Experimental studies on the serological relationships between Yersinia enterocolitica and Brucella abortus.

Yersinia enterocolitica O9 was shown to provoke O, OH and H antibody response in calves. Brucella abortus failed to generate Yersinia H antibody response and generated Brucella O antibodies that cross-reacted with Yersinia O and OH antigens. The presence of Yersinia H agglutinins along with a higher titre of Yersinia OH antibody than Brucella O antibody is suggestive of subclinical infection with yersiniosis rather than brucellosis. Cross-absorption of sera from calves with established Yersinia infections indicated that absorption of sera with Brucella O antigens, although completely removing Brucella O antibodies, failed to remove completely Yersinia O, OH and H antibodies, and thus provides an additional method of distinguishing between the two infections.     

Studies on the relationship between Yersinia enterocolitica IX and Brucella abortus agglutinins in naturally infected animals

Attempts were made to define the Brucella abortus and Yersinia enterocolitica IX infection status of animal populations by means of selected agglutination tests. The Brucella abortus O, the Yersinia enterocolitica IX OH and the Y enterocolitica IX H agglutinin titres were measured in a large number of cattle, goat and pig sera In the goats and, to a much lesser extent, the pigs, the relationships between these titres suggested that Yersinia infection was common. In contrast, the results from the cattle sera were complex and tended to indicate the presence of both Yersinia infection and brucellosis.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7

Sera from cattle naturally infected with Brucella abortus (n = 160), vaccinated with B. abortus S19 (n = 88) or immunized with Yersinia enterocolitica O:9 (n = 25) or Escherichia coli O157:H7 (n = 80) were collected. The sera were compared for antibody content to the same bacteria by indirect enzyme immunoassay (IELISA), fluorescence polarization assay (FPA) and competitive enzyme immunoassay (CELISA). Cattle sera (n = 523) collected randomly from across Canada were tested in the same tests. Sera from the B. abortus infected group reacted positively in the brucellosis IELISA (IELISA(Br)), CELISA and FPA (FPA(Br)) and the Y. enterocolitica IELISA (IELISA(Ye)) while the Y. enterocolitica FPA (FPA(Ye)) detected antibody in 93.8% and the E. coli IELISA (IELISA(Ec)) 86.9% and the E. coli FPA (FPA(Ec)) 48.1%. About 70% of the sera from B. abortus S19 vaccinated animals reacted in the three IELISAs, 45% in the CELISA, and 37.7% in the FPA(Ec), 21.6% in the FPA(Br) and 5.7% in the FPA(Ye). Sera from E. coli O:157 exposed cattle reacted mainly in the IELISA(Ec) and FPA(Ec) although surprisingly 87.5% reacted in the IELISA(Ye) and only 3.8% in the IELISA(Br). No reactions were observed with these sera in the FPA(Br) and FPA(Ye) but one serum gave a low positive reaction in the CELISA. All sera from Y. enterocolitica O:9 exposed cattle reacted in the IELISA(Br) and IELISA(Ye) and 80% in the IELISA(Ec). In the CELISA, 44% gave a positive reaction and 64% were positive in the FPA(Br), 28% in the FPA(Ye) and 12% in the FPA(Ec). Of the 523 Canadian sera, about 50% reacted in the E. coli tests with only minor reactions in the Y. enterocolitica O:9 and B. abortus assays. From the data, the cross reaction between E. coli O157:H7, Y. enterocilitica O:9 and B. abortus is dependent on the test used. Thus, extensive cross reaction was observed with the IELISA with much less reactivity in the FPA and the CELISA.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28 kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.     

Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella sp. and Yersinia enterocolitica O:9 in cattle and pigs

A rapid, inexpensive and rugged serological test that distinguishes cattle and swine infected with Brucella sp. or Yersinia enterocolitica O:9 is described. The test protocol, which is an indirect enzyme immunoassay uses a high concentration of divalent cation chelating agents to minimize binding of Y. enterocolitica O:9 antibody to rough lipopolysaccharide antigen derived from B. abortus RB51. No false positive reactions were observed when testing 100 Canadian cattle and swine without any evidence of brucellosis. The assay detected 91.6% of cattle (n=155) and 93.5% (n=31) of swine infected with Brucella sp. Sera from 58 cattle and 38 swine exposed to Y. enterocolitica O:9 were negative while only 20 sera from 121 'false positive' reactors of unspecified origin gave low level positive reactions, eliminating 84% of the false positive reactions.     

