Failure to isolate Brucella abortus from embryos or ova from culture-positive superovulated cows

Nine, Brucella abortus culture positive 2-yr-old cows were used to test the hypothesis that embryos and ova collected from such cows are not infected. Superovulation was induced at varying times postpartum or postabortion with intramuscular injections of follicle stimulating hormone (FSH). The cows were artificially inseminated with B. abortus-negative semen. Superovulations and nonsurgical embryo collections nonsurgical embryo collections were attempted twice for each cow. Jugular blood, udder secretions, cervical swabs, uterine collections, embryos and ova were cultured bacteriologically from the nine cows simultaneously at nonsurgical embryro collections, and B. abortus was isolated only from the udder secretions of seven cows. Brucella abortus was not isolated from 15 uterine collections, 21 embryos, or 18 ova from the culture-positive cows. It was concluded that B. abortus was not present at the detection limits of the culture method employed, which supports the finding or view that embryos and ova collected from donor cows at 100 days or greater post partum or post abortion are not likely to harbor Brucella.     

In vitro exposure of ovine ova to (Brucella abortus )

Fifty-three zona pellucida-intact ova were collected surgically from superovulated, Brucella -free mixed-breed ewes. Groups containing two to seven ova were incubated in medium containing Brucella abortus . All groups of ova were then washed 10 times, and ova and sequential washes were cultured for the isolation of B. abortus . Brucella were not found beyond the fifth wash for any group of ova, but were isolated from one of 12 groups of ova. Results indicate that mechanical washing in the absence of antibiotics is advantageous, but alone, is not totally reliable for removing B. abortus from exposed, zona pelucida-intact ovine ova.     

A review of the epidemiologic aspects of embryo transfer from Brucella abortus -infected cows

Available information on the epidemiologic aspects of embryo transfer from Brucella -infected cattle is reviewed to provide a knowledgeable perspective upon which the risk of transmission of this agent by the embryo can be assessed. Accumulated evidence indicates that exposure of preimplantation embryos to Brucella abortus in the uteri of superovulated, infected cows is unlikely. Further, it has been shown that embryo-washing procedures insure freedom from B. abortus even without antibiotics. The use of antibiotics with the proper cryoprotectant provides additional insurance that Brucella will not be transferred with frozen-thawed embryos. Four hundred fifteen (415) ova collected in 74 nonsurgical recoveries from Brucella -infected cows were culture-negative when examined for the presence of Brucella . After reviewing studies conducted on embryo transfer from Brucella abortus -infected cows, the authors conclude that B. abortus will not be transmitted when emphasis is placed on proper handling of embryos between collection from donors and transfer to recipients.     

Seroconversion of recipient ewes after transfer of ova exposed to Brucella ovis in vitro

Zona pellucida-intact ova collected from ewes seronegative to Brucella ovis were exposed in vitro to B ovis and washed 10 times in medium that contained no antibiotics. After exposure and washing, nontransferable ova were cultured for isolation of Brucella , and the viable ova were transferred into seven B ovis seronegative ewes. No pregnancies resulted, thus recipient ewes were bred during the next breeding season, and blood samples were collected for bacteriological and serological examination until one month after lambing. Brucella ovis was isolated from all of the nontransferable ova, indicating that the transferred ova had viable organisms adhered to them. Although no recipient was found to be pregnant at Day 45, all seven ewes responded to the transferred ova by producing anti-Brucella antibodies. With the exception of a ewe that was euthanized early in the project due to a traumatic injury, all recipients lambed normally during the following breeding season. Brucella was not found in any sample collected from ewes or lambs. However, ELISA titers for B ovis remained in the suspicious range and a ewe was positive on the CF test within 2 wk of lambing.     

Adherence of Brucella ovis to preimplantation ovine ova

Seventy-three zona pellucida-intact ova were collected surgically from 15 superovulated, Brucella -free mixed-breed ewes. Twenty-one groups containing one to five ova were incubated in medium containing Brucella ovis . Subsequently, seven and five groups were incubated for 24 and 4 h, respectively, at 37 degrees C in medium containing penicillin and streptomycin, while nine groups were not treated with antibiotics. All groups of ova were washed 10 times, and ova and sequential washes were cultured for the presence of B. ovis . Brucella were isolated from seven of the nine groups of nontreated ova and from the 10th wash for six of these groups. While Brucella were detected in fewer washes after antibiotic treatment, the organism was still isolated from 11 of the 12 treated groups. Results indicate that standard washing techniques are not reliable for removing B. ovis from exposed, zona pellucida-intact, ovine ova.     

A Comparison of Various Selective Media, including a New Selective Medium for the Isolation of Brucellae from Milk

S ummary : A comparative trial of a new medium for brucella was made on samples of milk from individual cows and herd samples of milk. The new medium gave a higher isolation rate than any of the other 3 selective media tested. There was no evidence that the new medium inhibited any of the biotypes of Brucella abortus , especially biotype 2; it was the only medium suitable for isolating from samples of herd milk strains of biotype 2 which are sensitive to antibiotics and aniline dyes.     

