Impaired bacterial proliferation, and compromised immune response in diabetic db/db mice infected with Brucella abortus

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1 농업생명과학연구 47(2) pp Journal of Agriculture & Life Science 47(2) pp Impaired bacterial proliferation, and compromised immune response in diabetic db/db mice infected with Brucella abortus Dae-Geun Kim 1 Jin-Ju Lee 1 Hannah Leah Simborio 1 Alisha Wehdnesday Bernardo Reyes 1 Wongi Min 1 Hu-Jang Lee 1 Hong-Hee Chang 2 Suk Kim 1,2* 1 College of Veterinary Medicine, Gyeongsang National University, Jinju, , Korea 2 Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, , Korea Received: MAR , Revised: APR , Accepted: APR ABSTRACT Brucellosis is an important bacterial zoonosis in humans and domestic animals. Brucella spp. are taken up, and survive within non-professional and professional phagocytes. In common belief, diabetes mellitus increases susceptibility to pathogenic infection. In this study, Brucella (B.) abortus was inoculated into a diabetic animal model, db/db mice, in order to show the course of brucellosis in diabetic state. The liver proliferation, bacterial burden of the liver, level of cytokines in serum and macrophage migration into liver, were investigated at 14 days post-infection. In comparison with the uninfected control mice, the results revealed that the weight of the liver of infected db/db mice was higher but with lower bacterial load in this organ. The level of MCP-1 mrna expression in the liver was lower, the levels of IL-12p70, IL-10, TNF-α and IFN-γ in serum was significantly higher and the macrophages migration was significantly lower in infected mice than in the control group. In conclusion, this present study suggested that MCP-1 suppression by B. abortus infection may inhibit the macrophages migration, and consequently may induce to abrogate the bacterial survival in db/db mouse liver. Furthermore, the increased inflammatory cytokines may contribute to inhibition of B. abortus proliferation in diabetic mice. Key words - Brucella abortus, Diabetic db/db mice, Liver, Bacterial proliferation *Corresponding author: Suk kim Tel : Fax : kimsuk@gnu.ac.kr

2 66 Journal of Agriculture & Life Science 47(1) Ⅰ. Introduction Brucellosis is an important bacterial zoonosis that manifests as a serious debilitating disease in humans and abortion and sterility in domestic animals (Corbel, 1997). Brucella abortus, the causative agent of brucellosis, is a Gram-negative facultative intracellular pathogen that can replicate within professional and nonprofessional phagocytes (Kim et al., 2003). The most common portals of entry for Brucella are mucous membranes of the respiratory and digestive tracts, as well as the conjunctiva and membranes covering the sexual organs of its natural host. Brucella are eventually taken up by phagocytic cells and reach the regional lymph nodes, leading to subsequent systemic dissemination. These organisms can efficiently colonize cells of the monocyte/macrophage lineage and replicate to high numbers in the liver and spleen (Silva et al., 2011). Host protection against Brucella depends on cell-mediated immunity, involving mainly activated antigen-presenting cells (macrophages, dendritic cells) and CD4+, CD8+ T-lymphocytes (Gorvel, 2008). Brucella antigens induce the production of T-helper 1 (Th1) cytokines in humans, thus Th1 immune response is essential for the clearance of Brucella infection (Yingst and Hoover, 2003). However, Brucella can survive and replicate in host macrophages and dendritic cells subverting innate immunity and evading adaptive immune mechanisms (Gorvel, 2008). The number of patients with type 2 diabetes mellitus, who exhibit insulin resistance, is increasing recently all over the world (Bonora et al., 2004). Contrary to common belief, the association between diabetes mellitus and increased susceptibility to infection in general is not supported by strong evidence (Thornton, 1971; Wheat, 1980). Diabetes is often identified as an independent risk factor for infections (Monaghan et al., 2011). However, many specific infections are more common in diabetic patients, and some occur almost exclusively. In addition, some infections occur with increased severity and are associated with an increased risk of complications, such as foot and ankle infections, the most common causes of hospitalization in diabetic patients of which Staphylococcus aureus is a major pathogen implicated in these infections (Ramsey et al., 1999). The diabetic db/db mouse (relabeled as lepr db ) is one of the most requested mice in diabetes and obesity researches. This strain of mice originally derived from an autosomal recessive mutation on chromosome 4 in mice of C57BL/KsJ strain originating from Bar Horbor, Maine (Shafrir E, 2003) which showed a high susceptibility to bacterial infection. The mutation in this animal was traced to db gene, which encodes for leptin receptors. These animals rapidly succumbed to subcutaneous B. psedomallei infection, paralleled by severe hypoglycaemia and increased expression of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, in the spleen, despite comparable bacterial loads in the spleen of non-diabetic mice (Hodgson et al., 2011). Furthermore, these mice were highly susceptible to Listeria monocytogenes, with inhibited elimination of bacteria from the liver. In addition, db/db mice are more susceptible to staphylococcal infection than their non-diabetic littermates and that persistent hyperglycemia modulates innate immunity in the diabetic host (Park et al., 2009). However, the severity and pathogenic mechanism of brucellosis in diabetic mice are still unclear. In this study, the host resistance to B. abortus infection in type 2 diabetic db/db mouse, focusing on the mechanism of bacterial proliferation inhibition and

