge of these animals 6 months later with outer membrane proteins, revealed a late maximum (72 h) in the development of induration diameters for R sheep while S animals showed maximal induration diameters by 24 h. However, there was no significant difference in induration response between 24 h and 72 h within each group of sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of inoculation regimes for the experimental production of swine erysipelas arthritis. II. Serological findings in a gel diffusion precipitin test and enzyme-linked immunosorbent assay.

The serological response of pigs to Erysipelothrix rhusiopathiae inoculation was monitored by a gel diffusion precipitin test (GDPT) using a crude, serotype-specific, autoclaved antigen and an enzyme-linked immunosorbent assay (ELISA) using a heat-extracted, alcohol precipitated and molecular seived antigen previously shown to react with serum from pigs infected with serotypes 1 or 2. All pigs receiving 3 or 5 weekly intravenous inoculations of either a highly virulent (VRS 229) or a lowly virulent isolate (VRS 252) produced GDPT-reactive antibody within 3 weeks, but only 44% were still reactive at 8 to 9.5 weeks. The ELISA response was significantly higher in pigs inoculated with the highly virulent strain, and was similar in pigs receiving 3 or 5 doses of either strain. In a dose-response trial, after 3 doses of VRS 229, GDPT reactivity occurred earlier and was stronger in pigs given higher doses of E. rhusiopathiae, but the response peaked 3 to 5 weeks after the start of challenge and was short lived. GDPT reactivity correlated with dose, but not with the severity of arthritis. The ELISA demonstrated specific IgG antibody was present by 2 weeks, and persisted to at least 11 weeks. The ELISA reactivity was significantly higher in pigs with arthritis than in pigs that received low doses and were not arthritic. Within groups of pigs with arthritis a significant, dose dependent, linear ELISA response developed but did not correlate with the presence or degree of arthritis at slaughter. Non-arthritic pigs had similar low ELISA responses to uninoculated controls.

Peripheral blood lymphocyte subsets in fleece rot-resistant and -susceptible sheep.

OBJECTIVE: To compare haematological values and lymphocyte phenotypes in the peripheral blood of fleece rot-resistant and -susceptible sheep. PROCEDURE: Experiments were conducted on 2- and 3-year-old Merino rams, flock 1 (17 rams) and flock 2 (32 rams), respectively. Within each flock, individual rams were classified as fleece rot-resistant or -susceptible, based on established criteria. Total and differential white cell counts, and indirect fluorescent antibody tests specific for B cells and T cells were performed on all sheep. The concentration of various subsets of circulating lymphocytes was then determined in each sheep. RESULTS: There were no significant differences between fleece rot-resistant and -susceptible sheep from either flock in the mean total or differential white cell counts. However, fleece rot-resistant rams in flock 1 did have a significantly higher concentration of circulating SBU-T1+ cells than fleece rot-susceptible rams from the same flock. No such difference was noted in the rams from flock 2. While all rams in flock 1 were free of clinical fleece rot, 24 rams in flock 2 (comprising all 17 fleece rot-susceptible and 7 of 15 fleece rot-resistant animals) had clinical signs of the disease. Fleece rot-free rams in this flock (irrespective of their classification as fleece rot-resistant or -susceptible) had significantly higher concentrations of circulating SBU-T1+ cells compared with fleece rot-affected animals. They also had significantly higher concentrations of circulating B cells, and total lymphocytes. CONCLUSIONS: An examination of peripheral blood lymphocyte subsets in fleece rot-resistant and -susceptible sheep revealed a possible association between resistance to fleece rot and the concentration of circulating SBU-T1+ cells.

Evaluation of surface components of Brucella ovis as antigens for the detection of precipitin antibody in serums from artificially exposed rams.

Surface components of Brucella ovis obtained by gentle physical shearing were tested as a potentially useful source of reagent for selective serological diagnosis. These antigens were used in a radial immunodiffusion (RID) test against serum from rams which had been inoculated with infective semen containing B. ovis by one of 4 routes namely mating rams with ewes previously inoculated intravaginally with infective semen, or by direct inoculation in the prepuce, rectum or nasal passage. Loosely attached surface antigens in the RID test formed precipitin bands with serums collected from rams 2 and 10 weeks after inoculation. In contrast, a detergent extracted membrane antigen B developed precipitin bands only with serum collected 10 weeks after inoculation from rams confirmed bacteriologically to be infected with B. ovis in the genital tract. The route by which the rams were artificially exposed did not affect the outcome of the RID test using the membrane B antigen. However, all experimentally exposed rams had demonstrable CF titres when a heat extracted antigen was used.

Biological properties of phospholipase C purified from a fleecerot isolate of Pseudomonas aeruginosa.

A role for one of many exocellular enzymes produced by Pseudomonas aeruginosa--phospholipase C (PLC)--as a prime candidate virulence factor in fleecerot dermatitis has been examined. The addition of Tween 80 in tryptose minimal medium effectively perturbed the membrane system of a field isolate of P. aeruginosa, resulting in increased production and release of a periplasmic enzyme marker, alkaline phosphatase (AP), and also of PLC. PLC activity levels in the culture supernatant were 10- to 15-fold higher in the presence of Tween than in its absence. Apart from AP, the culture medium contained little or no detectable proteolytic enzyme activity, thereby facilitating the partial purification of a haemolytic form of PLC by anion-exchange chromatography. This enzyme, when injected intradermally into the skin of sheep, elicited histopathological lesions virtually identical to those seen in naturally occurring fleecerot. In addition, serum from each of eight sheep afflicted with fleecerot contained high levels of circulating anti-PLC antibody activity when assayed by ELISA. Since these antibodies did not affect the enzymic function of PLC, it is likely that they do not bind to, or are incapable of conformational modification of, the active site.

Profiles of serological reactivity against cytosoluble antigens of Brucella ovis in experimentally infected rams.

Sera from rams infected with and excreting Brucella ovis in the semen (shedders), as well as from animals which had recovered from previous experimental challenge with B. ovis, were analyzed for their serological reactivities against cytosolic antigens of the bacterium. Membrane vesicles, including outer and inner membrane components, were precluded from the analyses by subjecting French-pressed bacteria to ultracentrifugation. The resulting cytosolic supernatant was fractionated into four major antigenic fractions, fractions A, B, C, and D, by high-pressure liquid chromatography. Temporal enzyme-linked immunosorbent assays with the A antigen revealed that all shedder rams displayed a rise-and-surge response, while rams which recovered from experimental challenge showed a rise-and-fall profile. The B antigen was less discriminatory in detecting a difference between the two ram groups, while C and D antigens were serologically unreactive in the enzyme-linked immunosorbent assay. In contrast to the reactivity patterns shown by native high-pressure liquid chromatography-fractionated cytosolic supernatant antigens, immunoblotting of C and D polypeptides generated by boiling in the presence of sodium dodecyl sulfate and mercaptoethanol was particularly useful in distinguishing between sera collected at the mid-surge phase of infected rams from sera obtained at the mid-fall stage of recovered animals. It is likely that native or denatured antigens of different cytosolic fractions may provide useful serological reagents for differentiating between infected rams and those which have recovered from exposure to B. ovis.