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Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.
By using mice infected with strains of Brucella abortus and Brucella melitensis we examined the histological responses to infection, the relationship of histology to persistence of organisms, and the relation of persistence of organisms to the acquisition of acquired cellular resistance (ACR). Infection with B. abortus resulted in well-formed granulomas in the livers, which persisted for more than 30 days. In contrast, infection with B. melitensis produced microabscesses in the livers which resolved before 30 days. The clearance of organisms from the tissues was also different. A total of 30 days after infection, large numbers of viable bacteria were recovered from the tissues of B. abortus-infected mice whereas bacteria were no longer recoverable from B. melitensis-infected animals. ACR to Listeria monocytogenes, another intracellular pathogen, persisted for more than 30 days in B. abortus-infected mice but waned rapidly in B. melitensis-infected animals. This disappearance of ACR due to B. melitensis paralleled the clearance of bacteria from the tissues.
Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.
CBA/N mice have an X-linked immune defect in B lymphocyte function which leads to their inability to respond to several thymus-independent antigens. We report here that these mice and immunologically defective F1 male (CBA/N X DBA/2N) mice can respond to Brucella abortus and to 2,4,6-trinitrophenyl derivatives of Brucella abortus (TNP-BA). These responses can be obtained in vivo and in vitro and are thymus-independent by the criteria that (a) they can be transferred to irradiated recipients by bone marrow cells and anti-Thy-1.2 and complement-treated spleen cells; (b) that nu/nu BALB/c spleen cells respond to TNP-BA in vitro; and (c) that anti-Thy-1.2 and complement-treated (CBA/N X DBA/2N)F1 male spleen cells respond to TNP-BA in vitro. B. abortus and TNP-BA are poor polyclonal B cell activators (PBA) and poor B cell mitogens, unlike lipopolysaccharide which is both a powerful PBA and B cell mitogen. These results therefore indicate that mice with the CBA/N B cell defect can respond to some thymus-independent antigens, namely TNP-BA, and as shown previously, TNP-LPS, although not to other thymus-independent antigens. This, in turn, suggests that thymus-independent antigens may be subdivided on the basis of their ability or inability to stimulate responses by CBA/N B lymphocytes.
Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.
While both Brucella abortus and Yersinia enterocolitica IX have O antigens in common, they differ significantly with respect to motility. Thus Br abortus is always non-motile while Y enterocolitica is motile when grown at room temperature. The presence of yersinia H agglutinins in serum can be shown to be evidence of previous exposure to Y enterocolitica. These agglutinins are not generated by brucella infection. A rapid H agglutination test will serve to provide this differentiation without interference from cross-reacting O antigens.
Naturally acquired Brucella abortus infections were studied during consecutive pregnancies in eight sheep and in their lambs over a period of 40 months to evaluate epizootiologic aspects of natural infection in sheep. Brucella abortus was isolated from the ewes following 16 of 26 natural terminations of pregnancy: from 5 of 6 ewes in the first year, from six of eight ewes in the second year, from two of six ewes in the third year, and from three of six ewes in the fourth year. Vaginal swab samples and milk samples were the most consistent source of the brucella organisms. Brucella abortus was isolated from three ewes when standard tube test seroagglutination titers were less than 1:100. In contrast, results of supplemental tests (card, 2-mercaptoethanol, complement-fixation, and Rivanol) remained positive during the study. During the 40 months, B abortus was isolated from 4 of 4 aborted fetuses, 2 of 5 stillborn lambs, 10 of 37 living lambs, and as an indicator of continuing infection, from 6 of 12 lambs born during the fourth year. Although B abortus has a definite host preference for cattle, this study demonstrated that under appropriate management conditions, sheep may be naturally infected and may remain infected for more than 40 months. Epizootiologic evaluation of all factors, including husbandry practices and exposure potential, should be utilized in determining the need to test other species that may have been exposed to cattle infected with B abortus.
