The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.     

Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy

Vero cells were inoculated with the six species of Brucella (B. abortus, B. melitensis, B. suis, B. neotomae, B. canis, and B. ovis) and examined by fluorescence and electron microscopy. All Brucella spp. were internalized by Vero cells. In all cells except those inoculated with B. canis, the numbers of intracellular brucellae increased with time after inoculation. Intracellular brucellae were first seen within phagosomes and phagolysosomes. Subsequent localization within cisternae of the rough endoplasmic reticulum was seen with all species of Brucella, except B. canis, which was restricted to phagolysosomes. Although rough brucellae were more adherent and entered a greater number of Vero cells, intracellular replication occurred in a larger percentage of cells with smooth rather than with rough brucellae. These results suggest that phagocytosed Brucella spp. are transferred 1) to cisternae of the rough endoplasmic reticulum, where unrestricted bacterial replication takes place; or 2) to phagolysosomes in which Brucella spp. fail to replicate. The various strains of Brucella spp. differ in their ability to induce their own transfer to the rough endoplasmic reticulum.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

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.     

The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus.

Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system. A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state. The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay. Interaction of B. abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B. abortus was investigated. This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.     

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

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.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

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.     

Effect of milk stasis on Brucella abortus infection of the mammary gland in goats

To compare the effects of milk stasis and milk flow on Brucella abortus infection of the mammary gland under the same systemic conditions, primiparous goats (n = 5) were inoculated IV with B abortus on the day of parturition, and suckling by their neonates was restricted to one mammary gland. Goats were euthanatized and necropsied at 3 weeks after inoculation, and milk, mammary glands, and supramammary lymph nodes were evaluated by bacteriologic, histologic, and immunoenzymatic staining techniques. Nonnursed mammary glands had high titers of brucellae in milk, moderate interstitial mastitis, and brucellar antigen in macrophages located primarily in alveolar and ductal lumina. Brucellae often filled the macrophage cytoplasm. In contrast, nursed mammary glands had fewer brucellae in milk, minimal inflammatory changes, and no detectable brucellar antigen in histologic sections. Hyperplastic changes were only seen in supramammary lymph nodes draining nonnursed mammary glands; these contained more brucellae than lymph nodes draining nursed mammary glands. These studies show that milk stasis may be the sole cause of increased susceptibility of nonnursed mammary glands to B abortus infection.     

Effect of stage of gestation on efficacy of Brucella abortus strain-19 vaccination in cattle.

Seventy-nine cattle in all stages of gestation were inoculated with a low dose (2.5 x 10(8) colony-forming units) of Brucella abortus strain 19, then challenge exposed with pathogenic B abortus strain 2308 during the subsequent gestation. A brucellosis case was defined by isolation of strain 2308 from dam or calf samples. Cumulative incidence of brucellosis cases was 48, 33, 25, or 47% for cattle that were, respectively, not pregnant, or 19 to 87, 100 to 167, or 190 to 253 days in gestation at vaccination. The cumulative incidence was 56% in 27 nonvaccinated controls. The 95% confidence intervals for risk ratios included 1 in all cattle, except those that were 100 to 167 days in gestation at vaccination (ie, second trimester); the confidence interval for this group was 0.21 to 0.97. The prevented fraction (1-risk ratio) attributed to strain 19, in ascending order, was 0.14, 0.16, 0.4, or 0.55, respectively, for cattle that were not pregnant, or were 190 to 253, 19 to 87, or 100 to 167 days in gestation at vaccination. Potential confounders of breed, pen effect, and gestation days at challenge exposure did not significantly affect results. Results supported the hypothesis that stage of gestation at vaccination will affect the prevented fraction of brucellosis, or efficacy of strain 19, in cattle vaccinated with a low dose and, therefore, is one factor that may explain variation in strain 19-induced protection.     

Plasmid transfer and plasmid-mediated genetic exchange in Brucella abortus.

