Darla R. Ewalt

Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus)

A culture isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) was characterized. The isolate was a gram-negative coccobacillus, and the colonial morphology was typical of a smooth Brucella. The isolate was positive for catalase, oxidase, nitrate reduction, and urease. Hydrogen sulfide was not produced. It grew in air at 37 C but required 72 hours for good growth. There was growth on media containing basic fuchsin, thionin, thionin blue, penicillin, and erythritol. The M antigen was dominant, and the isolate was lysed by 4 of 10 brucellaphages tested. The oxidative metabolic profile of the isolate was similar to that for B. abortus but differed in utilization of L-asparagine, L-glutamic acid, and DL-citrulline. Whole-cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles were markedly different from the protein profiles of reference strains of Brucella species. Biochemical and oxidative metabolism profiles indicated that the isolate belongs in the genus Brucella but did not match the profiles of any established species or biovars. This isolate may be an atypical strain of a recognized Brucella species or a new biovar or species of Brucella.

Evidence of Brucella infection in Parafilaroides lungworms in a Pacific harbor seal (Phoca vitulina richardsi).

Multiple isolates of Brucella sp. that differ from the recognized species within this genus have recently been isolated from viscera of 4 harbor seals (Phoca vitulina), 2 harbor porpoises (Phocoena phocoena), 1 common dolphin (Delphinus delphis), an Atlantic white-sided dolphin (Lagenorhynchus acutus), 2 striped dolphins (Stenella coeruleoalba), a hooded seal (Cystophora cristata), and a gray seal (Halichoerus grypus), 9,18 all from the Scottish coast. A similar Brucella sp. was isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) from the coast of Califomia. During routine capture operations, 18 of 102 Pacific harbor seals (Phoca vitulina richardsi) and 4 of 50 California sea lions (Zalophus californianus) from Puget Sound, Washington, had been reported with positive titers to Brucella sp. In this report, we describe the isolation, tissue location, and immunohistochemical and ultrastructural features of Brucella sp. infection in a pacific harbor seal. These findings suggest that transmission of brucellosis by infected lungworms is a possibility. On March 28, 1996, a 7-9-month-old 17.5-kg male Pacific harbor seal (105 cm length, snout to tail tip) was collected dead from Restoration Point on Bainbridge Island in the Puget Sound, Washington. The seal had been dead for 2-5 days, and there was slight decomposition. The carcass was transported to the Washington Department of Fish and Wildlife Marine Mammal Investigations Laboratory in Tacoma and necropsied. Blood drawn from the heart was separated by centrifugation, and serum was sent to the Washington Department of Agriculture Laboratory to screen for antibodies to Brucella sp. Values were compared with the standards outlined in the Brucellosis Eradication Uniform Methods and Rules (1992, revised 1994, USDA, APHIS). The sample was tested using B. abortus antigens provided by the National Veterinary Services Laboratory (NVSL), Ames, Iowa. Official protocols for

Brucella Suis Biovar 1 in Naturally Infected Cattle: A Bacteriological, Serological, and Histological Study

and immunodiffusion for serodiagnosis of paratuberculosis. Can J Vet Res 53:405–410. 20. Wright PF, Kelly WA, Gall DEJ: 1985, Application of a timing protocol to the reduction of interplate variability in the indirect enzyme immunoassay for detection of anti-Brucella antibody. J Immunoassay 6:189–205. 21. Yokomizo Y, Merkal RS, Lyle PAS: 1983, Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis. Am J Vet Res 44:2205–2207. 22. Yokomizo Y, Yugi H, Merkal RS: 1985, A method for avoiding false-positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine paratuberculosis Jpn J Vet Sci 47:111–119.