3 Kim et al : Impaired bacterial proliferation, and compromised immune response in diabetic db/db mice infected with Brucella abortus 67 host immune responses were investigated. Ⅱ. Material And Methods 2.1 Mice Female 7-week-old BKS.Cg-m +/+ Lepr db /J (db/db) and specific pathogen free (SPF) C57BL/6J mice (control) were purchased from the Korea Biotechnology Research Association. Mice were allowed to acclimate for 1 week before use. The animals were housed under controlled environmental conditions of temperature (22 ) and a 12 h light/dark cycle. They were maintained on a standard diet with no restriction to access of food and water. All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Gyeongsang national university (Permit number: GNU M0008), and were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Gyeongsang national university. 2.2 Bacteria culture and media B. abortus strains were derived from 544 (ATCC 23448), a smooth, virulent B. abortus biovar 1 strains. The B. abortus organisms were maintained as frozen glycerol stocks and cultured in Brucella broth (BD) or Brucella broth containing 1.5% agar without antibiotics for 3 days at 37. Bacteria were grown at 37 with vigorous shaking until they reached the stationary phase, and bacterial growth rates were measured spectrophotometrically at 600 nm. 2.3 Bacterial infection B. abortus 544 (ATCC 23448) was grown in Brucella broth for 24 h, and harvested in phosphate-buffered saline (PBS), adjusted spectrophotometrically at 600 nm and diluted to 10 5 colonyforming units (CFU)/ml. Mice were infected by intraperitoneal (i.p.) route with 1 x 10 4 CFU of viable bacteria in 0.1 ml PBS. At 14 days post-infection, the infected mice were sacrificed under anesthesia, livers and spleens were removed and then weighed. The numbers of viable B. abortus in the liver and spleen of infected animals were established by plating serial 10-fold dilutions of organ homogenates in on Brucella agar. The colonies were routinely counted after 48 h. 2.4 Immunohistochemistry The liver tissues from db/db and control mice were snap-frozen by quenching in liquid nitrogen. Twenty micrometer thick sections were prepared, and immunostained using the avidin-biotin-peroxidase complex (ABC) method with a ImmucoCruz rat ABC Staining System ABC kit (Santa cruz). These were then incubated with rat anti-mouse macrophage marker (RM H3; diluted 1:50; Santa cruz) overnight at 4. Diaminobenzidine was used as the chromogen and then the immunolabeled sections were counterstained with hematoxylin. Quantitative analysis of macrophages density was performed by counting the number of macrophages using the specific antibodies (RM H3; diluted 1:50; Santa cruz). Macrophages were counted in random high-power (x 200) fields. 2.5 Quantitative RT-PCR Total RNA was extracted using RiboEx reagent (GeneAll, Korea), in accordance with the manufacturer's instructions. Total RNA was reverse transcribed into cdna using a reverse transcriptase. Cytokine-specific primers were synthesized by BIONEER, according to sequence designs previously described (Bohn et al., 1994; Ehlers et al., 1992;

