Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse

Brucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial growth curves and resistance to environmental stress factors showed that Pyk plays an important role in B. abortus growth, especially under the conditions of nutrition deprivation, and resistance to oxidative stress. Additionally, cell infection assay showed that Pyk is necessary for B. abortus survival and evading fusion with lysosomes within RAW264.7 cells. Moreover, animal experiments exhibited that the Pyk deletion significantly reduced B. abortus virulence in a mouse infection model. Our results elucidated the role of the Pyk in B. abortus virulence and provided information for further investigation of Brucella virulence associated carbon metabolism.     

Protection of palmitic acid treatment in RAW264.7 cells and BALB/c mice during Brucella abortus 544 infection

BackgroundWe previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus.ObjectivesWe aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model.MethodsCell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O₂^-) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model.ResultsPA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O₂^-, and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance.ConclusionsOur results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids.     

Comparative histopathology in BALB/c mice infected with virulent and attenuated strains of Brucella abortus

The course of infection in BALB/c mice of virulent Brucella abortus strain 2308 (S-2308) and attenuated strain 19 (S-19) varies markedly. Whereas S-19 is eliminated at an exponential rate beginning at 2 weeks post infection (p.i.), strain 2308 assumes a steady state or plateau during the first 6 weeks p.i. and thereafter is eliminated very slowly over a period exceeding 6 months. Here we compared the initiation and maintenance of inflammatory reactions in spleens and livers of mice infected with either of the two strains of B. abortus for the first 6 weeks p.i. Histological changes in the liver were similar in response to either strain and were characterized by the development of small granulomas and an influx of polymorphonuclear leukocytes (PMN) and monocytes. Tissue reactions in the spleen were similar at weeks 1 and 2 p.i. At 3 weeks p.i. and thereafter, focal granulomatous responses in S-2308-infected mice exceeded those in mice infected with S-19. Numbers of nonspecific esterase (NSE) positive mononuclear leukocytes in S-19-infected spleens had increased by 3 weeks p.i. and remained elevated. No comparable increase in NSE positive cells occurred in mice infected with S-2308, and numbers were significantly lower. At 4 weeks p.i. the influx of mature neutrophils and the intensity of extramedullary hematopoiesis were significantly greater in S-19-infected spleens. A profound depletion of periarteriolar lymphoid tissue was noted in both infections for the first 3 weeks p.i. However, repopulation of lymphoid sheaths in S-19-infected spleens became significantly greater by 4 weeks p.i. and continued to increase at significantly higher levels during the next 2 weeks. This study demonstrates quantitative differences in splenic inflammatory responses which are temporally related to the more rapid elimination of S-19. Based upon the lower susceptibility of strain 2308 to the protective effects of immune serum it is hypothesized that the different patterns of infection and inflammation displayed by the 2 strains may related to the differential capacities of antibody opsonized S-19 and S-2308 to survive in activated macrophages.     

Unresponsiveness of vaccinated BALB/c mice to a second inoculation of lipopolysaccharide from Brucella abortus strain 2308

A study was conducted to determine whether the protection induced in mice by a primary inoculation of lipopolysaccharide from Brucella abortus would be enhanced by a second inoculation given at different time intervals. Protection was challenged by exposure of the mice to a virulent culture of B. abortus strain 2308. Reduced mean viable count and/or splenic weights were the criteria of protection. There was no significant difference (P greater than 0.05) in the protective responses among mice given a single inoculation. Vaccinated mice were significantly (P less than 0.05) better protected than were nonvaccinated mice. Mice given vaccinal inoculations simultaneous with challenge exposure were less protected (P less than 0.001) than were mice vaccinated prior to challenge, but were better protected (P less than 0.010) than were nonvaccinated mice.     

评价牛型布鲁氏菌疫苗免疫保护力BALB/c鼠模型的建立

为建立评价流产布鲁氏菌疫苗株免疫保护力的小鼠模型,选取6周龄雌性BALB/c小鼠为试验动物,随机分为3组(n=40):A19免疫攻毒组、非免疫攻毒组和PBS对照组。A19免疫组腹腔接种BALB/c鼠A19 5.0×10⁴ CFU,非免疫攻毒组和PBS对照组均接种PBS液0.2 mL。免疫后45 d,A19免疫攻毒组和非免疫攻毒组BALB/c鼠以3.0×10⁴ CFU剂量的2308强毒株攻击,攻毒后15和45 d分别剖杀小鼠,取小鼠脾脏称重、细菌分离、病理组织学检测。结果表明,攻毒后15 d,A19免疫攻毒组与未免疫攻毒组和PBS对照组之间克脾指数差异显著(P<0.05);攻毒后45 d,A19免疫攻毒组与PBS对照组的克脾指数差异不显著(P>0.05),与未免疫攻毒组克脾指数差异显著(P<0.05);免疫攻毒组的小鼠组织病理变化明显轻于未免疫攻毒组。结果表明,用BALB/c鼠为试验动物,以A19为疫苗参考株建立动物实验模型可以应用于牛型布鲁氏菌疫苗免疫保护力评价。     