Brucella abortus in wildlife on selected cattle farms in Alabama

Two studies of brucellosis in wildlife on farms where the brucellosis infection prevalence in cattle was known are reported. On a research farm, 233 feral animals of 22 mammalian species and 12 of seven avian species were trapped during three time periods. Sixty were studied before cattle were introduced, 128 were studied while 501 cattle infected with Brucella abortus were calving and aborting, and 60 specimens were collected 20 mo after the last infected cow calved. Selected tissues from 229 wild animals were cultured and sera from 138 were examined using the brucellosis card, standard tube agglutination (STA), 2-mercaptoethanol (2-ME) and rivanol (RIV) tests. Brucella abortus was not recovered from any animals sampled prior to cattle being introduced and all sera collected were negative. Brucella abortus was isolated from four opossums (Didelphis virginiana) and one raccoon (Procyon lotor) in the group of animals trapped during the calving period. Three serums were tested and had STA titers ranging from 1:100 to 1:200. Of 68 sera only one had antibodies. Brucella were not isolated from 59 animals trapped after the calving period and only one of 42 serums had antibodies. On regional cattle farms, 243 wild animals were trapped. Brucellae were not isolated from 223 animals which were cultured. No serums had significant titers. The data from this study suggest opossums and raccoons can be infected from cattle but are unlikely to maintain the infection.     

Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle

Two groups of six, non-brucellosis vaccinated, brucellosis seronegative pregnant American bison (Bison bison) were individually challenged with 1 x 10(7) colony forming units (CFU) of Brucella abortus strain 2308. Three days after challenge, each bison group was placed in a common paddock with six non-vaccinated, brucellosis susceptible, pregnant domestic heifers. In a parallel study, two groups of six susceptible, pregnant cattle were simultaneously challenged with the identical dose as the bison and each group was placed with six susceptible cattle in order to compare bison to cattle transmission to that observed in cattle to cattle transmission. Blood samples were collected from bison and cattle weekly for at least 1 mo prior to exposure to B. abortus and for 180 days post-exposure (PE). Sera from the bison and cattle were evaluated by the Card, rivanol precipitation, standard plate agglutination, standard tube agglutination, cold complement fixation tube, warm complement fixation tube, buffered acidified plate antigen, rapid screening, bovine conjugated enzyme linked immunosorbent assay, bison or bovine conjugated enzyme linked immunosorbent assay, and the hemolysis-in-gel techniques for the presence of antibodies to Brucella spp. At the termination of pregnancy by abortion or birth of a live-calf, quarter milk samples, vaginal swabs, and placenta were collected from the dam. Rectal swabs were collected from live calves, and mediastinal lymph nodes, abomasal contents and lung were taken at necropsy from aborted fetuses for culture of Brucella spp. These tissues and swabs were cultured on restrictive media for the isolation and identification of Brucella spp. Pathogenesis of brucellosis in bison was studied in an additional group of six pregnant bison which were challenged with 1 x 10(7) CFU of B. abortus strain 2308. One animal was euthanatized each week PE. Tissues were collected at necropsy and later examined bacteriologically and histologically. Lesions of brucellosis in bison did not significantly differ grossly or histologically from those in cattle. There were six abortions and two nonviable calves in the bison group, as compared to nine abortions in the 12 similarly inoculated cattle. As determined by bacterial isolations, transmission of B. abortus from bison to cattle (five of 12 susceptible cattle became infected) did not differ statistically from cattle to cattle transmission (six of 12 susceptible cattle became infected) under identical conditions. No single serologic test was constantly reliable to diagnosing B. abortus infected bison for 8 wk PE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brucella abortus synthesizes phosphatidylcholine from choline provided by the host

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-beta-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process.     

Evaluation of the differences between biofilm and planktonic Brucella abortus via metabolomics and proteomics

Functional & Integrative Genomics - This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in...     

Kinetic study of cytokines production by human peripheral blood mononuclear cells in response to Brucella DNA

In spite of reports on cytokines induction by the Brucella DNA in murine model, there is no comparison between pathogenic and appropriate vaccine strains in human. We investigated the cytokines profile of human peripheral blood mononuclear cells (PBMCs) induced by DNA extracted from pathogenic isolates of Brucella melitensis and B. abortus as well as Rev1 and S19; the appropriate vaccine strains. It was observed that despite differential induction of Interleukin(IL)-12 and IL-10 production, identical IL-12/IL-10 concentration ratio was obtained by all Brucella strains DNAs that was 2 after 24h and 4 after 5 days of incubation. In addition, IL-2 and Interferon(IFN)-gamma production were profoundly increased compared to the medium at day 3 and 5 respectively but IFN-alpha was not induced. Therefore, Brucella strains DNAs are Th1 inducing component with similar pattern in human PBMCs.     

