Human exposures to Brucella canis from a pregnant dog during an international flight: Public health risks, diagnostic challenges and future considerations.

AIMS: This report documents the exposure of passengers and crew of a commercial international flight to the zoonotic pathogen Brucella canis after an infected dog aborted in the passenger cabin of the aircraft. This case demonstrates the challenges associated with brucellosis screening and the risks that airline personnel, airport employees and travellers face when animals with unrecognized zoonotic infections are transported. METHODS/RESULTS: The public health investigation of this case was conducted by the Centers for Disease Control, the Illinois Department of Health and the Illinois Department of Agriculture, in collaboration with a local veterinary clinic and several academic and federal diagnostic laboratories. It included an extensive diagnostic evaluation of the dam and aborted foetuses to confirm a diagnosis of canine brucellosis. Passengers, airline personnel and staff from the veterinary clinic where the dogs were treated underwent risk assessments, and clinic staff also received detailed guidance regarding infection prevention practices. CONCLUSIONS: Animal shelters and breeding programs are recommended to screen dogs routinely for brucellosis, but it is not unusual for domestic or imported animals to have unknown health histories, including the dog's brucellosis status, at the time of purchase, adoption, or re-homing. Testing recommendations and requirements vary by state, making it challenging for state public health and animal health agencies to monitor and respond appropriately. This case highlights the importance of Brucella spp. screening in sexually intact dogs prior to breeding, purchase, or domestic or international transportation of the dogs. The transportation of pregnant dogs may present a previously unrecognized public health threat in addition to contributing to unnecessary stress and health risks for pregnant animals.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.

Global phylogenomic diversity of Brucella abortus: spread of a dominant lineage.

Brucella abortus is a globally important zoonotic pathogen largely found in cattle hosts and is typically transmitted to humans through contaminated dairy products or contact with diseased animals. Despite the long, shared history of cattle and humans, little is known about how trade in cattle has spread this pathogen throughout the world. Whole genome sequencing provides unparalleled resolution to investigate the global evolutionary history of a bacterium such as B. abortus by providing phylogenetic resolution that has been unobtainable using other methods. We report on large-scale genome sequencing and analysis of B. abortus collected globally from cattle and 16 other hosts from 52 countries. We used single nucleotide polymorphisms (SNPs) to identify genetic variation in 1,074 B. abortus genomes and using maximum parsimony generated a phylogeny that identified four major clades. Two of these clades, clade A (median date 972 CE; 95% HPD, 781-1142 CE) and clade B (median date 150 BCE; 95% HPD, 515 BCE-164 CE), were exceptionally diverse for this species and are exclusively of African origin where provenance is known. The third clade, clade C (median date 949 CE; 95% HPD, 766-1102 CE), had most isolates coming from a broad swath of the Middle East, Europe, and Asia, also had relatively high diversity. Finally, the fourth major clade, clade D (median date 1467 CE; 95% HPD, 1367-1553 CE) comprises the large majority of genomes in a dominant but relatively monomorphic group that predominantly infects cattle in Europe and the Americas. These data are consistent with an African origin for B. abortus and a subsequent spread to the Middle East, Europe, and Asia, probably through the movement of infected cattle. We hypothesize that European arrival to the Americas starting in the 15th century introduced B. abortus from Western Europe through the introduction of a few common cattle breeds infected with strains from clade D. These data provide the foundation of a comprehensive global phylogeny of this important zoonotic pathogen that should be an important resource in human and veterinary epidemiology.

Bayesian latent-class modelling of quarantine testing procedures for American Bison (Bison bison) in the Greater Yellowstone Area to determine Brucella abortus freedom.

OBJECTIVE: American bison (Bison bison) quarantine protocols were established to prevent transmission of brucellosis outside the Greater Yellowstone Area, while allowing for distribution of wild bison for conservation and cultural purposes. Quarantine standards require rigorous testing over 900 days which has led to the release of over 200 bison to Native American tribes. Standards were evaluated using 15 years of laboratory and management data to minimize the burden of testing and increase the number of brucellosis-free bison available for distribution. ANIMALS: All bison (n = 578) from Yellowstone National Park were corralled by the National Park Service and United States Department of Agriculture. PROCEDURES: A statistical and management evaluation of the bison quarantine program was performed. Bayesian latent-class modeling was used to predict the probability of nondetection of a seroreactor at various time points, as well as the probability of seroconversion by days in quarantine. RESULTS: At 300 days, 1 in 1,000 infected bison (0.0014 probability) would not be detected but could potentially seroconvert; the seroconversion model predicted 99.9% would seroconvert by day 294, and 12.8% of bison enrolled in quarantine would seroconvert over time. Using a 300-day quarantine period, it would take 30 years to potentially miss 1 seroreactor out of over 8,000 bison enrolled in the quarantine program. CLINICAL RELEVANCE: Reducing the quarantine program requirements from over 900 days to 300 days would allow management of quarantined bison in coordination with seasonal movement of bison herds and triple the number of brucellosis-free bison available for distribution.

Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish.

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals ( Phoca vitulina richardii) and North Atlantic hooded seals ( Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS) 711 gene and sequence type (ST)27 primer-probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS 711 and ST27 primer-probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS 711 primer-probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS 711 primer-probe was used to test Atlantic cod ( Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS 711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

Characterisation of North American Brucella isolates from marine mammals.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock.

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations.

Identification of Source of Brucella suis Infection in Human by Using Whole-Genome Sequencing, United States and Tonga.

Brucella suis infection was diagnosed in a man from Tonga, Polynesia, who had butchered swine in Oregon, USA. Although the US commercial swine herd is designated brucellosis-free, exposure history suggested infection from commercial pigs. We used whole-genome sequencing to determine that the man was infected in Tonga, averting a field investigation.

Brucella papionis sp. nov., isolated from baboons (Papio spp.).

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T)).

Identification of Brucella suis from feral swine in selected states in the USA.

Serologic tests currently available for brucellosis diagnosis detect antibodies to Brucella but do not distinguish between species of Brucella. Although Brucella suis is known to circulate within various feral swine (Sus scrofa) populations, our objective was to determine the primary species of Brucella circulating in feral swine populations in areas of the US with high brucellosis prevalence. We cultured lymph nodes from 183 feral swine. We identified 22 isolates from 21 animals, and all isolates were genotyped as B. suis. Most isolates were B. suis biovar 1, with the exception of two genetically distinct isolates from one feral swine in Hawaii, which were identified as B. suis biovar 3. Serum from each feral swine was also tested by the fluorescence polarization assay when possible, but only 52% (95% CL = 29.8-74.3) of culture-positive animals were antibody positive. Our results indicate that brucellosis infections in feral swine within the US are typically caused by B. suis. However, improved serologic tests are needed to more accurately determine exposure to Brucella spp. and to monitor disease trends in feral swine populations.

Transmission of brucellosis from elk to cattle and bison, Greater Yellowstone area, U.S.A., 2002-2012.

Bovine brucellosis has been nearly eliminated from livestock in the United States. Bison and elk in the Greater Yellowstone Area remain reservoirs for the disease. During 1990-2002, no known cases occurred in Greater Yellowstone Area livestock. Since then, 17 transmission events from wildlife to livestock have been investigated.

Molecular epidemiology of Brucella abortus isolates from cattle, elk, and bison in the United States, 1998 to 2011.

A variable-number tandem repeat (VNTR) protocol targeting 10 loci in the Brucella abortus genome was used to assess genetic diversity among 366 field isolates recovered from cattle, bison, and elk in the Greater Yellowstone Area (GYA) and Texas during 1998 to 2011. Minimum spanning tree (MST) and unweighted-pair group method with arithmetic mean (UPGMA) analyses of VNTR data identified 237 different VNTR types, among which 14 prominent clusters of isolates could be identified. Cattle isolates from Texas segregated into three clusters: one comprised of field isolates from 1998 to 2005, one comprised of vaccination-associated infections, and one associated with an outbreak in Starr County in January 2011. An isolate obtained from a feral sow trapped on property adjacent to the Starr County herd in May 2011 clustered with the cattle isolates, suggesting a role for feral swine as B. abortus reservoirs in Starr County. Isolates from a 2005 cattle outbreak in Wyoming displayed VNTR-10 profiles matching those of strains recovered from Wyoming and Idaho elk. Additionally, isolates associated with cattle outbreaks in Idaho in 2002, Montana in 2008 and 2011, and Wyoming in 2010 primarily clustered with isolates recovered from GYA elk. This study indicates that elk play a predominant role in the transmission of B. abortus to cattle located in the GYA.

A novel Brucella isolate in association with two cases of stillbirth in non-human primates - first report.

BACKGROUND: Brucellosis is veterinary and human health problem. METHODS: A 13-year-old wild caught multiparous and an 8-year-old colony-born nulliparous baboon had stillbirths in the second trimester of pregnancy. Culture isolates from both postpartum uteruses were characterized using traditional biochemical analysis, PCR, and multilocus sequencing. RESULTS: The isolates morphologically resembled Brucella although their phenotypic characteristics were not consistent with any currently described species. The isolates represent a novel lineage within the genus with unique alleles, not previously seen in surveys of greater than 300 isolates representing the known diversity of the genus, present at 5/9 loci examined. CONCLUSIONS: The described cases are to the best of our knowledge the first presentation of a naturally acquired Brucella infection in non-human primates associated with stillbirths from the same colony where Brucella seropositivity in the baboons was described 45 years ago. The organism appears to represent a previously undescribed Brucella species.