4 68 Journal of Agriculture & Life Science 47(1) Table 1. Cytokine specific primer pair sequences used in RT-PCR Cytokine MCP-1 ß-Actin IFN-γ TNF-α IL-1β IL-2 IL-12p35 Oligonucleotide sequence (5'-3') GGA AAA ATG GAT CCA CAC CTT GC TCT CTT CCT CCA CCA CCA TGC AG TGG AAT CCT GTG GCA TCC ATG AAA C TAA AAC GCA GCT CAG TAA CAG TCC G AGC GGC TGA CTG AAC TCA GAT TGT AG GTC ACA GTT TTC AGC TGT ATA GGG GGC AGG TCT ACT TTG GAG TCA TTG C ACA TTC GAG GCT CCA GTG AAT TCG G TCA TGG GAT GAT GAT GAT AAC CTG CT CCC ATA CTT TAG GAA GAC ACG GAT T TGA TGG ACC TAC AGG AGC TCC TGA G GAG TCA AAT CCA GAA CAT GCC GCA G GCA AGA GAC ACA GTC CTG GG TGC ATC AGC TCA TCG ATG GC Size (bp) Reference 582 Bongers et al., Murray et al., Murray et al., Murray et al., Ehlers et al., Murray et al., Romani et al., 1994 Murray et al., 1990; Romani et al., 1994) (Table 1). The optimal MgCl 2 concentration for each primer pair was determined experimentally. The thermal cycling parameters were as follows: 95 for 2 min, followed by 30 cycles each of denaturation at 94 for 50 s, annealing of primer and fragment at 60 for 50 s, and primer extension at 72 for 1 min. A final extension of 72 for 4 min was included. In preliminary experiments, amplification for 30 cycles was shown to lie in the linear portion of the curve for the amount of PCR product produced. The PCR products were visualized by UV light after electrophoresis using a 2.0% agarose gel containing 0.5 mg of ethidium bromide/ml. As the DNA size marker, a 100 bp ladder (ElpisBiotech, Korea) was used. PCR using primers for β-actin was performed on each individual sample as an internal positive-control standard. As a negative control, PCR omitting cdna (water as the substitute) was run concurrently. PCR using primers for β-actin was also performed using non-reverse-transcribed total RNA for each sample to confirm that amplification was based solely on cdna. PCR-assisted mrna amplification was repeated twice for three separately prepared cdna samples. Results shown were representative of triplicate cdna samples. 2.6 Cytokine measurement in serum Cytokine assessment was carried out using BD cytometric bead array mouse inflammation kit (BD Bioscience, San Jose, CA, USA) for detection of cytokines in plasma diluted (1/10) with appropriate diluent. The cytometric bead array (CBA) technique utilizes micro particles or beads labeled with discrete fluorescence intensity. The maximum emission of capture beads is at 650 nm on random early detection (RED) parameter. Cytokine specific capture antibody is covalently attached to beads. The captured cytokines are detected using specific antibodies with phycoerythrin (PE) fluorochrome which emits at 585 nm on yellow parameter. The intensity of fluorescence