Brucella clearance as a sensitive method for the detection of cross reactions of Brucella abortus with Yersinia enterocolitica 03, 06, 09 and Salmonella urbana and Salmonella abony

Determination of the Brucella clearance rate has proved to enable assessment of Brucella immune reaction in rat, even after vaccination with Yersiniae and Salmonellae. Vaccination with Yersinia (Y.) enterocolitica O6 and O9 produced 95 per cent of "high responders", whereas 65 per cent of "high responders" and 25 per cent of "non-responders" were recorded in the wake of O3. Salmonella (S.) urbana vaccination gave 50 per cent of "high responders" and 27 per cent of "non-responders", while 100 per cent "non-responders" resulted from S. dublin. Vaccination, using Brucella abortus Buck 19, gave 100 per cent "high responders". The differentiated nature of immune reactions to Y. enterocolitica O3, S. urbana, and S. abony has been attributed to an individual genetic capability of reaction to the cross-reactive antigen.     

Yersinia enterocolitica associated with third trimester abortion in buffaloes

A group of buffaloes aborted at the third trimester of their gestational period. Yersinia enterocolitica was isolated from all cases. All aborted animal sera had high antibody titres against Y. enterocolitica antigen which crossreacted significantly with Brucella abortus antigen. All animals were culturally negative after intrauterine infusion of gentamycin sulphate (160 mg/day) for three consecutive days.     

Non specific serological reactions in the diagnosis of bovine brucellosis: experimental oral infection of cattle with repeated doses of Yersinia enterocolitica O:9

Eight heifers were orally infected with 4 x 10(9) colony forming units of a field cattle strain of Yersinia enterocolitica O:9 in a capsule, 5 days a week, for about 9 weeks (day 0-day 64 (D0-D64). The faecal shedding of Y. enterocolitica O:9 began on D5 for seven out of the eight challenged cattle with a high level of excretion during the first month, followed by a decrease till the day of slaughter (D76). Y. enterocolitica O:9 was not isolated from organs collected at slaughter. No clinical symptoms were observed. Hyperplasia of intestinal lymph formations was the sole microscopic lesions observed. Five animals showed a serological reaction against Brucella antigens in at least one of the following tests: Rose-Bengal test, complement fixation test, tube agglutination test or indirect ELISA (iELISA) tests. Only one animal showed a high level of serological response and a positive reaction in the dithiothreitol-microagglutination test. The observed variability in terms of individual sensitivity to the Y. enterocolitica O:9 infection is in agreement with the low individual prevalence rate and the transient serological reaction and faecal Y. entercolitica O:9 shedding observed in herds showing false positive serological reactions in brucellosis.     

The application of PCR to the identification of selected virulence markers of Yersinia genus

The subject of this study was thirty nine strains of Yersinia enterocolitica, isolated from faeces of humans who showed symptoms typical of intestinal yersiniosis, and seventy strains of Y enterocolitica, four strains of Y. pseudotuberculosis, and one strain of Y. kristensenii from healthy pigs. In the population tested the following serogroups appeared: O3, O9, O2, O5. A PCR was used to detect the presence of pathogenic chromosomal markers, such as myfA and inv genes of the tested Yersinia species. Among Y. enterocolitica strains isolated from humans and belonging to serogroup O3 (thirty four strains) and serogroup O9 (five strains) thirty three Y. enterocolitica O3 strains and four Y. enterocolifica O9 strains, gave a positive reaction to the nmyfA gene, yielding a fragment of 280 base pairs (bp). Among seventy Y. enterocolitica strains isolated from pigs forty strains belonging to serogroup O3 and fifteen strains belonging to serogroup O9 gave a positive reaction to the myfA gene. The presence of 390 bp amplified products, corresponding to the inv gene fragment, was detected in PCR products of three Y pseudotlluberculosis strains from pigs and only in one Y. enterocolitica O3 strain from humans, which had no myfA gene. The results obtained show that the myfA gene is only present in the strains that belong to pathogenic serotypes of Y. enterocolitica. The myfA gene prevailed in the Y. enterocolitica O3 and O9 strains from humans but was less common in the Y. enterocolitica O3 and O9 strains from pigs.     