Naturally-occurring plasmids and gene transfer mechanisms have not yet been reported in brucellae. Here we show that Brucella abortus is capable of maintaining and transferring the broad-host-range plasmids pTH10 (IncP), pSa (IncW) and R751 (IncP), and describe pTH10-mediated transfer of B. abortus chromosomal genes to Escherichia coli. All three plasmids transferred by conjugation from E. coli to B. abortus S19, and from B. abortus S19 to B. abortus 292 (biovar 4). They were stably maintained with no effect on biotyping characteristics. Plasmid pTH10 is a Tn1-containing derivative of RP4. It confers temperature-sensitive resistance to kanamycin, tetracycline and ampicillin to E. coli, but its tetracycline resistance and temperature sensitivity were poorly expressed in B. abortus. Plasmids pTH10 and pSa both transferred from B. abortus to E. coli DP50, a strain that is auxotrophic for diaminopimelic acid (DAP) Plasmid pTH10 (but not pSa) mobilized Brucella chromosomal gene(s) for DAP synthesis to DP50, yielding non-DAP-requiring (NDR) transconjugants. Neither plasmid transferred the NDR marker from their original E. coli host strains, nor did pTH10 transfer it from NDR transconjugants. Escherichia coli NDR transconjugant EP8.11 was cured of pTH10 by passage at the nonpermissive temperature, but retained the NDR marker and the Tn1-encoded resistance to ampicillin, indicating Tn1-mediated integration of Brucella chromosomal DNA into the E. coli chromosome.     

Prevalence of Brucella abortus and Brucella canis antibodies in dogs in Nigeria

Serum samples collected from dogs brought for routine physical examination, vaccination and other complaints at the Small Animal Clinic of Ahmadu Bello University, Zaria, Nigeria were tested for Brucella abortus and Brucella canis antibodies. Ninety-five (38-2 per cent) of 249 dogs studied were positive for B. abortus agglutinins by the Rose Bengal plate test (RBPT) but none was sero-positive by the standard agglutination test (SAT). The antibody prevalence for B. canis by the SAT was 28-6 per cent for 224 dogs tested. Exotic breeds of dogs had a prevalence of 34-9 per cent for B. canis agglutinins while 28-1 per cent of local dogs were sero-positive. Twenty-two per cent of dogs older than 2 years were sero-positive compared to a prevalence of 33-3 per cent found amongst dogs younger than 1 year. A similar B. canis infection rate was observed amongst male (29-6 per cent) and female (26-7 per cent) dogs.     

Effect of nursing on Brucella abortus infection of mammary glands of goats

Eight 1-year-old, goats were inoculated intravenously with Brucella abortus (B. abortus) on the day of parturition and necropsied at 28 days after inoculation. Four nursed their kids and four did not (milk was not removed from the udders). Tissues and fluids were examined by bacterial isolation, light microscopy, and serologic methods. Nonnursing goats had high titers of brucellae (less than or equal to 10(8) organisms/ml) in milk (brucellae were isolated from four of four udders), had marked enlargement of supramammary lymph nodes, and had lymphoplasmacytic and histiocytic interstitial mastitis. Immunoperoxidase staining revealed that brucellae were primarily in macrophages and neutrophils of the mammary alveolar and ductal lumens and in macrophages of the subcapsular sinuses of the supramammary lymph node. In contrast, nursing goats excreted brucellae intermittently at low concentrations (less than 10(3) organisms/ml) in milk; brucellae were isolated at necropsy from one of four udders; supramammary lymph nodes were not enlarged; and mammary lesions were not seen. Brucellae were detected in more tissues other than the udder, and serum anti-Brucella antibody titers were higher in nonnursing goats than in nursing goats. The present study indicates that the failure to nurse or release milk enhances localization and replication of B. abortus in mammary glands of goats after parturition, and that mammary gland infection may result in increased systemic spread and persistence of brucellae in the host.     

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.