Evaluation of the goat cellular immune response to rBtuB-Hia-FlgK peptides from Brucella melitensis.

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out.

Protective efficacy and safety of Brucella melitensis 16MΔmucR against intraperitoneal and aerosol challenge in BALB/c mice.

Brucellosis is a zoonosis of nearly worldwide distribution. Vaccination against this pathogen is an important control strategy to prevent the disease. Currently licensed vaccine strains used in animals are unacceptable for human use due to undesirable side effects and modest protection. Substantial progress has been made during the past 10 years toward the development of improved vaccines for brucellosis. In part, this has been achieved by the identification and characterization of live attenuated mutants that are safer in the host but still can stimulate an adequate immune response. In the present study, the identification and characterization of the mucR mutant (BMEI 1364) as a vaccine candidate for brucellosis was conducted. BALB/c mice were vaccinated intraperitoneally at a dose of 10(5) CFU with the mutant to evaluate safety and protective efficacy against intraperitoneal and aerosol challenge. All animals vaccinated with the vaccine candidate demonstrated a statistically significant degree of protection against both intraperitoneal and aerosol challenge. Safety was revealed by the absence of Brucella associated pathological changes, including splenomegaly, hepatomegaly, or granulomatous disease. These results suggest that the 16MΔmucR vaccine is safe, elicits a strong protective immunity, and should be considered as a promising vaccine candidate for human use.

Amplified fragment length polymorphism reveals specific epigenetic distinctions between Mycobacterium avium subspecies paratuberculosis isolates of various isolation types.

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle.

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 DeltavjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 DeltavjbR::Kan at a dose of 1 x 10(5) CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 DeltavjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.

Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction.

Brucella usually carry two highly homologous genes (omp2a and omp2b) for porin-like proteins. In several B. abortus biovars the omp2a gene has a large deletion compared to other Brucella omp2's. In this study we have measured Omp2 pore activity in planar bilayers. Omp2b exhibits well-defined trimeric channel activity whilst Omp2a forms monomeric pores of variable size which are smaller than Omp2b. No sequence homology exists between Omp2 and porins of known structure, so hydrophobic moment analysis has been used to model their membrane topology. From this it appears likely that the deletion removes the crucial L3 internal loop.

SDS-soluble and peptidoglycan-bound proteins in the outer membrane-peptidoglycan complex of Brucella abortus.

Outer membrane-peptidoglycan complex from Brucella abortus was separated from cytoplasmic membrane and cytosol by either sucrose density gradient fractionation or differential (rate) centrifugation of surface labeled cells disrupted by sonication without the use of detergents. The outer membrane-peptidoglycan complex had a buoyant density of 1.22 gm/ml and contained 67 labeled SDS-soluble proteins when examined by SDS-PAGE. Included were four major bands exhibiting molecular masses of 88k, 40k, 35.7k and 26k daltons corresponding to previously described group 1, 2 and 3 outer membrane proteins. Lysozyme treatment of outer membrane-peptidoglycan complex increased its buoyant density to 1.25 gm/ml and released eight additional peptidoglycan-linked proteins.

The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity.

Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 microg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to beta-lactam antibiotics. PBP2 complements the activity of PBP1. New beta-lactam antibiotics and improved vaccines might be developed based on this property.

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain.

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.

Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus.

The outer membrane-peptidoglycan complex (OM-PG) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400 micrograms, 1200 micrograms, and 4000 micrograms at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400 micrograms or 8000 micrograms. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains of B. abortus.

Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative responses.

Five groups of heifers were immunized with various subcellular fractions of Brucella abortus and tested for their responsiveness in lymphocyte proliferative responses in vitro. The five subcellular fractions used as immunogens were: (1) a mixture of recombinant outer membrane proteins fused to Escherichia coli beta-galactosidase, (2) a mixture of outer membrane proteins BaomI, BaomIIB1, and BaomIII1, (3) a mixture of outer membrane proteins 7.5 kDa and 8.8 kDa, (4) a complex of smooth lipopolysaccharide and proteins, and (5) a complex of outer membranes and peptidoglycan (OM-PG complex) from a rough strain. All immunogens were emulsified in adjuvant and administered twice at a 61-day interval. Two other groups of cows were included; one immunized with strain 19 and the other with adjuvant only. Strain 19 and the rough OM-PG complex induced responsiveness in lymphocyte proliferation assays in a high percentage of immunized cows. The smooth lipopolysaccharide-protein complex induced responsiveness in fewer cows. The lowest frequencies of responding cows were found in groups that received either recombinant proteins or purified protein mixtures. Based on these results, we concluded: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with subcellular fractions of B. abortus and (2) the more complex the immunogen, the greater the frequency of responding cows.

Of mice, calves, and men. Comparison of the mouse typhoid model with other Salmonella infections.

Numerous Salmonella typhimurium virulence factors have been identified and characterized using experimental infection of mice. While the murine typhoid model has been used successfully for Salmonella typhi vaccine development and to infer virulence mechanisms important during typhoid fever, information derived from infection of mice has been of limited value in elucidating the mechanisms by which S. typhimurium causes enteritis in humans. Progress in our understanding of virulence mechanisms contributing to diarrheal disease comes from recent studies of bovine enteritis, a S. typhimurium infection, which manifests as acute gastroenteritis. This review compares virulence genes and mechanisms required during murine typhoid, typhoid fever, and bovine enteritis. Comparison of illnesses caused in different animal hosts identifies virulence mechanisms involved in species specific disease manifestations. The determination of the relative importance of virulence factors for disease manifestations in different host species provides an important link between the in vitro characterization of genes and their role during host pathogen interaction.