Serological cross-reactions between different brucella species and Yersinia enterocolitica. Agglutinating activity of 19 S and 7 S antibodies aginst somatic antigen of Brucella abortus and Yersinia enterocolitica type IX

Antisera from rabbits immunized with acetone-killed whole cells and with lipopolysaccharides from Brucella abortus (B.a.) and Yersinia enterocolitica type IX (Y.e.) were gel-filtered on Sephadex G-200 columns.All the antisera had serologically cross-reacting agglutinins against B.a. and Y.e. both in the 19 S and in the 7 S antibody fractions.The homologous and the heterologous agglutinating activity of 19 S and of 7 S antibodies was tested before and after treatment with dithiothreitol (DTT) and subsequent alkylation. Unlike 19 S antibodies, 7 S antibodies were resistant to the DTT reduction.The results from heterologous absorptions of the respective 19 S and 7 S antibody fractions indicated that 19 S antibodies both to B.a. and to Y.e. had a greater tendency towards being absorbed to the cross-reacting antigenic determinants than had the corresponding 7 S antibodies. The agglutination titres for 19 as well as 7 S antibody fractions derived from the immunizations with B.a. (both whole cells and LPS) fell more markedly after heterologous absorptions than did the analogous titres for corresponding Y.e. antibody fractions. Possible explanations of these differences are discussed.     

Differentiation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: the use of specific lymphocyte transformation and brucellin skin tests

The lymphocyte transformation test (using an in vitro whole-blood lymphocyte stimulation procedure) and the Brucellin skin test were applied to five heifers infected with virulent Brucella abortus strain 544, five cows inoculated with Yersinia enterocolitica serotype 09, and four non-exposed cows. Lymphocytes from Brucella-inoculated animals persistently gave very high blastogenic reactions indicative of active Brucella infection. The test was persistently negative in Yersinia-infected and non-exposed cattle. Four of the five cows infected with Yersinia enterocolitica type 09 and all four control cattle were persistently negative to the delayed hypersensitivity skin reaction with brucellin. All cattle infected with Yersinia enterocolitica type 09 were strongly positive to the Rose Bengal, Serum agglutination, Complement fixation and Antibovine globulin tests using Brucella abortus antigens. One lactating cow infected with Yersinia enterocolitica type 09 was positive to Brucella milk ring test. These results indicate that standard Brucella serological tests are unreliable in differentiating the two infections in cattle and that both the Lymphocyte transformation and brucellin skin tests could be used to differentiate bovine brucellosis from yersiniosis.     

Fecal immunoglobulin A (IgA) antibodies to Yersinia enterocolitica in mice orally vaccinated and infected

Fecal immunoglobulin-A (IgA) antibodies to Yersinia enterocolitica serovar O3 strain were detected in the mice orally immunized with formalin-killed organisms. Y. enterocolitica O3 organisms were inhibited to colonize in the intestines of the mice producing fecal IgA. The fecal IgA antibodies were detected in the mice orally infected with the bacteria. When IgA was produced in the mice infected, they ceased shedding the organisms in their feces.     

Immuno-magnetic separation and agar layer methods for the isolation of freeze-injured Yersinia enterocolitica O:8 from water

To develop an effective method to isolate an injured pathogenic Yersinia enterocolitica O:8 organism from environmental samples, we compared the isolation of freeze-injured and non-injured Y. enterocolitica O:8 and found that the isolation was more successful when immuno-magnetic separation (IMS) with anti-Y. enterocolitica O:8 antibody was used. Plating onto cefsulodin-irgasan-novobiocin (CIN) agar and Virulent Yersinia enterocolitica (VYE) agar by means of the agar layer method was found to be effective in isolating the injured cells. The alkali treatment which is generally used for selective detection of Yersinia organism failed to isolate freeze-injured pathogenic Y. enterocolitica O:8 cells. Recovery methods without using the alkali treatment were superior for detecting freeze-injured Y. enterocolitica O:8. Our results demonstrate that the IMS and the agar layer methods should be used to isolate injured pathogenic Yersinia organisms from environmental samples such as water.     