Evaluation of the Brucella Abortus Species–Specific Polymerase Chain Reaction Assay, an Improved Version of the Brucella AMOS Polymerase Chain Reaction Assay for Cattle

In a blind test, 344 samples representing 80 bacterial isolates were analyzed by the Brucella abortus species–specific polymerase chain reaction (BaSS PCR) assay for the identification and discrimination of B. abortus field strains (wild-type biovars 1, 2, and 4) from 1) B. abortus vaccine strains, 2) other Brucella species, and 3) non-Brucella bacteria. Identical samples were tested in 2 laboratories. Half the samples were fully viable, and half were bacteria that had been killed by methanol fixation. The results in 1 laboratory correctly identified 100% of the samples, resulting in a predictive value of 100% for all categories and 100% sensitivity and specificity under the prescribed conditions. The second laboratory misidentified 31 samples, resulting in a range of 66.7–100% sensitivity, 93.2–99.7% specificity, and 77.3–98.2% predictive values depending on the category. There was no significant difference in viable versus fixed bacteria for either laboratory. Subsequent review of the protocol indicated that contamination was the likely cause of 26 of the 31 erroneous identifications. The results show that the BaSS PCR assay has the potential to be a very reliable screening tool for B. abortus identification. However, the data also provide a cautionary reminder of the importance of preventing contamination in diagnostic PCR.

Evaluation of the HOOF-Print assay for typing Brucella abortus strains isolated from cattle in the United States: results with four performance criteria.

BackgroundA fundamental question that arises during epidemiological investigations of bacterial disease outbreaks is whether the outbreak strain is genetically related to a proposed index strain. Highly discriminating genetic markers for characterizing bacterial strains can help in clarifying the genetic relationships among strains. Under the auspices of the European Society of Clinical Microbiology and Infectious Diseases, the European Study Group for Epidemiological Markers (ESGEM) established guidelines for evaluating the performance of typing systems based of a number of criteria. Recently, HOOF-Print genotype analysis, a new method for typing Brucella abortus strains based on hypervariability at eight tandem repeat loci, was described. This paper evaluates the HOOF-Print assay by four of the criteria set out by the ESGEM: typeability, reproducibility, power of discrimination, and concordance with other typing methods.ResultsThe HOOF-Print Assay was evaluated with a test population composed of 97 unrelated field isolates and 6 common laboratory strains of B. abortus. Both typeability and reproducibility of the assay were excellent. Allele diversity and frequency varied widely among the eight loci, ranging from 1 to 13 alleles. The power of discrimination, measured by the Hunter-Gaston discrimination index (HGDI), varied by locus ranging from 0 to 0.89, where a maximal value of 1.0 indicates discrimination of all strains. The HGDI values calculated for subgroups sorted by biovar were similar to the values determined for the whole population. None of the individual loci achieved the recommended HGDI threshold of 0.95, but the HGDI of the composite profiles was 0.99 (93 unique genotypes from 97 field strains evaluated), well above the recommended threshold. By comparison, the HGDI value for biovar typing was 0.61 in a test population biased with disproportionate numbers of the less common biovars. Cluster analysis based on HOOF-Print genotypes assembled the strains into hierarchical groups with no apparent association with the time or location of strain isolation. Likewise, these hierarchical groups were not homogeneous with regard to biotype. In one extreme case, two field isolates with identical fingerprints were identified as different biovars by conventional methods.ConclusionThe main purpose of this study was to assess the ability of HOOF-Print genotyping to discriminate unrelated field strains of B. abortus, and whether the assay met established requirements for bacterial strain typing methods. The discriminatory power of the assay was remarkable, considering the genetic homogeneity found among species within the genus. The assay met or exceeded all of the recommended levels for the performance criteria of typeability, reproducibility, and power of discrimination, however some inconsistencies with conventional biovar typing were observed. Nevertheless, the results indicate that with cautious interpretation, multilocus genotyping of polymorphic tandem repeats by HOOF-Print analysis could be a valuable complement to routine epidemiological investigations into localized B. abortus outbreaks.

Brucellosis in Ringed Seals and Harp Seals from Canada

A novel Brucella sp. was isolated from lymph nodes of four ringed seals (Phoca hispida) collected near Pangnirtung (Baffin Island, Canada) in January and February 1995 and in one harp seal (Phoca groenlandica) collected near the Magdalen Islands (Gulf of St. Lawrence, Canada) in March 1996. Bacteriological characteristics were the same for all five isolates. The colonies were typical of Brucella spp., but took 2 to 5 days longer than the traditional species to appear on primary isolation media. Biotyping results did not match any of the known biovars of Brucella, but were similar to isolates of the genus Brucella previously reported from marine mammals inhabiting other areas of the northern hemisphere. This is the first confirmed report of brucellosis in marine mammals from Canada, and the first report of this organism in ringed and harp seals.

