Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.

Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

Towards single screening tests for brucellosis.

This paper describes an indirect enzyme-linked immunosorbent assay (I-ELISA) and a fluorescence polarisation assay (FPA), each capable of detecting antibody in several species of hosts to smooth and rough members of the genus Brucella. The I-ELISA uses a mixture of smooth lipopolysaccharide (SLPS) and rough lipopolysaccharide (RLPS) as the antigen, and a recombinant protein A/G conjugated with horseradish peroxidase as the detection reagent. When using individually determined cutoff values, the SLPS/RLPS combined-antigen I-ELISA detected antibody in slightly more animals exposed to SLPS or to RLPS than did I-ELISA procedures using each individual antigen separately. Similarly, the assay using combined antigens detected antibody in slightly fewer animals not exposed to Brucella sp. When a universal cutoff of 10% positivity was used (relative to strongly positive control sera of each species), the overall performance index (percentage sensitivity plus percentage specificity) value decreased by 1.0 (from 199.4 to 198.4). In the FPA, it was not possible to use a universal cutoff without significant loss of performance. The overall sensitivity value for the FPA using the combined FPA antigen was 1.0% lower than using the O-polysaccharide (OPS) from SLPS and 9.1% higher than using the core antigen (CORE) from RLPS. When the combined antigen was used, the FPA specificity was slightly higher (1.2%) than from only the OPS, and considerably higher (12.6%) than the CORE. Overall, both the I-ELISA and the FPA with combined antigens were suitable as screening tests for all species of Brucella in the animal species tested.

Enzyme immunoassay for the diagnosis of brucellosis: chimeric Protein A-Protein G as a common enzyme labeled detection reagent for sera for different animal species.

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody-enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.

Characterization of the caprine model for ruminant brucellosis.

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.

Brucella vaccines in wildlife.

Brucellosis has been known to exist in populations of wildlife since the early part of the 20th century. At the beginning of this century in the US, Brucella abortus is a problem in elk and bison in the Greater Yellowstone Area, B. suis is prevalent in millions of feral swine in most of the southern states, and caribou/reindeer in Alaska are infected with B. suis biovar 4. Brucellosis has been virtually eliminated in domestic livestock in the US after decades of expensive governmental disease prevention, control and eradication programs. Now the most likely source of transmission of brucellosis to humans, and the risk of reintroduction of brucellosis into livestock is from infected populations of free-ranging wildlife. Brucellosis was eradicated from livestock through a combination of testing, vaccination, and removal of infected animals. The use of vaccines to control brucellosis in populations of wildlife and therefore reducing the risk of transmission to humans and livestock has been proposed in several instances. This manuscript reviews research on the use of Brucella vaccines in species of wildlife with emphasis on safety and efficacy.

Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry.

Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice. The infection nevertheless persists for months. It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae. This would allow B. abortus to elude detection by the host's immune system. To test this, B. abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection. When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures. Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells. Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells. The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen. Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.

Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant.

The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (P<0.001, n=6). BM 25 also colonised fewer adults (P<0.05, n=6) and kids (P<0.01, n=6) than strain 16M. The Deltaomp25 mutant was found capable of transient in vivo colonisation of non-pregnant goats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (P<0.05, n=7). However, unlike strain Rev. 1, BM 25 does not appear to cause abortions in late gestation based on this study with a small number of animals. The B. melitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.

Risk factors for Brucella seropositivity in goat herds in eastern and western Uganda.

Cross-sectional prevalences and risk factors for Brucella seropositivity in goats in eastern and western Uganda were investigated. Serum was collected from 1518 goats randomly selected from 145 herds which had been identified using multistage sampling. The brucellosis card test (CT) and the Brucella melitensis tube-agglutination test (TAT) were used in parallel to detect antibodies against B. abortus and B. melitensis, respectively. Interviewer-administered questionnaires were used to collect information on goat health and management. This information was used in multivariable logistic-regression models to determine the risk factors for Brucella seropositivity in goat herds. For each analysis, a herd was considered positive if at least one goat in the herd tested positive for antibodies against Brucella and negative if none was positive. Four percent (55/1480) of the goats screened with the CT had antibodies against Brucella. The reactors were distributed in 13% (19/145) of the herds. The most-important herd-level risk factors identified were use of a hired caretaker as the primary manager of the operation compared to owner/family members (adjusted odds ratio (OR)=8.1; 95% CI 1.6, 39.7), keeping sheep in addition to goats (OR=6.0; CI 1.5, 23.7) compared to having no sheep, and free browsing (OR=4.7; 95% CI 1.0, 20.7) when compared to tethering or zero-grazing. Using the TAT, 10% (141/1446) of the goats tested positive. The positives were distributed in 43% (63/145) of the herds. Free browsing (OR=6.7; 95% CI 2.7, 16.9) when compared to tethering or zero-grazing and lack of veterinary care (OR=2.9; CI 1.3, 6.7) were the most-important factors identified in the multivariable model for B. melitensis herd seropositivity. To explore/reduce the risk of misclassification in a secondary analysis, herds were reclassified as positive if at least one goat tested positive on both tests and negative if none of the goats was positive on any of the two tests. Using this classification, 2% (30/1320; 95% CI 2, 3%) of the goats tested positive resulting in 13% (12/93) of the herds being positive. The distribution of the above risk factors by brucellosis herd-status (as defined by the second criterion) is also presented.

