Isolation of Potentially Novel Brucella spp. from Frogs (original) (raw)
Related papers
Journal of Zoo and Wildlife Medicine
Two novel and distinct Brucella strains were recovered from 5 of 10 adult, sex undetermined, captive waxy tree frogs (Phyllomedusa sauvagii) and two of five adult, sex undetermined, captive Colorado river toads (Incilius alvarius) held in a zoologic collection with clinical and pathologic findings of bacterial disease. These amphibians originated from three separate private breeding facilities over several years and exhibited disease 9-49 mo following release from quarantine. Common presenting signs were vague but included focal abscessation, weight loss, change in coloration, anorexia, and decreased perching. Two waxy tree frogs and one Colorado river toad recovered with supportive care and antimicrobial treatment based on susceptibility testing. Microgranulomatosis, subcutaneous and renal abscessation, femoral osteomyelitis, and multicentric infection were the most common histologic findings. The organisms were identified antemortem in samples from subcutaneous abscesses, cloaca, and skin and from a variety of organ systems postmortem, and demonstrated a consistent susceptibility pattern. Initial isolates were misidentified as Ochrobactrum anthropi. Polymerase chain reaction and sequencing of the 16S rRNA gene identified the two organisms as novel Brucella strains similar to Brucella inopinata-like sp. and other novel organisms within the emerging ''BO clade.'' Brucella strain oaks (isolated from waxy tree frogs) and Brucella strain leathers (isolated from Colorado river toads) differed from each other by 16 of 571 base pairs in a region of chromosome 2, and did not closely match any previous GenBank entries. This report describes the clinicopathologic features of infection by these bacteria in two amphibian species and expands the range of novel Brucella organisms from amphibian reservoirs.
Frontiers in Veterinary Science, 2018
Several Brucella isolates have been described in wild-caught and "exotic" amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentified as Ochrobactrum anthropi using a commercial identification system, prior to being identified as Brucella spp. by MALDI-TOF assay. Classical phenotypic identification confirmed the Brucella genus, but did not make it possible to conclude unequivocally on species determination. Conventional and innovative bacteriological and molecular methods concluded that the investigated strain was very close to B. microti species, and not B. inopinata-like strains, as expected. The methods included growth kinetic, antimicrobial susceptibility testing, RT-PCR, Bruce-Ladder, Suis-Ladder, RFLP-PCR, AMOS-ERY, MLVA-16, the ectoine system, 16S rRNA and recA sequence analyses, the LPS pattern, in silico MLST-21, comparative whole-genome analyses (including average nucleotide identity ANI and whole-genome SNP analysis) and HRM-PCR assays. Minor polyphasic discrepancies, especially phage lysis and A-dominant agglutination patterns, as well as, small molecular divergences suggest the investigated strain should be considered a B. microti-like strain, raising concerns about its environmental persistence and unknown animal pathogenic and zoonotic potential as for other B. microti strains described to date.
PloS one, 2016
The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome...
Brucella microti‐ like prevalence in French farms producing frogs
Transboundary and Emerging Diseases, 2019
Brucellosis affects a wide range of animal hosts, generally causing reproductive disorders resulting in significant economic losses in farmed species. It is recognized as one of the most important bacterial zoonoses worldwide with 500,000 human cases per year (Godfroid et al., 2005). The genus Brucella comprises twelve species historically differentiated by host specificity and phenotypic characteristics (OIE, 2016). Brucella spp. have been evidenced in various hosts belonging to different ecosystems. Beside the six classical species (B. abortus, B melitensis, B. suis, B. canis, B. neotomae and B. ovis) affecting terrestrial mammals (OIE, 2016), novel members have been lately added to this group (B. pinnipedialis, B.ceti and B. papionis), thus extending the variety of Brucella animal hosts to marine mammals and primates (Foster, Osterman, Godfroid, Jacques, & Cloeckaert, 2007; Whatmore et al., 2014). Three other novel species genetically related to an atypical group of the genus (Scholz & Vergnaud, 2013) were first isolated in warm-blooded hosts: B. microti in common voles and foxes (
Scientific reports, 2017
Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains re...