The effects of superovulation on Brucella abortus infection in the bovine uterus

Uterine flushings were collected three times at predetermined intervals from 11 mixed-breed beef cows and cultured for Brucella abortus . Prior to sampling, all cows had aborted fetuses from which brucellae had been isolated. Initial collections were made between 21 and 34 days following abortion. The second flushing was conducted at the onset of injections used for inducing superovulation and the third flushing was conducted 6 to 8 days after the ensuing estrus. The latter two flushes were conducted between 60 and 120 days following abortion. Brucellae were isolated from uterine flushings collected from 6 of the 11 cows on the initial round of sampling. Cultures of all subsequent uterine flushings collected before and after injections for superovulation were negative. It was concluded that the superovulatory treatment is not likely to reactivate the release of brucellae into the uterine lumen during the period when embryos are normally collected.     

Experimental Brucella abortus infection in wolves

Four juvenile male wolves (Canis lupus) each received an oral dose of 1.6-1.7 x 10(12) colony-forming units of Brucella abortus biovar 1 isolated from a bison (Bison bison) in Wood Buffalo National Park (Canada), and two others served as negative controls. Infected wolves did not show clinical signs of disease but did develop high Brucella antibody titers. Small numbers of B. abortus were excreted sporadically in feces until day 50 postinoculation (PI). Very small numbers of the bacterium were isolated from urine of only one wolf late on the same day that it was infected, and very small numbers of colonies of B. abortus were obtained from buccal swabs of three wolves for up to 48 hr PI. Two infected wolves euthanized 6 mo after the start of the experiment had no lesions, and colonies of B. abortus were isolated from thymus and most major lymph nodes. The other two infected wolves euthanized 12 mo after the start of the experiment had no lesions, and smaller numbers of brucellae were recovered from fewer lymph nodes compared with the wolves killed 6 mo earlier. The sporadic excretion of very small numbers of brucellae by the wolves was insignificant when compared with the infective dose for cattle. Brucella abortus, brucellosis, Canis lupus, pathogenesis, serology, wolf.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

VirB3 to VirB6 and VirB8 to VirB11, but not VirB7, are essential for mediating persistence of Brucella in the reticuloendothelial system

The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system. Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice. While a nonpolar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a nonpolar deletion of virB1 only reduces survival in macrophages, whereas virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during survival in macrophages and virulence in mice. Mutants carrying nonpolar deletions of the virB3, virB4, virB5, virB6, virB7, virB8, virB9, virB10, or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3, virB4, virB5, virB6, virB8, virB9, virB10, or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2, virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11 are essential for virulence of B. abortus in mice, while functions encoded by the virB1, virB7, and virB12 genes are not required for persistence in organs with this animal model.     

Serologic survey of Brucella spp. antibodies in some marine mammals of North America

A serologic survey of anti-Brucella spp. antibodies was undertaken on 2,470 samples of 14 North American marine mammal species collected between 1984-97. Serum or blood from eight species of cetaceans and six species of pinnipeds was sampled from Pacific, Atlantic, and Arctic oceans. Two competitive enzyme-linked immunosorbent assays (C-ELISA's), using specific monoclonal antibodies to Brucella abortus cell wall components, were used to detect anti-Brucella spp. antibodies in the samples. Sera from 33 cetaceans and 61 pinnipeds gave inhibition values, in one or both of the tests, which exceeded the threshold that indicates Brucella spp. exposure in cattle. Seropositive animals were identified from Pacific, Atlantic, and Arctic oceans. While Brucella spp. was not isolated, differences in the response of seropositive cetacean and pinniped sera in the two assays suggest that two antigenically distinct species or biovars of Brucella spp. are present. No pathology consistent with clinical brucellosis was noted in any of the animals tested although detailed examination was not conducted on all carcasses.     

Pathology of brucellosis in bison from Yellowstone National Park

Between February 1995 and June 1999, specimens from seven aborted bison (Bison bison) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2-wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus. Lesions in the B. abortus-infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six seropositive cattle from a B. abortus-infected herd in July and August, 1997. Results of the bison and cattle studies were similar.     

Counter-immunoelectrophoresis for the detection of Brucella antigen and antibodies in the diagnosis of brucellosis in buffaloes

Counter-immunoelectrophoresis was employed for the detection of Brucella antigen in stomach contents of aborted buffalo fetuses and antibody in aborted as well as apparently healthy in contact buffaloes. Five of 16 aborted cases were serologically positive for brucellosis but isolation of Brucella abortus was successful in only two cases. By counter-immunoelectrophoresis, Brucella antigen was detected in the fetal stomach contents of four serologically positive cases.
Of the 68 serum samples from in contact healthy buffaloes, 10 were positive with counter-immunoelectrophoresis: more than were detected by tube agglutination, Rose Bengal plate agglutination, complement fixation and agar gel precipitation test.