Attenuation of a Brucella abortus mutant lacking a major 25 kDa outer membrane protein in cattle.

OBJECTIVE: To determine the virulence of a Brucella abortus mutant, BA25, lacking a major 25 kd outer membrane protein (Omp25) in cattle. ANIMALS: 20 mixed-breed heifers in late gestation. PROCEDURE: 10 heifers were inoculated with 1 x 10(7) colony-forming units of the Omp25 mutant via the conjunctival sac, and an equal number were infected with the virulent parental strain B. abortus 2308. The delivery status of the dams was recorded, and colonization was assessed following necropsy. The ability of BA25 to replicate inside bovine phagocytes and chorionic trophoblasts was also evaluated in vitro because of the propensity of virulent brucellae to replicate inside these cells in vivo. RESULTS: The parental strain induced abortions in 5 of 10 inoculated cattle, whereas only 1 of 10 dams exposed to BA25 aborted. Brucella abortus strain 2308 colonized all of the cow-calf pairs and induced Brucella-specific antibodies in 100% of the dams. In contrast, BA25 was isolated by bacteriologic cultural technique from 30% of the calves and 50% of the inoculated dams (n = 10). Of the 10 heifers inoculated with BA25, 4 did not develop Brucella-specific antibodies nor were they colonized by the mutant strain. In bovine macrophages and chorionic trophoblasts, BA25 replicated in significantly lower numbers than the virulent parental strain (n = 3). CONCLUSIONS AND CLINICAL RELEVANCE: The 25 kd outer membrane protein may be an important virulence factor for B. abortus in cattle. The attenuation of the Omp25 mutant in cattle may involve the inability of BA25 to replicate efficiently in bovine phagocytes and chorionic trophoblasts.

Fusobacteriosis in captive wild-caught pronghorns (Antilocapra americana).

An outbreak of Fusobacterium necrophorum-induced septicemia occurred in a group of 40 captive wild-caught pronghorns (Antilocapra americana). Primary pododermatitis or necrotic stomatitis progressed to produce fatal septicemia with metastatic lesions in the forestomachs, lung, liver, and cecum in 38 of the animals. Two remaining animals were euthanatized because of chronic pododermatitis. Housing the animals in a pasture previously used by bovids and heavy rains with persistence of ground water pools in the pasture were contributing factors in the pathogenesis of this outbreak.

Oral vaccination of sexually mature pigs with Brucella abortus vaccine strain RB51.

OBJECTIVE: To develop a novel oral vaccine delivery system for swine, using the rough vaccine strain of Brucella abortus. ANIMALS: 56 crossbred pigs from a brucellosis-free facility. PROCEDURE: In 3 separate experiments, pigs were orally vaccinated with doses of 1 x 10(9) to > 1 x 10(11) CFU of strain RB51 vaccine. The vaccine was placed directly on the normal corn ration, placed inside a whole pecan, or mixed with cracked pecans and corn. RESULTS: Oral vaccination of pigs with vaccine strain RB51 resulted in a humoral immune response to strain RB51 and short-term colonization of the regional lymph nodes. CONCLUSIONS AND CLINICAL RELEVANCE: A viscous liquid such as Karo corn syrup in association with pecans that scarify the oral mucosa are necessary when placing the live vaccine directly onto corn or other food rations. Doses of > 1 x 10(11) CFU of RB51 organisms/pig in this mixture ensures 100% colonization of regional lymph nodes via the oral route. This method may allow an efficient and economical means to vaccinate feral swine for brucellosis.

Re-examination of the role of the Brucella melitensis HtrA stress response protease in virulence in pregnant goats.

Based on previously reported studies describing the experimental infection of pregnant goats with B. melitensis strain RWP5, we proposed that the HtrA protease plays an important role in the virulence of B. melitensis in its natural ruminant host. Subsequent studies, however, have shown that RWP5 is actually an htrA cycL double mutant. In order to definitively evaluate the role of the B. melitensis htrA in virulence, we constructed an authentic htrA mutant and examined this strain in pregnant goats. The findings of these studies indicate that the contribution of the htrA gene product to the virulence of B. melitensis in its natural host is not as great as was previously proposed.

Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle.

PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis. This report describes the results of studies conducted with this mutant in the natural bovine host. Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1 x 10(10) colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1. At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308. In a second experiment, eight heifers in mid-gestation were infected with 1 x 10(7) CFU of either strain via the conjunctival sac. The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves. Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain. These studies demonstrate that the B. abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308.

Validation of the fluorescence polarization assay and comparison to other serological assays for the detection of serum antibodies to Brucella abortus in bison.