Monophosphoryl lipid A-induced immune enhancement of Brucella abortus salt-extractable protein and lipopolysaccharide vaccines in BALB/c mice.

A study was conducted to determine the effect of monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) as adjuvants on the protective responses in BALB/c mice vaccinated with Brucella abortus salt-extractable protein (BCSP) or proteinase-K-treated B abortus lipopolysaccharide (PKLPS). Mice were vaccinated with different doses of BCSP or PKLPS given alone or in combination with MPL or TDM. Mice were challenge-exposed 4 weeks later with virulent B abortus strain 2308. Two weeks after challenge exposure, the number of B abortus colony-forming units (CFU) per spleen, spleen weights, and spleen cell interleukin 1 production were measured. Serum IgG and IgM concentrations specific for vaccinal immunogens were measured before and after challenge exposure with B abortus. Spleen weights and mean B abortus CFU per vaccine group were significantly lower in BCSP- and PKLPS-vaccinated mice, compared with those of nonvaccinated control mice. Monophosphoryl lipid A enhanced the suppression of splenic infection when given with the BCSP vaccine, but not when given with the PKLPS vaccine. Trehalose dimycolate had no effect on mean CFU when given with BCSP, but incorporation of TDM resulted in a significant increase in mean CFU when given with PKLPS. Spleen weights in BCSP- or PKLPS-vaccinated mice were not different when these vaccines were combined with MPL or TDM. Because of the wide variation in the results, we could not conclude that vaccination with BCSP or PKLPS alone, or in combination with MPL altered spleen cell interleukin-1 production in B abortus-infected mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Humoral immune response of BALB/c mice to a vaccinia virus recombinant expressing Brucella abortus GroEL does not correlate with protection against a B. abortus challenge

This work is a part of an ongoing effort to develop vaccinia virus recombinants expressing various Brucella abortus proteins. The B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination and expression confirmed by Western blotting. Female BALB/c mice inoculated with recombinant vaccinia virus/GroEL produced GroEL and vaccinia virus specific antibodies. Mice were challenged 8 weeks post-inoculation with virulent B. abortus strain 2308 and protection measured by the rate of clearance of live Brucella from spleens. Although induction of specific immune response to GroEL and vaccinia virus was demonstrated by the appearance of antibodies in mice, no significant level of protection was demonstrable.     

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.     

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.     

Experimental Brucella abortus infection in pigs

Sixteen pigs inoculated by the intra-conjunctival, intravenous or subcutaneous routes with Brucella abortus Strain 544 developed a short-lived infection usually accompanied by conjunctival and vaginal excretion of the organism for up to 99 days post-inoculation. Serological tests performed by the agglutination, complement fixation, Rose Bengal plate, antiglobulin, immunodiffusion or ELISA procedures with B. abortus antigens disclosed wide variations in the antibody responses of individual animals. In some cases the serological tests were negative even though the animal was shown to be excreting B. abortus. The intradermal test for delayed hypersensitivity to Brucella antigens gave more consistent results, especially when supported by histological evaluation of the skin reactions.     

Putative outer membrane autotransporter protein influences survival of Brucella suis in BALB/c mice