5 Kim et al : Impaired bacterial proliferation, and compromised immune response in diabetic db/db mice infected with Brucella abortus 69 of yellow parameter is proportional to the amount of cytokine present in test samples. Cytokines were determined in the test samples according to the manufacturer's instructions. Briefly, test samples (50 µ l) and PE detection antibody were incubated with capture bead reagent for 3 h in the dark at room temperature. All unbound antibodies were washed (1.0 ml wash buffer) and re-suspended in 300 µl before acquisition on BD FACS array bio-analyzer (BD Bioscience, San Jose, CA, USA). All six cytokines exhibited single, well separated, peaks. Six individual cytokine standard curves (range 20-5,000 pg/ml) were run in each assay. The range of detection was between 3 and 5,000 pg/ml calculated from curve estimation for an average of five assays using power fit and R 2 >0.99 for all cytokines. Fig. 1. Liver weight of uninfected and infected mice at 14 days postinfection. Control ( ) and diabetic ( ) mice was infected intraperitoneally with or without B. abortus. The result represents the mean ± SD of organ weight. (***P<0.001.) investigated. Diabetic db/db mice were infected with 2.7 Statistical analysis The results obtained were expressed as the mean ± standard deviation (SD) for the replicate experiments. Student's t-test was used to make a statistical comparison between the groups. Results with P<0.05 were considered statistically significant. Ⅲ. Results And Discussions 3.1 Bacterial proliferation in mice Diabetic db/db mice were susceptible to L. monocytogenes, and clearance of bacteria was suppressed especially in liver (Ikejima et al., 2005). Streptozotocin induced (Type 1) diabetes rat model of B. melitensis infections had a significantly higher number of bacteria in their spleen and liver than rats in control group (Yumuk et al., 2003). From these reasons, the host resistance to B. abortus infection in type 2 diabetic db/db mouse was Fig. 2. Bacterial proliferation (CFUs) in mice. Control ( ) and diabetic ( ) mice was infected intraperitoneally with B. abortus. The recovery of viable bacteria from the liver of mice at 14 days post-infection was determined. The result represents the mean ± SD of CFUs. (*P<0.05) 10 4 CFU of viable B. abortus intraperitoneally to determine bacterial growth in the liver. At 14 days post-infection, the mice were sacrificed, and liver weight (Fig. 1) and the bacterial load (Fig. 2) in the liver were determined. The weight of the liver of infected db/db mice group were higher than the control mice (P<0.001) (Fig. 1). On the other hand,

6 70 Journal of Agriculture & Life Science 47(1) the bacterial load in the liver of db/db mice group were significantly less than that of the control group (P<0.05) (Fig. 2). (A) 3.2 Expression of cytokine mrna in the liver of diabetic db/db mice Since cytokines play an important role for killing bacteria, we examined whether the impaired bacterial clearance exhibited by db/db mice is associated with the ability of cytokine expression. On the basic roles of MCP-1 for attracting and activating monocytes (Chensue, 2001), we investigated the MCP-1 expression in the liver of the db/db mice which showed that MCP-1 mrna expression was less than those of the control mice after B. abortus infection (Fig. 3A and B). The expression resulted in an up to 0.31-fold reduction at 14 days post-infection compared to that of the control mice, but this difference was not significant. On the other hand, IL-10 mrna expression in the liver of db/db mice was 2.2-fold upregulated than that of the control mice (Fig. 3B). However, IFNγ, TNF-α, IL-1β, IL-2 and IL-12p35 mrna expression did not show significant differences between db/db and control mice (Fig. 3B). In a study done by Hodgson et al. (2011), the expression of the proinflammatory cytokines, such as IL-1 and TNF-α was significantly higher in diabetic mice on day 3 post-infection with B. pseudomallei. The elevated expression of IL-1 and TNF-α potentially contributes to poor prognosis in diabetic mice following subcutaneous B. pseudomallei infection similar to BALB/c mice which rapidly succumb to B. pseudomallei infection following a hyper inflammatory response (Ulett et al., 2000). The present study suggested that MCP-1 suppression and IL-10 activation by B. abortus infection may have an important role for immune responses in db/db mice. (B) Fig. 3. Expression of cytokine mrna in the liver. The RT-PCR results (A) and the mrna expression comparison (B) in the liver of control ( ) and diabetic ( ) mice were determined at 14 days post-infection. The result (B) represents the mean ± SD of expression of cytokine mrna. 3.3 Macrophage populations of liver after B. abortus infection Quantitative analysis of cell density was performed

