Bacterial survival, lymph node pathology, and serological responses of bison (Bison bison) vaccinated with Brucella abortus strain RB51 or strain 19

From August 1993 to June 1994, 3 month-old bison (Bison bison) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6), strain 19 (S19, n = 3), or with saline (n = 1) and serologic responses and persistence of vaccine strains within lymph nodes were monitored. Bison vaccinated with S19 had granulomatous lymphadenitis and greater peak numbers of B. abortus than those vaccinated with SRB51. Bison vaccinated with RB51 had similar histological lesions and B. abortus were still present in lymph nodes at 16 weeks. Although antibodies against RB51 were produced, standard tube agglutination test responses of RB51-vaccinates remained negative. The histological lesions of B. abortus infections in bison were similar to those observed in cattle, but bison did not clear SRB51 as rapidly as cattle.     

Attitudes of oncologists, family doctors, medical students and lawyers to euthanasia

OBJECTIVE: To evaluate clearance of the vaccine strain, immunologic responses, and potential shedding of Brucella abortus strain RB51 organisms after vaccination of bison calves. ANIMALS: Fourteen 7-month-old female bison calves. PROCEDURE: 10 bison calves were vaccinated SC with 1.22 x 10(10) colony-forming units of B abortus strain RB51. Four bison calves were vaccinated SC with 0.15M NaCl solution. Rectal, vaginal, nasal, and ocular swab specimens were obtained to evaluate potential shedding by vaccinated bison. The superficial cervical lymph node was biopsied to evaluate clearance of the vaccine strain. Lymphocyte proliferative responses to strain RB51 bacteria were evaluated in lymph node cells obtained from biopsy specimens and also in peripheral blood mononuclear cells. RESULTS: Strain RB51 was recovered from superficial cervical lymph nodes of vaccinates examined 6, 12, and 18 weeks after vaccination (4/4, 3/4, and 1/4, respectively) but not in vaccinates examined at 24 weeks (0/3) after vaccination or nonvaccinates examined at all sample collection times (n = 1 bison/sample period). Serologic, immunologic, and bacterial culture techniques failed to reveal shedding of strain RB51 by vaccinates or infection of nonvaccinated bison. Lymphocyte proliferative responses were evident in lymph node cells and blood mononuclear cells from strain RB51-vaccinated bison beginning 12 weeks after vaccination. CONCLUSION: Strain RB51 was cleared from bison by 18 to 24 weeks after vaccination. Bison vaccinated with strain RB51 did not shed the vaccine strain to nonvaccinated bison housed in close proximity. Strain RB51 did not induce antibody responses in bison that would interfere with brucellosis surveillance tests, but did stimulate cell-mediated immunity.     

Mechanomyography and oxygen consumption during incremental cycle ergometry

Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture. Currently available serologic surveillance tests for B. abortus do not detect seroconversion following SRB51 vaccination. The purpose of this study was to evaluate a dot-blot assay using gamma-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51. Dot-blot titers of sera at a recommended dosage (10(10) colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B. abortus strain 19 and greater (P < 0.05) than titers of nonvaccinated cattle. In the first 12 weeks after vaccination with 10(10) colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater. Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 10(10) colony-forming units of strain RB51. Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (10(9) colony-forming units) did not differ (P > 0.05) from titers of sera from nonvaccinated cattle. Following intraconjunctival challenge with B. abortus strain 2308, titers on the RB51 dot-blot assay did not differ (P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.     

Experimental infection of pregnant cattle with the vaccine candidate Brucella abortus strain RB51: pathologic, bacteriologic, and serologic findings

To determine the placental tropism and abortigenicity of the vaccine candidate Brucella abortus strain RB51 (SRB51), a rough mutant of the virulent strain 2308, ten Polled Hereford heifers were inoculated intravenously in the 6th month of gestation. Heifers were euthanatized and examined at postinoculation week (PIW) 8 (n = 5) or at full term (n = 5). Four of five infected heifers sampled at PIW 8 and three of four infected heifers at term had placentitis, whereas reproductive tissues of three normal cows used for comparison had no placentitis. Numerous macrophages, immunoreactive for SRB51 antigen, as well as neutrophils, fibrin, and cell debris filled the arcade zone between chorion and maternal septae. Trophoblastic epithelium of the placentomal arcade zone had intracellular bacteria that were immunoreactive for SRB51 antigen. The tips of maternal septa had a lymphoplasmacytic infiltrate with small multifocal erosions and ulcerations of maternal epithelium. SRB51 was cultured from all tissues in which lesions were seen. Placentae of one cow from each group had no placentitis and contained no SRB51. In mammae, interstitial lymphoplasmacytic infiltrates and suppurative infiltrates within alveoli and intralobular ductules were seen in two of five heifers at PIW 8. SRB51 was cultured from liver, spleen, lung, and bronchial lymph nodes in four of five calves at PIW 8 and three of four full-term calves, but no lesions were seen. One near-term heifer had disseminated infection, placentitis, and lymphoplasmacytic endometritis, and delivered a premature weak calf. These results establish that SRB51 is less abortifacient than previously published reports with strain 19, in that only one of four heifers delivered prematurely following intravenous inoculation with SRB51, whereas intravenous inoculation with strain 19 leads to 100% abortion. However, it also shows that SRB51 can infect the bovine placenta, mammary gland, and fetus, can induce placentitis, and, in some cases, can lead to preterm expulsion of the fetus.     