Isolation of Brucella from a White’s tree frog (Litoria caerulea)
JMM Case Reports, 2015
Introduction: Brucellosis is a zoonotic disease that has a significant economic, social and public health impact in many parts of the world. The causative agents are members of the genus Brucella currently comprising 11 species and with an expanding known host range in recent years. Case presentation: One of a pair of White's tree frogs (Litoria caerulea) developed skin lesions from which a pure growth of a haemolytic organism was obtained. The isolate was identified as Brucella melitensis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, although the colony morphology was inconsistent with this identification. Applying the classical biotyping approach used to subdivide members of the genus Brucella, the isolate did not correspond to any known Brucella sp. However, PCR targeting of genes specific for members of the genus Brucella was strongly positive and 16S rRNA gene sequencing revealed a close relationship with extant Brucella spp. In order to place the isolate more accurately, a multilocus sequencing approach was applied, which confirmed that the isolate represented a novel member of the emerging 'atypical' Brucella group, which includes isolates from human disease, from rodents and, more recently, reported isolations from frogs in Germany. Conclusion: This case represents the first report of isolation of a Brucella sp. from frogs outside Germany and suggests that these isolates may be widespread. Whilst there is no evidence to date that these isolates represent a zoonotic threat, the association of other 'atypical' Brucella sp. with human disease suggests that appropriate measures should be taken to avoid unnecessary contact with potentially infected amphibians until the zoonotic potential of this group is better understood.
Brazilian Journal of Animal and Environmental Research, 2021
Ambystoma velasci, is a natural inhabitant of the crater lakes Atexcac, La Preciosa and Quechulac, in the Eastern Basin, Puebla, Mexico. Currently, their populations are at risk from the loss and microbiological contamination of their habitat. Brucella spp., is a zoonotic bacterium, which has been expanding to new taxa as reservoirs. The objective of this project was to detect and isolate Brucella sp., in Ambystoma velasci as a new host. Between February 2014 and April 2017, 25 specimens of A. velasci were collected. The primo-isolation were recovered from samples of the digestive, reproductive and excretory system (cloaca), and identified by standard microbiological and molecular tests, obtaining (40%) of positive results. They were compared by PCR, with two reference vaccine strains (BM16 and BS19), the bp 26 (IS711) gene was amplified, which is specific
Journal of Medical Microbiology, 2008
Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. are phenotypically and genetically closely related pathogens that may cause disease with similar clinical presentation. Consequently, difficulties in their identification and differentiation have been reported. In this study, a sensitive recA gene-based multi-primer single-target PCR (MP-ST-PCR) was developed that allowed the specific detection and differentiation of these clinically relevant pathogens. The specificity of the assay was evaluated using a representative panel of 50 O. anthropi and 16 O. intermedium strains and the type strains of all Brucella spp. Detection limits for purified DNA from O. anthropi, O. intermedium and Brucella melitensis were 100, 10 and 100 fg, respectively. Brucella DNA was also successfully detected in various clinical specimens from a human patient with culture-proven brucellosis and from a Brucella-infected sheep and its aborted fetuses. The sensitivity of the MP-ST-PCR was comparable to that of an evaluated in-house Brucella real-time PCR assay. The developed assay closes a diagnostic gap and provides a simple but robust tool for the sensitive detection and correct identification of O. anthropi, O. intermedium and Brucella spp.
Brucella suis bacteremia misidentified as Ochrobactrum anthropi by the VITEK 2 system
The Journal of Infection in Developing Countries, 2016
Ochrobactrum and Brucella are genetically related genera of the family Brucellaceae, sharing 98.8% rRNA similarity. Because of their phenotypic similarity, Ochrobactrum can be miscoded as Brucella by automated identification systems. The misidentification on blood cultures (BCs) of B. suis as O. anthropi by the VITEK 2 system is herein described. A 67-year-old male with a prosthetic mitral valve and fever was admitted with bacteremia due to a Gram-negative coccobacillus identified as O. anthropi by VITEK 2. The patient’s fever persisted along with positive blood cultures despite specific antimicrobial treatment. Due to this adverse outcome, the patient was interrogated again and admitted having domestic swine. Serological tests were positive for acute brucellosis. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of BC strains identified B. suis biovar 1. Timely identification of Brucella is essential for providing proper treatment to the patient and for ...