7 Kim et al : Impaired bacterial proliferation, and compromised immune response in diabetic db/db mice infected with Brucella abortus 71 via counting the number of macrophages using the specific antibody at 14 days post-infection. The macrophage cells in the liver were counted in random high-power fields of each immunohistochemically stained section. The present study revealed that the population of macrophages in db/db mice was significantly lower than those of the control mice (Fig. 4). Fig. 4. Macrophage populations in liver of control ( ) and diabetic ( ) mice at 14 days post-infection. The result represents the mean ± SD for a group of five mice. Statistically significant differences between macrophage populations in liver of diabetic mice and that of the control are indicated by an asterisk (**P<0.01). phagocytic cells. Brucella has been proposed to develop a stealth strategy through pathogen-associated molecular patterns (PAMPs) reduction, modification and hiding, to ensure low stimulatory activity and toxicity for cells allowing Brucella to reach its replication niche before the activation of antimicrobial mechanisms by adaptive immunity (Barquero-Calvo et al., 2007). 3.4 Cytokine levels in serum Cytokines play an important role in Brucella infection as shown in the protective immunity against B. abortus mediated by acquired cellular resistance, of which the gamma interferon (IFN-γ) - producing T cells play a key role (Zhan and Cheers, 1995). The addition of either IL-2 or IFN-γ to macrophage cultures results in a reduced intracellular CFU of the virulent B. abortus (Jiang and Baldwin, 1993). Furthermore, the treatment of Brucella-susceptible mice with IL-12 increases both primary and secondary In this study, the db/db mice infected with B. abortus yielded a lower bacterial load in the liver than the control group. Macrophages have been shown to constitute an important site for Brucella intracellular replication within tissues. Despite the fact that over 90% of Brucella internalized by macrophages are killed soon after phagocytosis, a few bacteria can be protected within vacuoles that exclude proteases and oxidants via blocking phagosome-lysosome fusion and establishing an intracellular niche permissive for replication without affecting the survivability of these Fig. 5. Cytokine levels in plasma of control ( ) and diabetic ( ) mice. Serum IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10, IL-6 were measured at each time points. The result represents the mean ± SD for a group of five mice. Statistically significant differences between cytokine levels of diabetic mice and that of control are indicated by an asterisk (*P<0.05, **P<0.01, ***P<0.001.)

8 72 Journal of Agriculture & Life Science 47(1) immunity (Sathiyaseelan et al., 2006). In the study, the concentrations of IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10 and IL-6 in serum of db/db mice were measured to confirm the levels of circulating inflammatory cytokines in the blood. The results showed that the concentrations of IL-12p70, TNF-α, IFN-γ and IL-10 of db/db mice were significantly higher than those of the control mice at 14 days post-infection (Fig. 5). In conclusion, this study suggested that the decreased macrophage population and the increased inflammatory cytokines have resulted in the bacterial population inhibition in the liver of the infected db/db mice. However, further study should be done on bacterial pathogenicity and host immune response for chronic brucellosis in diabetic state which may help to understand the pathogenic mechanisms of bacterial infection in humans with diabetes mellitus. Ⅳ. Acknowledgements The present work was supported by ipet (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries) ( ), Ministry for Food, Agriculture, Forestry and Fisheries, Korea.»Literature cited Barquero-Calvo, E., Chaves-Olarte, E., Weiss, D.S., Guzman-Verri, C., Chacon-Diaz, C., Rucavado, A., Moriyon, I., Moreno, E., Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection. PLoS One 2 (7), e631. Bohn, E., Heesemann, J., Ehlers, S., Autenrieth, I.B., Early gamma interferon mrna expression is associated with resistance of mice against Yersinia enterocolitica. Infect. Immun. 62, Bongers M, Liehl E, Barsig J. One-step RT-PCR to detect cytokine/ chemokine induction in macrophages. Focus 1999; 21: 668. Bonora, E., Kiechl, S., Willeit, J., Oberhollenzer, F., Egger, G., Meigs, J.B., Bonadonna, R.C., Muggeo, M., Population-based incidence rates and risk factors for type 2 diabetes in white individuals: the Bruneck study. Diabetes 53, Chensue, S.W., Molecular machinations: chemokine signals in host-pathogen interactions. Clin. Microbiol. Rev. 14, , table of contents. Chomczynski, P., Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, Corbel, M.J., Brucellosis: an overview. Emerg. infect. dis. 3, Detilleux, P.G., Deyoe, B.L., Cheville, N.F., 1990b. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro. Infect. Immun. 58, Dilworth, D.D., McCarrey, J.R., Single-step elimination of contaminating DNA prior to reverse transcriptase PCR. PCR methods and applications 1, Ehlers, S., Mielke, M.E., Blankenstein, T., Hahn, H., Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity. J. Immunol. 149, Gorvel, J.P., Brucella: a Mr "Hide" converted

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