PATHOGENESIS AND EPIDEMIOLOGY OF BRUCELLOSIS IN YELLOWSTONE BISON: SEROLOGIC AND CULTURE RESULTS FROM ADULT FEMALES AND THEIR PROGENY

Our objective in this prospective study was to determine the natural course of Brucella abortus infection in cohorts of seropositive and seronegative, female bison (Bison bison) and their offspring in Yellowstone National Park (YNP) for 5 yr. We collected specimens from 53 adult females and 25 calves at least once and from 45 adults and 22 calves more than once. Annual seroconversion rates (negative to positive) were relatively high (23% for calves and juvenile bison, 6% in the total sample of adult female bison in our study, and 11% in the adult females that began the study as seronegatives). Antibody was not protective against infection, even for calves that passively received antibody from an infected mother’s colostrum. Antibody levels stayed remarkably constant, with only a slow decline over time. We found only two seroconversions from a weak positive status to negative. Infected bison aborted and shed viable bacteria. Risk of shedding infective Brucella was highest for bison in the 2 yr following seroconversion from negative to positive. In one bison, we detected shedding for 3 yr following seroconversion. Regardless of serostatus of dams and neonates, most calves were seronegative by 5 mo of age. There was no relationship between the antibody status of the dam and the tendency of a calf to seroconvert to positive during the duration of the study.

Seroconversion and abortion in cattle experimentally infected with Brucella sp. isolated from a Pacific harbor seal (Phoca vitulina richardsi).

Previously unrecognized Brucella species have been isolated from a number of marine mammals, including harbor seals (Phoca vitulina richardsi) in the Puget Sound area of the state of Washington. Because of the presence of dairy herds in proximity to the harbor seal populations, a study was conducted to determine the effects of the harbor seal Brucella isolate in experimentally inoculated cattle. Six pregnant cattle were exposed by intravenous injection (n = 3) or intraconjunctival inoculation (n = 3). Two pregnant cows were intravenously injected with saline and served as controls. All of the cows receiving the Brucella seroconverted on 1 or more tests commonly used for the detection of Brucella abortus infection. Two of the cattle receiving the intravenous inoculation aborted, and brucellae were demonstrated in the fetuses and dams immediately following abortion. The remaining 4 Brucella-inoculated animals and their fetuses were culture negative for the organism at 14 weeks postinoculation. Results of this study indicate the marine mammal Brucella is capable of producing seroconversion and abortion in cattle but is less pathogenic in that species than B. abortus.

Abortion Caused by Brucella abortus Biovar 1 in a Free-ranging Bison (Bison bison) from Yellowstone National Park

A near-term aborted bison (Bison bison) fetus was collected near Old Faithful geyser in Yellowstone National Park, Wyoming (USA). On necropsy, the fetus liver had a small capsular tear, and there was a small quantity of blood in the peritoneal cavity. Microscopic lesions included mild, purulent bronchopneumonia and mild, multifocal, interstitial pneumonia. Brucella abortus biovar 1 was isolated from fetal abomasal contents, lung, and heart blood.

Evidence of Brucella sp. infection in marine mammals stranded along the coast of southern New England.

Abstract After recent isolations of Brucella sp. from pinnipeds and cetaceans, a survey was initiated to investigate the prevalence of Brucella sp. infections and serologic evidence of exposure in marine mammals stranded along the coasts of Connecticut and Rhode Island. One hundred and nineteen serum samples from four species of cetaceans and four species of pinnipeds were collected from 1985 to 2000 and tested for antibodies to Brucella sp. using the brucellosis card test, buffered acidified plate antigen test, and rivanol test. In addition, 20 of these were necropsied between 1998 and 2000, with lymphoid and visceral tissues cultured for Brucella sp. Three of 21 (14%) harbor seals (Phoca vitulina) and four of 53 (8%) harp seals (Phoca groenlandica) were seropositive. Brucella sp. was isolated from two of four (50%) harbor seals and three of nine (33%) harp seals. Of the five animals with positive cultures, two were seropositive and three seronegative. Brucella sp. was most frequently cultured from the lung and axillary, inguinal, and prescapular lymph nodes. Tissues from which Brucella sp. was isolated showed no gross or histopathologic changes. These results indicate that marine mammals stranded along the coast of southern New England can be exposed to and infected with Brucella sp.