Pathogenicity and Its Implications in Taxonomy: The Brucella and Ochrobactrum Case
Pathogens
The intracellular pathogens of the genus Brucella are phylogenetically close to Ochrobactrum, a diverse group of free-living bacteria with a few species occasionally infecting medically compromised patients. A group of taxonomists recently included all Ochrobactrum organisms in the genus Brucella based on global genome analyses and alleged equivalences with genera such as Mycobacterium. Here, we demonstrate that such equivalencies are incorrect because they overlook the complexities of pathogenicity. By summarizing Brucella and Ochrobactrum divergences in lifestyle, structure, physiology, population, closed versus open pangenomes, genomic traits, and pathogenicity, we show that when they are adequately understood, they are highly relevant in taxonomy and not unidimensional quantitative characters. Thus, the Ochrobactrum and Brucella differences are not limited to their assignments to different “risk-groups”, a biologically (and hence, taxonomically) oversimplified description that, ...
Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR
BMC Research Notes, 2009
Background Bovine brucellosis caused by Brucella abortus is endemic in most large commercial and smallholder cattle farms of Zimbabwe, while brucellosis in other domestic animals is rare. The diagnosis of brucellosis is mainly accomplished using serological tests. However, some Brucella spp. have been isolated from clinical cases in the field and kept in culture collection but their biochemical profiles were not documented. We report biochemical profiling and AMOS-PCR characterization of some of these field isolates of Brucella originating from both commercial and smallholder cattle farming sectors of Zimbabwe. Findings Fourteen isolates of Brucella from culture collection were typed using biochemical profiles, agglutination by monospecific antisera, susceptibility to Brucella-specific bacteriophages and by AMOS-PCR that amplifies species- specific IS711. The results of the biochemical profiles for B. abortus biovar 1 (11 isolates) and biovar 2 (2 isolates) were consistent with those of reference strains. A single isolate from a goat originating from a smallholder mixed animal farm was identified as B. melitensis biovar 1. The AMOS-PCR produced DNA products of sizes 498 bp and 731 bp for B. abortus (biovar 1 and 2) and B. melitensis biovar 1, respectively. Conclusion We concluded that the biochemical profiles and AMOS-PCR characterization were consistent with their respective species and biovars. B. abortus biovar 1 is likely to be the predominant cause of brucellosis in both commercial and smallholder cattle farms in Zimbabwe.
Molecular characterization of Brucella species from Zimbabwe
PLOS Neglected Tropical Diseases, 2019
Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.
Assays for Identification and Differentiation of Brucella Species: A Review
Microorganisms
Brucellosis is one of the most important and widespread bacterial zoonoses worldwide. Cases are reported annually across the range of known infectious species of the genus Brucella. Globally, Brucella melitensis, primarily hosted by domestic sheep and goats, affects large proportions of livestock herds, and frequently spills over into humans. While some species, such as Brucella abortus, are well controlled in livestock in areas of North America, the Greater Yellowstone Ecosystem supports the species in native wild ungulates with occasional spillover to livestock. Elsewhere in North America, other Brucella species still infect domestic dogs and feral swine, with some associated human cases. Brucella spp. patterns vary across space globally with B. abortus and B. melitensis the most important for livestock control. A myriad of other species within the genus infect a wide range of marine mammals, wildlife, rodents, and even frogs. Infection in humans from these others varies with geog...