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.

DNA homology of Brucella abortus strains 19 and 2308.

The restriction endonuclease digestion DNA patterns from Brucella abortus strains 19 and 2308 were examined with 11 restriction enzymes (AvaI, BamHI, BglII, BstEII, DdeI, EcoRI, HindIII, KpnI, PstI, XbaI, and SalI). The DNA electrophoretic banding patterns between the 2 strains were highly similar, using this restriction enzyme analysis. Differences were not discernable between B abortus strains 19 and 2308 in any of the restriction banding patterns examined. Methylation at CCGG or GATC sites was not detectable on the basis of digestion with isoschizomers (HpaII and MspI, and DpnI, Sau3AI and MboI). Homology between B abortus strains 19 and 2308 was assessed, using solution-hybridization techniques followed by S1 nuclease assays. Results of these reassociation experiments indicated 98.6 to 99.3% homology between B abortus strains 19 and 2308 with 13.5 to 18.6% homology between B abortus (strains 19 and 2308) and the E coli HB101 control. We concluded that any DNA differences between the 2 B abortus strains are small and will require analysis at the DNA sequence level.

Differential reactivity of bovine lymphocytes to species of Brucella.

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis.

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.

Brucella abortus in Bison. II. Evaluation of strain 19 vaccination of pregnant cows.

Protection against Brucella abortus induced abortion and infection provided by strain 19 (S19) vaccination was evaluated in American bison (Bison bison). Forty-eight pregnant bison were manually inoculated (MI) with S19 vaccine, 44 were ballistically inoculated (BI) with an absorbable hollow pellet containing lyophilized S19, and 46 were manually injected with buffered saline as non-vaccinated controls (NVC). All bison were Brucella spp. seronegative prior to the experiment, in the second trimester of pregnancy, and were randomly assigned to experimental groups. Approximately 60 days post-vaccination, abortions were observed in the vaccinated bison. Brucella abortus strain 19 was recovered from a bison that had recently aborted, her fetus, and from 11 of 12 other aborted fetuses. Fifty-eight percent (53 of 92) of vaccinated bison aborted, and no abortions were observed in the NVC bison. One cow aborted during her second post-vaccinal pregnancy and S19 was identified from the dam and fetus indicating that chronic S19 infections can occur in bison. Positive antibody titers were present 10 mo post-vaccination in 73% (66 of 91) of the bison. Thirteen mo post-vaccination, 30 MI vaccinates, 27 BI vaccinates, and 30 NVC bison were challenged during the second trimester of pregnancy with 1 x 10(7) CFU of B. abortus strain 2308 via bilateral conjunctival inoculation. Protection against abortion was 67% (P less than or equal to 0.0001) for vaccinated bison compared to 4% in NVC. Protection against B. abortus infection was determined to be 39% (P greater than or equal to 0.001) for vaccinates and 0% (zero of 30) for NCV. Persistent antibody titers, vaccine induced abortions, and chronic S19 infections indicate that the S19 vaccine doses used in this study are not suitable for pregnant bison.

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)

Effects of stage of gestation and breed on bovine responses to vaccination with Brucella abortus strain 19.

Eighty-eight cattle were injected SC with 2.5 x 10(8) viable cells of Brucella abortus strain 19. All but 1 heifer became seropositive on the basis of the results of 7 brucellosis tests, and the proportion positive decreased with time. The proportion of cattle that were seropositive during a 20- to 67-week period after vaccination was as follows, in decreasing order: hemolysis-in-gel, 59%; buffered-acid plate antigen, 39%; ELISA, 16%; card, 10%; rivanol, 8%; cold complement-fixation, 7%; and automated complement-fixation, 5%. Using the serologic classification in Uniform Methods and Rules for brucellosis eradication, 7 cattle tested brucellosis-positive (2 suspects and 5 reactors). None of the 27 nonpregnant heifers tested positive. Of 18 heifers that were 84 to 135 days in gestation when vaccinated, 6 (33%) tested positive for brucellosis, compared with 0 of 13 and 1 (3%) of 30 heifers that were 11 to 78 and 145 to 253 days in gestation at vaccination, respectively (X2 = 12.07; 2 df; P less than 0.01). Neither breed (Angus, Hereford, Jersey, and Brahman) nor calf survival was related to brucellosis-positive results. Postpartum milk samples from 61 heifers and 24 tissues from 2 reactor cattle were culture-negative for B abortus.

Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus).

Two bottlenose dolphins (Tursiops truncatus) aborted fetuses that died as a result of Brucella infection. Brucella placentitis occurred in both cases. Infected placenta and vaginal/uterine fluids may transmit Brucella species to other cetaceans. In a third case, an identical organism was cultured from lung necropsy tissue of an adult female T. truncatus. Microbiology, specific polymerase chain reaction, and pulsed-field gel electrophoresis results supported the designation of an additional genomic group(s), Brucella delphini, for isolates adapted to T. truncatus. Current serologic diagnostic tests reliable for known Brucella species are unreliable in detecting dolphin brucellosis. Our findings, together with previous reports, suggest that dolphin brucellosis is a naturally occurring disease that can adversely impact reproduction in cetaceans. The zoonotic significance of cetacean brucellosis is unknown, although the disease has not been reported in people who have frequent contact with dolphins. Further studies on the zoonotic aspects, distribution, prevalence, virulence, and impact of this disease in cetaceans and other marine mammal species are needed.