Lymphocyte proliferative responses of goats vaccinated with Brucella melitensis 16M or a delta purE201 strain

The response to a Brucella melitensis purEK deletion mutant, delta purE201 (referred to as strain 201), was compared with the response to its parental strain, 16M, in juvenile goats. Proliferative responses to gamma-irradiated bacteria were detected earlier in strain 201-infected goats. Lymphocytes from strain 16M- or 201-infected goats proliferated in response to one-dimensional polyacrylamide gel electrophoresis-separated proteins of similar mass isolated from strain 16M or Brucella abortus RB51. Data from this study suggest that some antigens stimulating cell-mediated responses are conserved among Brucella species, as 201- and 16M-infected goats recognized similar proteins expressed by RB51 and 16M.     

Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide dismutase protects mice against virulent B. abortus

Vaccination of mice with Escherichia coli expressing Brucella Cu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortus vaccine strain RB51. In addition, vaccination with E. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.     

Increasing incidence and comparison of nalidixic acid-resistant Salmonella enterica subsp. enterica serotype typhimurium isolates from humans and animals

We determined the resistance to quinolone of 309 Salmonella enterica subsp. enterica serotype Typhimurium strains isolated from humans and animals (cattle, pigs, or poultry) in 1995 or 1996. Nalidixic acid resistance increased from 8.5% in 1995 to 18.6% in 1996. The highest resistance levels correlated with a mutation at Ser-83 (or Asp-82). All strains remained ciprofloxacin susceptible. Human and animal isolates were compared by pulsed-field gel electrophoresis, and the banding patterns of the human isolates most closely matched those of the bovine isolates.     

Stability of strain genotypes of Burkholderia pseudomallei from patients with single and recurrent episodes of melioidosis

The constancy of strain genotypes of multiple isolates of Burkholderia pseudomallei from 13 patients with melioidosis was examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of DNA. Seven of 8 patients with single episodes of melioidosis each yielded genetically identical isolates and only one of five patients with recurrent episodes was infected with a new strain clearly distinct from the original primary strain. Variation was observed in PFGE patterns of primary and relapse isolates of another patient but this was insufficient to define genetically distinct strains. We conclude that most patients with single or multiple episodes of melioidosis retain a single strain.     

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus

Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in the bacteria grown in the cell culture medium have been induced during intracellular growth in macrophages. In contrast, approximately 50 proteins that were expressed during growth in cell culture medium were completely repressed during intracellular growth. The level of expression of 19 proteins increases while that of 54 proteins decreases during intracellular growth. To understand these results, the protein synthesis pattern of B. abortus during intracellular growth was compared with those during other stress conditions. Under each stress condition studied, several new proteins were induced that were not present during regular growth conditions. Comparison of the protein synthesis pattern of B. abortus during intracellular growth with those obtained under various stress conditions has indicated that the response to intracellular growth was not just a simple sum of stress conditions studied so far.     

Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup 0157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans.     

Simultaneous identification of antibodies to Brucella abortus and Staphylococcus aureus in milk samples by flow cytometry

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureus antibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortus or S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-microm-diameter latex particles. The difference in size between B. abortus and S. aureus permits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.     

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

OBJECTIVES: To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis. DESIGN: Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates. PROCEDURE: DNAs from 35 Australian isolates of M paratuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared. RESULTS: The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales. CONCLUSION: Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis, and could be used to study the transmission of strains in Australia.     

Molecular epidemiology provides evidence of genotypic heterogeneity of multidrug-resistant Pseudomonas aeruginosa serotype O:12 outbreak isolates from a pediatric hospital

Ribotyping randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis were used for the epidemiologic evaluation of eight Pseudomonas aeruginosa O:12 isolates obtained from eight children and two P. aeruginosa O:12 environmental isolates from a hematology ward. Randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis were able to discriminate isolates that were indistinguishable by biochemical typing, O serotyping or ribotyping.     

Molecular cloning and characterization of cgs, the Brucella abortus cyclic beta(1-2) glucan synthetase gene: genetic complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB mutants

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic beta(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic beta(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic beta(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic beta(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic beta(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic beta(1-2) glucan may be a virulence factor in Brucella infection.     