In Gram-negative bacteria, autotransporters are secreted proteins able to translocate themselves through the inner- and outer-membranes to the cell surface or to the extracellular environment. The influence of the putative outer membrane autotransporter (OmaA) protein to the persistence of Brucella suis was investigated. Sequence analyses revealed that the OmaA protein of B. suis strain 1330 consists of a signal peptide, a passenger alpha-domain, and a transporter beta-domain, which are the characteristic components of an autotransporter protein. The transporter beta-domain consists of 14 individual amphipathic beta-strands, and a 46-amino acid long alpha-helix lies upstream of the transporter domain, indicating that the B. suis OmaA is a type-I classical autotransporter. BLAST search and phylogenetic analyses revealed that the B. suis OmaA protein shares more similarities with adhesin autotransporter proteins than with protease autotransporter proteins of other bacteria. An OmaA-deficient strain (1330DeltaomaA) was generated by disrupting the DNA region encoding the passenger alpha-domain of the OmaA protein of B. suis wild type strain 1330. The omaA gene encoding the full-length OmaA protein was cloned and used to complement the OmaA-deficient strain. The OmaA-deficient strain did not differ from the wild type strain in terms of persistence in J774 macrophage cell line 24 and 48 h after inoculation, or clearance from the spleens of BALB/c mice at 1 week after intraperitoneal inoculation. These observations suggest that the function of the OmaA protein is dispensable during the acute phase of B. suis infection. However, the OmaA-deficient strain was cleared from the spleens of BALB/c mice faster than the wild type strain between the third and the ninth week after intraperitoneal inoculation, indicating that the OmaA may be important during the chronic phase of B. suis infection. Relative to the BALB/c mice injected with saline, those vaccinated with the OmaA-deficient strain exhibited 3.0-3.9log10 colony forming units protection against a challenge with B. suis strain 1330. This study is the first report correlating an autotransporter protein deficiency with persistence of B. suis in vitro and in vivo.     

Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.     

Brucella abortus infection in indigenous Korean dogs

Three dogs reared on a dairy farm with a high incidence for Brucella abortus were serologically positive for B. abortus and no other Brucella spp. The identity of the organism was confirmed to be B. abortus by AMOS (abortus melitensis ovis suis)-polymerase chain reaction with specific primers for B. canis. One hundred percent homology of the canine isolate and the bovine pathogen isolated from the farm was demonstrated. The only possible source of infection was infected cattle on the same farm. It is suggested that dogs be routinely included in brucellosis surveillance and eradication programs.     

Brucella abortus infection in 14 farm dogs

Fourteen dogs were obtained from 10 farms with Brucella-infected cattle and were studied for periods ranging from 2 to 81 days. At necropsy, Brucella abortus biovar 4 was isolated from all 14 dogs. Mandibular, medial retropharyngeal, tracheobronchial, and mesenteric lymph nodes yielded the highest rate of recovery. Urogenital infection with active shedding was seen in a single aged bitch. Fecal samples (291 from 13 dogs) were B abortus culture negative. Ten dogs monitored serologically over time had standard tube agglutination test titers that fluctuated between 1:50 with incomplete reaction and greater than or equal to 1:200 with positive reaction. At necropsy, the magnitude of seroconversion was not directly related to the number of culture-positive tissues. The maximal duration of infection, based on the time interval between the last possible exposure to infected cattle and recovery of the organism at necropsy, was 464 days. If it were assumed that infection occurred at about the same time as seroconversion in the cattle herd of origin, maximal observed duration of infection was 539 days. The actual maximal duration of infection could not be determined from this study, but would have exceeded the values reported here. The data indicate that dogs have the potential to infect cattle and could pose a threat for longer duration of disease transmission than had previously been assumed. Although the risk of transmission appears small, infection of human beings or cattle associated with Brucella-infected dogs would cause unfavorable political and economic consequences, particularly in low-incidence areas. Removal of contact dogs from infected herds should prevent such transmission.     

Experimental infection of opossums with Brucella abortus

Thirteen opossums (Didelphis virginiana) trapped in east central Alabama were fed approximately 1.5 X 10(9) Brucella abortus colony forming units. Serologic responses to at least 1 of 3 tests developed in 8 of the 13 opossums. Brucella abortus was recovered from 18 of 159 blood samples from 4 of the 13 opossums and from 7 of 159 fecal samples from 6 of them. All culture-positive feces had been excreted within 4 days after exposure. Sixty-four urine, 123 saliva, and 78 vaginal samples were culture-negative. Eleven baby opossums, in their mothers' pouches at the time of capture, were culture-negative. Brucella abortus was isolated from 10 of 13 adult opossums. Ten additional opossums were trapped and tested for brucellosis. One had a tube agglutination titer of 1:25, and B abortus was isolated from the liver and spleen. Brucella abortus was isolated from lung and spleen of 8 seronegative opossums. The remaining 8 opossums were negative to all tests.     