Brucella suis Bacteremia Misidentified as Ochrobactrum antropii by Vytek 2 Automated System
2014
Ochrobactrum and Brucella are genetically related genera of the family Brucellaceae, sharing 98.8% rRNA similarity. Because of their phenotypic similarity, Ochrobactrum can be miscoded as Brucella by automated identification systems. The misidentification on blood cultures (BCs) of B. suis as O. anthropi by the VITEK 2 system is herein described. A 67-year-old male with a prosthetic mitral valve and fever was admitted with bacteremia due to a Gram-negative coccobacillus identified as O. anthropi by VITEK 2. The patient's fever persisted along with positive blood cultures despite specific antimicrobial treatment. Due to this adverse outcome, the patient was interrogated again and admitted having domestic swine. Serological tests were positive for acute brucellosis. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of BC strains identified B. suis biovar 1. Timely identification of Brucella is essential for providing proper treatment to the patient and for advising safe handling of laboratory cultures in biological safety cabinets to prevent laboratory-acquired infection. Countries where brucellosis is endemic must be aware of this possibility.
Whole-Genome-Based Phylogeny and Divergence of the Genus Brucella
Journal of Bacteriology, 2009
Brucellae are worldwide bacterial pathogens of livestock and wildlife, but phylogenetic reconstructions have been challenging due to limited genetic diversity. We assessed the taxonomic and evolutionary relationships of five Brucella species-Brucella abortus, B. melitensis, B. suis, B. canis, and B. ovis-using whole-genome comparisons. We developed a phylogeny using single nucleotide polymorphisms (SNPs) from 13 genomes and rooted the tree using the closely related soil bacterium and opportunistic human pathogen, Ochrobactrum anthropi. Whole-genome sequencing and a SNP-based approach provided the requisite level of genetic detail to resolve species in the highly conserved brucellae. Comparisons among the Brucella genomes revealed 20,154 orthologous SNPs that were shared in all genomes. Rooting with Ochrobactrum anthropi reveals that the B. ovis lineage is basal to the rest of the Brucella lineage. We found that B. suis is a highly divergent clade with extensive intraspecific genetic diversity. Furthermore, B. suis was determined to be paraphyletic in our analyses, only forming a monophyletic clade when the B. canis genome was included. Using a molecular clock with these data suggests that most Brucella species diverged from their common B. ovis ancestor in the past 86,000 to 296,000 years, which precedes the domestication of their livestock hosts. Detailed knowledge of the Brucella phylogeny will lead to an improved understanding of the ecology, evolutionary history, and host relationships for this genus and can be used for determining appropriate genotyping approaches for rapid detection and diagnostic assays for molecular epidemiological and clinical studies.
Molecular characterization of Brucella strains isolated from marine mammals
Journal of clinical microbiology, 2000
Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus, B. melitensis, B. canis, and B. suis. IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new Brucella species may be appropriate.
Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification
Journal of Applied Microbiology, 1996
M. DA COSTA, J.-P. GUILLOU, B. GARIN-BASTUJI, M. THIÉBAUD AND G. DUBRAY. 1996. DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3′-labelled specific probe and by PCR using 98 non-Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after EcoRI digestion.
Circulating Brucella species in wild animals of the Serengeti ecosystem, Tanzania
One Health Outlook, 2021
Brucellosis is a bacterial zoonosis of public health and economic importance worldwide. It affects a number of domestic animals, wild animals and humans. Human brucellosis originates from either livestock or wildlife. The species of Brucella circulating in wild animals in Tanzania is largely unknown due to insufficient surveillance. This study was carried out to identify Brucella species found in selected wildlife hosts in the Serengeti ecosystem. The study used a total of 189 archived samples that were obtained from cross-sectional studies previously conducted between 2000 and 2017 in the Serengeti ecosystem in Tanzania. Whole blood, serum and amniotic fluid collected from buffalos, lions, wildebeest, impala, zebra and hyena were available for DNA extraction. Multiplex polymerase chain reaction for B. abortus, B. melitensis, B. ovis and B. suis (AMOS PCR) and quantitative real-time PCR (qPCR) targeting the bcsp31 and IS711 genes for Brucella genus detection and the IS711 targets al...