The effects of dexamethasone immunosuppression on turkey osteomyelitis complex in an experimental Escherichia coli respiratory infection

The results of a field trial conducted in Latin America with two indirect enzyme-linked immunosorbent assays (ELISAs) and two competitive ELISAs (CELISAs) for the detection of bovine antibody to Brucella abortus are reported. One of the CELISA formats performed most accurately. The percentage of positive reactions in the CELISA relative to the selected positive rose bengal agglutination test (RBT) and complement fixation test (CFT) results was 97.47%, the percentage of negatives relative to the selected negative RBT and CFT results for unexposed cattle was 98.32%, and the percentage of negatives in cattle vaccinated with B. abortus 19 was 96.51%. The same assay format under Canadian conditions had an actual sensitivity of 100%, a specificity of 99.90% in nonvaccinates, and a specificity of 97.7% in a strain 19-vaccinated population. Overall, the CELISA performed as expected and the results were not dissimilar from the results obtained in the Canadian study. This provided further evidence that this CELISA can in many instances differentiate infected cattle from those that are vaccinated or infected with a cross-reacting organism while still giving very few false-positive or false-negative results.     

Endotoxic activity of rough organisms of Brucella species

A rough-specific antigen extracted from the rough species Brucella ovis and lipopolysaccharide extracted from smooth Brucella abortus demonstrated equivalent levels of activity in tests for mouse lethality and limulus lysate clotting activity. Acetone-extracted whole cells of B. ovis and of B. canis and of a rough mutant of B; abortus had the same toxicity for mice, but it was not possible to extract endotoxin from B. canis by the methods used.     

Phagocytosis, killing, and oxidant production by bovine monocyte-derived macrophages upon exposure to Brucella abortus strain 2308

Phagocytosis and intracellular survival of Brucella abortus, and oxidant production by monocyte-derived macrophages from ten B. abortus-naive cows were studied. Phagocytosis of bacteria opsonized with naive-autologous sera or reactor serum was significantly less than phagocytosis of bacteria opsonized with fetal bovine serum. After phagocytosis, intracellular survival of bacteria opsonized with naive-autologous or reactor sera was significantly less than survival of bacteria opsonized with fetal bovine serum. Production of oxidant by macrophages stimulated with B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera was not significantly different. Although macrophages from one animal showed significantly less phagocytic activity, intracellular killing and oxidant production by macrophages from the ten individual cows toward B. abortus opsonized with naive-autologous, reactor, and fetal calf sera were homogeneous. The abilities of the macrophages to phagocytize and to kill B. abortus were not associated with each other or with oxidant production. Innate resistance or sensitivity to B. abortus was not identified in the cows based on macrophage function.     

A prolonged outbreak of Shigella sonnei infections in traditionally observant Jewish communities in North America caused by a molecularly distinct bacterial subtype

During 1994-1996, Shigella sonnei outbreaks occurred in 8 North American traditionally observant Jewish communities. These communities remain relatively separate from neighboring populations while maintaining close contact by travel with coreligionists in other cities. Epidemiologic investigations suggested community-to-community transmission via travel. Outbreak-related and control isolates of S. sonnei from each city were subtyped by pulsed-field gel electrophoresis (PFGE) to confirm an epidemiologic linkage between outbreaks. Forty-three (94%) of 46 outbreak-related isolates had closely related PFGE patterns, constituting a single subtype; 33 (94%) of 35 control isolates demonstrated unrelated PFGE patterns. Several patterns differing by < or = 3 bands were identified within the outbreak subtype; one of these accounted for 65% of outbreak isolates. Hence, a single subtype of S. sonnei caused an international outbreak involving 8 traditionally observant Jewish communities, but not neighboring populations, over a 2-year period, suggesting sustained propagation of the epidemic strain between communities.     

An ethical walk through the ICU

Brucella cultures isolated from sick dogs have not been properly studied in Russia up to the present time. In 1994 a culture has been isolated from aborted fetus of a dog. Investigations by the traditional methods referred it to Brucella genus, species canis (strain K-01). Reference strain B. canis RM6/66 and B. canis K-01 were identical by the profiles of protein antigens in immunoblotting with a set of antibrucellosis sera. B. canis, B. suis, B. abortus, and B. melitensis. However, immunoblotting with sera to B. canis showed the similarity of B. canis cultures with the reference strain B. suis 1330, and use of sera to B. suis, B. abortus, and B. melitensis helped differentiate between the reference B. suis 1330 strain and B. canis strains. All the antisera used permitted the differentiation of Brucella strains from Yersinia enterocolitica 0:9, Escherichia coli 0:157, and Salmonella typhimurium cross reacting with Brucella in serological tests. Immunoblotting is a promising taxonomic criterion for identification of newly detected representatives of the Brucella genus.