猪圆环病毒2型感染裸鼠及BALB/c小鼠的研究

用猪圆环病毒2型（PCV2）经腹腔注射BALB/c小鼠及裸鼠各15只,每隔2周1次,共感染3次.BALB/c小鼠初次、再次感染后均未出现明显的临床症状,仅有2只小鼠出现病理变化;从血清中可检测到PCV2核酸及其ORF2抗体;同时淋巴细胞增殖反应显著增强,NK和CTL细胞杀伤率显著升高;CD4^＋、CD8^＋、CD3^＋T淋巴细胞及CD19^＋B淋巴细胞数量显著减少.裸鼠也未出现明显的临床症状,从血清中可检测到PCV2核酸,但未检测到PCV2 ORF2抗体;除NK细胞杀伤率显著升高外,其余免疫学指标无显著改变.结果表明,BALB/c小鼠比裸鼠对PCV2易感,各项免疫学指标变化与PCV2感染猪一致,但不表现临床症状,仅个别BALB/c小鼠出现病理变化,说明BALB/c小鼠对PCV2不如猪易感.     

Experimental infection of dogs with Brucella abortus

Thirteen female dogs, which included eight principals that were fed approximately 4.4 X 10(10) colony forming units (cfu) of Brucella abortus strain 2308 and five sentinels that were housed with the principals, were examined for serologic responses, blood culture, tissue distribution of the organisms and pathologic lesions. Serum samples from each dog were tested on the day of exposure and on post exposure days 5, 7, 10, 14, 21, 28, 35, 42 and 49 for antibodies to B. abortus, using the brucellosis card (BC), standard tube agglutination (STA), 2-mercaptoethanol (ME) and rivanol (RIV) tests. Antibodies were detected in the principals by day 5 and increased through day 21. The STA test was the first to become positive, followed by the BC, ME and RIV tests. After 28 days, the serologic titers receded. From day 14 through day 42, all principals had greater than or equal to 1:50 STA titers. On day 49, seven principals had greater than or equal to 1:50 STA titers and one had a 1:25 STA titer. The sentinels were negative for all tests, except sentinel number 9 which had STA titers ranging from 1:25 to 1:50 on day 14 through day 35. Blood cultures that were obtained from each principal at intervals from one hour after exposure through 49 days were negative. Brucella abortus was isolated from various lymph nodes of the eight principals and from sentinel number 9, which was apparently infected by ingesting brucellae contaminated feces from the principals. Microscopic lesions were not observed in the culture-positive tissues examined.     

Experimental infection of sheep with Brucella abortus

A case of brodifacoum poisoning is described in a six-year-old male Kelpie cross working dog. The clinical features were severe exercise intolerance, haemorrhage from the oral and nasal cavities, dyspnoea and pale mucous membranes. Diagnosis was confirmed by demonstrating an abnormally long whole blood clotting time. The dog was treated successfully by administering 1 litre of whole blood intravenously, intramuscular vitamin K₁ and a three week course of oral vitamin K₃.

Experience at the Massey University Small Animal Clinic and Hospital has indicated that poisoning of dogs with the newer long acting anticoagulant rodenticides is becoming more common.     

Establishment of dose-response relationships in BALB/c mice, using Brucella cell surface protein and lipopolysaccharide

A study was conducted to determine the immune (increased antibody) and protective (reduced colony-forming units) responses induced in mice by a: (i) single vaccinal inoculation, using various concentrations of Brucella cell surface protein (BCSP) or lipopolysaccharide (LPS); (ii) primary inoculation, using various concentrations of BCSP, followed by a secondary inoculation, using a standard concentration of BCSP; and (iii) primary inoculation, using 1 concentration of BCSP or LPS, followed by a secondary inoculation, using various concentrations of BCSP or LPS. Four weeks after the primary inoculation, mice were challenge exposed with approximately 1 x 10(4) colony-forming units of Brucella abortus strain 2308 and all mice were euthanatized at 6 weeks. Reduced splenic weights and reduced colony-forming units in the spleens of vaccinated mice, compared with nonvaccinated mice, were the criteria of protection. Increase in serum IgM and IgG was defined as immunity. Both BCSP and LPS induced protective and immune responses that were proportional to the dose given up to an optimal limit. However, concentrations higher than optimal decreased the protective and immune responses. This was true for mice given either 1 or 2 vaccinal inoculations. Enhanced secondary protective responses were seen only when suboptimal doses were used in the primary inoculation. Excessive or optimal doses in the secondary inoculations prevented or obscured the protectiveness and immunity by primary inoculations. The protective effects appeared to be additive when suboptimal doses were used in the primary and secondary inoculations.(ABSTRACT TRUNCATED AT 250 WORDS)