Scientific Reports, 2020
Brucellosis is a neglected zoonotic disease caused by alpha proteobacterial genus Brucella comprising of facultative intracellular pathogenic species that can infect both animals and humans. In this study, we aimed to identify genome-wide unique insertion sequence (IS) elements among Brucella abortus, B. melitensis, B. ovis, B. suis and B. canis for use in species differentiation by conducting an intensive in silico-based comparative genomic analysis. As a result, 25, 27, 37, 86 and 3 unique ISs were identified respectively and they had a striking pattern of distribution among them. To explain, a particular IS would be present in four species with 100% identity whereas completely absent in the fifth species. However, flanking regions of that IS element would be highly identical and conserved in all five species. Species-specific primers designed on these flanking conserved regions resulted in two different amplicons grouping the species into two: one that possesses IS and the other that lacks it. Seeking for species-specific amplicon size for particular species was sufficient to identify it irrespective of biovar. A multiplex PCR developed using these primers resulted in successful differentiation of the five species irrespective of biovars with significant specificity and sensitivity when examined on clinical samples.
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Applied and Environmental Microbiology, 2014
Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative. . 2015. Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions. Appl Environ Microbiol 81:578 -586.
FEBS Open Bio, 2015
In orally acquired bacteria, the ability to counteract extreme acid stress (pH 6 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamatedependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports c-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter.
BMC research notes, 2014
The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was ...
PLoS ONE, 2013
A high prevalence of Brucella pinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal (Cystophora cristata); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B. pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B. pinnipedialis reference strain (NCTC 12890) from harbor seal (Phoca vitulina), B. ceti reference strain (NCTC 12891) from harbor porpoise (Phocoena phocoena) and a B. ceti Atlantic white-sided dolphin (Lagenorhynchus acutus) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B. pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal.
PLOS ONE, 2016
Pathology has not been observed in true seals infected with Brucella pinnipedialis. A lack of intracellular survival and multiplication of B. pinnipedialis in hooded seal (Cystophora cristata) macrophages in vitro indicates a lack of chronic infection in hooded seals. Both epidemiology and bacteriological patterns in the hooded seal point to a transient infection of environmental origin, possibly through the food chain. To analyse the potential role of fish in the transmission of B. pinnipedialis, Atlantic cod (Gadus morhua) were injected intraperitoneally with 7.5 x 10 7 bacteria of a hooded seal field isolate. Samples of blood, liver, spleen, muscle, heart, head kidney, female gonads and feces were collected on days 1, 7, 14 and 28 post infection to assess the bacterial load, and to determine the expression of immune genes and the specific antibody response. Challenged fish showed an extended period of bacteremia through day 14 and viable bacteria were observed in all organs sampled, except muscle, until day 28. Neither gross lesions nor mortality were recorded. Anti-Brucella antibodies were detected from day 14 onwards and the expression of hepcidin, cathelicidin, interleukin (IL)-1β, IL-10, and interferon (IFN)-γ genes were significantly increased in spleen at day 1 and 28. Primary mononuclear cells isolated from head kidneys of Atlantic cod were exposed to B. pinnipedialis reference (NCTC 12890) and hooded seal (17a-1) strain. Both bacterial strains invaded mononuclear cells and survived intracellularly without any major reduction in bacterial counts for at least 48 hours. Our study shows that the B. pinnipedialis strain isolated from hooded seal survives in Atlantic cod, and suggests that Atlantic cod could play a role in the transmission of B. pinnipedialis to hooded seals in the wild.
Scientific reports, 2017
Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains re...