A number of serological tests were compared for the detection of antibodies to Brucella abortus in bison (Bison bison). The performance of the fluorescence polarization assay (FPA) in both the preliminary evaluation and a subsequent blind validation indicated that this test was the most suitable for serological diagnosis of brucellosis in bison. The sensitivity and specificity in the preliminary evaluation were 92.1% and 99.4%, respectively. The sensitivity and specificity in a subsequent blind study were 96.3% and 97.6%, respectively. In a double blind study conducted on bison vaccinated with B. abortus strain 19, the data suggests that the FPA can differentiate bison infected with B. abortus from bison vaccinated with B. abortus strain 19. Both the indirect immunoassay (IELISA) and the competitive immunoassay (CELISA) performed nearly as well as the FPA. The buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT) did not perform as well as the FPA, CELISA or the IELISA in both studies. The FPA is a homogeneous assay eliminating the washing steps and reducing incubation to minutes rather than hours saving on time, equipment, materials, reagents and cost. These attributes, together, with its excellent sensitivity and specificity make the FPA an attractive test for the detection of serum antibodies to Brucella abortus in bison.

Safety of Brucella abortus strain RB51 in bull elk.

Some of the elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (Wyoming, Idaho, Montana; USA) are infected with Brucella abortus, the bacterium that causes bovine brucellosis. Brucella abortus strain RB51 vaccine is being considered as a means to control B. abortus induced abortions in cow elk. However, the most probable vaccination strategies for use in free-ranging elk might also result in some bull elk being inoculated, thus, it is important to insure that the vaccine is safe in these animals. In the winter of 1995, 10 free-ranging bull elk calves were captured, tested for B. abortus antibodies, and intramuscularly inoculated with 1.0 x 10(9) colony forming units (CFU) of B. abortus strain RB51. Blood was collected for hemoculture and serology every 2 wk after inoculation for 14 wk. Beginning 4 mo postinoculation and continuing until 10 mo postinoculation elk were serially euthanized, necropsied, and tissues collected for culture and histopathology. These elk cleared the organism from the blood within 6 wk and from all tissues within 10 mo. No lesions attributable to B. abortus were found grossly and only minimal to mild lymphoplasmacytic epididymitis was found in a few elk on histologic examination. In a separate study, six adult bull elk from Wind Cave National Park (South Dakota, USA) were taken to a ranch near Carrington (North Dakota, USA). Three were orally inoculated with approximately 1.0 x 10(10) CFU of RB51 and three were inoculated with corn syrup and saline. Ninety days post-inoculation semen was examined and cultured from these bulls. Strain RB51 was not cultured from their semen at that time. There were no palpable abnormalities in the genital tract and all elk produced viable sperm. Although they contain small sample sizes, these studies suggest that B. abortus strain RB51 is safe in bull elk.

Safety and efficacy of Brucella abortus strain RB51 vaccine in captive pregnant elk.

Brucella abortus strain RB51 is a laboratory-derived rough mutant of virulent B. abortus strain 2308 used as a vaccine because it induces antibodies that do not react on standard brucellosis serologic tests. Strain RB51 vaccine was evaluated in pregnant captive elk (Cervus elaphus) to determine (1) if it induced abortion and (2) if it protected against abortion following subsequent challenge. The time period of this study (February-June, 1998) was similar to field conditions where elk are vaccinated and possibly exposed to B. abortus. Fourteen elk were randomly and equally divided into vaccinated and control groups. The vaccinated group was vaccinated intramuscularly with 1.03 x 10(10) colony-forming units (CFU) of strain RB51 and seroconverted postvaccination. Antibodies to strain RB51 were detected by a modification of an existing dot-blot assay. Both groups were challenged 40 days postvaccination with 9.8 x 10(6) CFU of B. abortus strain 2308 administered intraconjunctivally. The first abortion occurred 38 days postchallenge. Abortion occurred in all control elk and in five of seven vaccinated elk 5 to 12 wk postchallenge (P = 0.23). Mixed strain RB51 and 2308 infections were present in fetuses and vaginas from the vaccinated group whereas only strain 2308 was cultured from control group fetuses and vaginal swabs. Further evaluation of strain RB51 will be necessary to determine if it will be safe and efficacious in free-ranging pregnant elk.

A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis.

Sera from Canadian pigs (brucellosis free, n = 14037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.

Use of western immunoblot analysis for testing moose serum for Brucella suis biovar 4 specific antibodies.

To determine if 12 moose (Alces alces) from northern Alaska with agglutinating antibodies specific for Brucella spp. had been exposed to either B. suis biovar 4 or B. abortus biovar 1, western immnnoblot serologic analysis was performed. Differential serologic responses to strain specific A and M antigenic variances of the lipopolysaccharide O-polysaccharide sugar allowed strain identification. Prior to examination, test sera were absorbed with killed whole cells from either B. abortus biovar 1, containing predominately A antigen (A+ M-); B. melitensis biovar 1, containing essentially M antigen (A- M+); or B. suis biovar 4, containing both antigenic tyes (A+ M+). The resulting sera were then examined by western immunoblot for recognition of either B. abortus biovar 1, B. melitensis biovar 1, or B. suis biovar 4 cell lysates. The results of this study indicate that these moose were exposed to B. suis biovar 4, a known pathogen of caribou (Rangifer tarandus) from arctic Alaska.