The development of acquired resistance to Brucella abortus was studied in mice infected intravenously with small numbers of live CO2-dependent B. abortus 544 used as vaccinal strain. CO2-independent B. abortus was used as challenge strain. The influence of several parameters in the development of acquired resistance was examined: vaccination--challenge interval, challenge route, dose of challenge, challenge-sacrifice interval. The behavior of the challenge strain in normal mice depended on the size of the inoculum. After an intravenous inoculum of about 1 X 10(6) bacteria, the level of infection in the liver or the spleen followed three phases: a phase of increase of the viable counts, a phase of decrease, and a phase of plateau. The decreasing phase was more pronounced in liver than in spleen. After an intraperitoneal injection of the same dose of bacteria, an infection developed following approximately the same pattern, except during the first day of infection. One month after vaccination by an inoculum of about 1 X 10(4) viable B. abortus, an accelerated appearance of the decreasing phase prevented almost completely the multiplication of an intravenous challenge of about 1 X 10(6) B. abortus in the spleens. In the livers the same phenomenon took place after a small increase of the bacterial population. After an intraperitoneal challenge, the numbers of Brucella isolated from the spleens and livers of vaccinated mice first fell sharply to practically nothing around the sixth day, and then rose to reach a phase of slow increase or of plateau until the twenty-fourth day. In our experimental conditions, the vaccine population remained undisturbed by the reinfecting inoculum.
When Brucella abortus strain 19 is given intraperitoneally to mice it protects them against subsequent infection with large doses of Babesia microti. The protection obtained was more effective when B. abortus was given intraperitoneally than when it was injected subcutaneously. This non-specific protection seems to be best explained by the stimulation of macrophages so as to release a mediator which limits the intracellular replication of the parasites.
Cell extracts of Brucella abortus (British 19) catabolized erythritol through a series of phosphorylated intermediates to dihydroxyacetonephosphate and CO-2. Cell extracts required adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), Mg2+, inorganic orthophosphate, and reduced glutathione for activity. The first reaction in the pathway was the phosphorylation of mesoerythritol with an ATP-dependent kinase which formed d-erythritol 1-phosphate (d-erythro-tetritol 1-phosphate). d-Erythritol 1-phosphate was oxidized by an NAD-dependent dehydrogenase to d-erythrulose 1-phosphate (d-glycero-2-tetrulose 1-phosphate). B. abortus (US-19) was found to lack the succeeding enzyme in the pathway and was used to prepare substrate amounts of d-erythrulose 1-phosphate. d-Erythritol 1-phosphate dehydrogenase (d-erythro-tetritol 1-phosphage: NAD 2-oxidoreductase) is probably membrane bound. d-Erythrulose 1-phosphate was oxidized by an NAD-dependent dehydrogenase to 3-keto-l-erythrose 4-phosphate (l-glycero-3-tetrosulose 4-phosphate) which was further oxidized at C-1 by a membrane-bound dehydrogenase coupled to the electron transport system. Either oxygen or nitrate had to be present as a terminal electron acceptor for the oxidation of 3-keto-l-erythrose 4-phosphate to 3-keto-l-erythronate 4-phosphate (l-glycero-3-tetrulosonic acid 4-phosphate). The beta-keto acid was decarboxylated by a soluble decarboxylase to dihydroxyacetonephosphate and CO-2. Dihydroxyacetonephosphate was converted to pyruvic acid by the final enzymes of glycolysis. The apparent dependence on the electron transport system of erythritol catabolism appears to be unique in Brucella and may play an important role in coupling metabolism to active transport and generation of ATP.
The growth of Brucella abortus (US-19) in a complex tryptose-yeast extract medium containing D-glucose is inhibited by 10 mM erythritol. The enzymes of the erythritol pathway, except for D-erythrulose 1-phosphate dehydrogenase (D-glycero-2-tetrulose 1-phosphate:nicotinamide adenine dinucleotide (NAD+) 4-oxidoreductase) were detected in the soluble and membrane fractions of cell extracts. Glucose catabolism by cell extracts was inhibited by erythritol, whereas, phosphorylated intermediates of the hexose monophosphate pathway were converted to pyruvic acid with oxygen consumption. Erythritol kinase (EC 2.7.1.27; adenosine 5'-triphosphate (ATP): erythritol 1-phosphotransferase) was found to be eightfold higher in activity than the hexokinase in cell extracts. In vivo, ATP is apparently consumed with the accumulation of D-erythrulose 1-phosphate (D-glycero-2-tetrulose 1-phosphate) and no substrate level phosphorylation. ATP levels dropped 10-fold in 30 min after addition of erythritol to log phase cells in tryptose-yeast extract medium with D-glucose as the carbon source. These data suggest bacteriostasis in the presence of erythritol results from the ATP drain caused by erythritol kinase.
A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.