Brucella titers in swine caused by Yersinia enterocolitica, serotype 0:9

Antibody to Brucella abortus developed in two thirds of all gilts kept on a pig breeding station. Systematic tests taken for the purpose of detecting clinical symptoms and of isolating Brucella were negative. however, Yersinia (Y.) enterocolitica, Serotype 0:9, was cultured from rectal swab samples which had been obtained from 31 to 78 gilts. The clinical, bacteriological, and serological tests gave rise to the assumption that the Brucella titres have been caused by Yersinia enterocolitica infection. Such conclusion, however, could be drawn only as a result of a complex investigation. Detection of Yersinia enterocolitica alone is not sufficient a proof by which to rule out the possibility of concomitant Brucella infection. The question is discussed to what extent swine may be considered to be a potential source of infection of man.     

Comparative immune responses to native cell envelope antigens and the hot sodium dodecyl sulfate insoluble fraction (PG) of Brucella abortus in cattle and mice

Brucella abortus vaccines composed of native cell envelopes or outer membrane proteins of smooth strain 2308 were compared with a vaccine (PG) composed of the insoluble residue of strain 2308 cell envelopes which had been extracted with hot sodium dodecyl sulfate. Vaccines were given by injection in an oil base adjuvant containing trehalose dimycolate and muramyl dipeptide or without adjuvant. Mice vaccinated with 30 micrograms native cell envelopes or PG and challenged 4 weeks later with virulent B. abortus strain 2308 displayed equivalent levels of protective immunity at 1 and 4 weeks post-infection. Heifers were vaccinated with 5 mg of antigens in adjuvant; PG was also administered without adjuvant. Humoral and cell mediated immune (CMI) responses were tested at monthly intervals. PG without adjuvant induced negligible immune responses. Native cell envelope antigens induced significantly higher titers of whole cell agglutinins over a 3-month period than did PG, although revaccination with PG in adjuvant enhanced the production of agglutinins and both vaccines induced antibodies to the O polysaccharide. Lymphocyte blastogenesis responses and delayed hypersensitivity reactions to porin and group 3 proteins were stimulated by both native and PG vaccines, and the magnitude of the responses did not differ significantly between the treatment groups. These vaccines were therefore comparable in their capacity to induce protective immunity in mice and CMI responses in cattle, whereas antibody responses induced by PG in cattle were generally lower. These findings provide a basis for evaluation of nonliving B. abortus vaccines in cattle.     

Arthropathy associated with Brucella abortus strain 19 vaccination in cattle. I. Examination of field cases

Thirty cows presenting with lameness and persistent serological reactions to Brucella abortus had chronic granulomatous arthropathy of the femorotibial and occasionally other joints. Attempts to culture Brucella or other pathogens gave negative results but organisms of Brucella morphology were seen in fluorescent antibody-stained cryostat sections of synovial tissue. The synovial fluids contained high titres of antibodies to B. abortus and Yersinia enterocolitica O:9 and had elevated total protein and immunoglobulin concentrations showing an oligoclonal electrophoretic profile. Immune complexes and rheumatoid factor were detected in some of the fluids.     

Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A, serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD). METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5 x 10(9) colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR). RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non-pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain. CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

A rapid method for detection of Yersinia enterocolitica serotype O:3 in pig feces using monoclonal antibodies.

A simple colony immunoblotting method using monoclonal antibodies (MAbs) was developed to detect Y. enterocolitica serotype O:3 in pig feces. One of the MAbs studied was able to detect single colonies of the organism in the presence of calculated 3.1 x 10(8) heterologous organisms in pig feces. The MAb was found to be specific for the lipopolysaccharide (LPS) O-antigens of Y. enterocolitica serotype O:3. No significant cross-reactivity was found against a variety of closely related serotypes and Gram-negative organisms. The MAb could also be used in a slide agglutination test and an indirect fluorescence antibody assay for rapid identification of Y. enterocolitica serotype O:3.