Two hundred and eight strains of Brucella isolated from cattle and 5 isolated from man in various areas of France, during a two-year period from 1972 to 1974, were examined by all methods recommended by the Subcommittee on Taxonomy of the genus Brucella and were found to have the same characteristics that identify and define the species Brucella abortus. Of the 213 strains, 105 (49,29 p. 100) were biotype I, 103 strains (48,36 p. 100) biotype 3, three strains (1,41 p. 100) biotype 4 and 2 strains (0,94 p. 100) biotype 2. The distribution of Brucella abortus biotypes isolated from cattle and man in France is reviewed and compared with the data from our sample. It corroborates the epidemiologic importance of Brucella abortus biotype 3, just after type 1, and the absence of biotypes 5 to 9(1) in France. Five strains of Brucella abortus biotype 3, originating from a well-defined geographical area, are ureaseless. This new biochemical character distinguishes these strains from the main group of biotype 3 and is discussed from an epidemiological and methodological point of view. Data on the oxydative rates obtained on 12 substrates by the 213 strains are presented in graphic form which reveals a metabolic profile for the species Brucella abortus. The interest of this graphic presentation is discussed and its use should be recommended.
Cells producing antibody to brucella lipopolysaccharide were detected in spleens of mice infected with Brucella abortus 19 by a hemolytic plaque assay. The appearance of immunoglobulin M-producing cells preceded humoral antibodies. The primary plaques were observed 5 days after inoculation, and they were still present by day 70.
The susceptibility to brucella infection (Brucella abortus 544) of different inbred mice and outbred CD1 strains has been studied. The degree of infection was determined on days 7 and 11 after challenge. The CBA and DBA2 strains showed greater susceptibility and homogeneity compared to the other strains (C57B16, C3H, C57B16 x DBA2) and to the CD1 mice. The kinetics of infection in DBA2 and CBA, compared to those of the CD1 for a longer period, show that the degree of infection decreases rapidly after 11 days. However, 90 days after challenge, the level of infection in DBA2 mice remains higher than in CBA and CD1. The DBA2 strain seems able to provide an improvement for anti-brucella vaccine control.
The biotypes in the species Brucella abortus were arranged according to the sequential flow in the alterations of their characteristics. This reordering of the biotypes indicated that the organism currently classified as B abortus type 2 is the parent organism of all the biotypes in this species. However, laboratory-selected mutants from B abortus type 2 had characteristics that were mimetic, but not duplicative, of their naturally occuring counterparts. Evidence which indicates this phenomenon is related to permeability of the cell wall is presented and discussed.
The electron transport system in Brucella abortus has been characterized. Spectral studies of membrane preparations have indicated the presence of cytochromes a + a3 (maxima at 612 nm), cytochrome b (maxima at 560, 530, and 428 nm), cytochrome c (maxima at 552 and 522 nm), cytochrome o (maxima of carbon monoxide complex at 418 nm), and flavoproteins (minimum at 582 and 450 nm). Cytochromes a + a3 appeared only after cells had reached late log phase, possibly due to lowered oxygen tension in the medium. Dehydrogenases were shown to be present for D-erythritol 1-phosphate, L-lactate, reduced nicotinamide adenine dinucleotide, and succinate. All of the above substrates reduced the electron transport chain and at least some of the flavoproteins, indicating similar pathways of electron transport. N-ethylmaleimide, p-chloromercuribenzoate, and KCN were the only electron transport inhibitors that blocked electron transport by 100%. The system seemed to be uniquely resistant to other electron transport inhibitors.
The B 19S strain of Brucella abortus is found to act as an adjuvant to the anti-sheep red blood cell (SRBC) reaction in some congenic strains of mice but not in others. If the recipient is H-2b, there is no adjuvant effect (B 19S); neither is there (thymus-dependent anti-B 19S reaction as measured by thymocyte activation and change of electrophoretic mobility. In contrast, there is a thymus-independent anti-B 19S reaction (production of haemagglutinins) as good in the H-2b mice as in the others. Experiments with T cell deprived mice show that the adjuvant action of B 19S is thymus-dependent. As the anti-SRBC reaction without adjuvant is also thymus-dependent, it is difficult to distinguish anti-SRBC and anti-B 19S reactions from the adjuvant action of B 19S on the anti-SRBC reaction. Several explanations are possible, all involving H-2 (Ir?) controlled thymus dependent mechanisms.