Brucella Antibodies in Alaskan True Seals and Eared Seals—Two Different Stories
Frontiers in Veterinary Science
Brucella pinnipedialis was first isolated from true seals in 1994 and from eared seals in 2008. Although few pathological findings have been associated with infection in true seals, reproductive pathology including abortions, and the isolation of the zoonotic strain type 27 have been documented in eared seals. In this study, a Brucella enzyme-linked immunosorbent assay (ELISA) and the Rose Bengal test (RBT) were initially compared for 206 serum samples and a discrepancy between the tests was found. Following removal of lipids from the serum samples, ELISA results were unaltered while the agreement between the tests was improved, indicating that serum lipids affected the initial RBT outcome. For the remaining screening, we used ELISA to investigate the presence of Brucella antibodies in sera of 231 eared and 1,412 true seals from Alaskan waters sampled between 1975 and 2011. In eared seals, Brucella antibodies were found in two Steller sea lions (Eumetopias jubatus) (2%) and none of the 107 Northern fur seals (Callorhinus ursinus). The low seroprevalence in eared seals indicate a low level of exposure or lack of susceptibility to infection. Alternatively, mortality due to the Brucella infection may remove seropositive animals from the population. Brucella antibodies were detected in all true seal species investigated; harbor seals (Phoca vitulina) (25%), spotted seals (Phoca largha) (19%), ribbon seals (Histriophoca fasciata) (16%), and ringed seals (Pusa hispida hispida) (14%). There was a low seroprevalence among pups, a higher seroprevalence among juveniles, and a subsequent decreasing probability of seropositivity with age in harbor seals. Similar patterns were present for the other true seal species; however, solid conclusions could not be made due to sample size. This pattern is in accordance with previous reports on B. pinnipedialis infections in true seals and may suggest environmental exposure to B. pinnipedialis at the juvenile stage, with a following clearance of infection. Furthermore, analyses by region showed minor differences in the probability of being seropositive for harbor seals from different regions regardless of the local seal population trend, signifying that the Brucella infection may not cause significant mortality in these populations. In conclusion, the Brucella infection pattern is very different for eared and true seals.
PloS one, 2016
The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome...
Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages
BMC genomics, 2017
Swine brucellosis caused by B. suis biovar 2 is an emergent disease in domestic pigs in Europe. The emergence of this pathogen has been linked to the increase of extensive pig farms and the high density of infected wild boars (Sus scrofa). In Portugal and Spain, the majority of strains share specific molecular characteristics, which allowed establishing an Iberian clonal lineage. However, several strains isolated from wild boars in the North-East region of Spain are similar to strains isolated in different Central European countries. Comparative analysis of five newly fully sequenced B. suis biovar 2 strains belonging to the main circulating clones in Iberian Peninsula, with publicly available Brucella spp. genomes, revealed that strains from Iberian clonal lineage share 74% similarity with those reference genomes. Besides the 210 kb translocation event present in all biovar 2 strains, an inversion with 944 kb was presented in chromosome I of strains from the Iberian clone. At left ...
Brucella Genomics: Macro and Micro Evolution
International Journal of Molecular Sciences, 2020
Brucella organisms are responsible for one of the most widespread bacterial zoonoses, named brucellosis. The disease affects several species of animals, including humans. One of the most intriguing aspects of the brucellae is that the various species show a ~97% similarity at the genome level. Still, the distinct Brucella species display different host preferences, zoonotic risk, and virulence. After 133 years of research, there are many aspects of the Brucella biology that remain poorly understood, such as host adaptation and virulence mechanisms. A strategy to understand these characteristics focuses on the relationship between the genomic diversity and host preference of the various Brucella species. Pseudogenization, genome reduction, single nucleotide polymorphism variation, number of tandem repeats, and mobile genetic elements are unveiled markers for host adaptation and virulence. Understanding the mechanisms of genome variability in the Brucella genus is relevant to comprehe...
PLoS ONE, 2014
Ochrobactrum intermedium is considered as an emerging human environmental opportunistic pathogen with mild virulence. The distribution of isolates and sequences described in literature and databases showed frequent association with human beings and polluted environments. As population structures are related to bacterial lifestyles, we investigated by multi-locus approach the genetic structure of a population of 65 isolates representative of the known natural distribution of O. intermedium. The population was further surveyed for genome dynamics using pulsed-field gel electrophoresis and genomics. The population displayed a clonal epidemic structure with events of recombination that occurred mainly in clonal complexes. Concerning biogeography, clones were shared by human and environments and were both cosmopolitan and local. The main cosmopolitan clone was genetically and genomically stable, and grouped isolates that all harbored an atypical insertion in the rrs. Ubiquitism and stability of this major clone suggested a clonal succes in a particular niche. Events of genomic reduction were detected in the population and the deleted genomic content was described for one isolate. O. intermedium displayed allopatric characters associated to a tendancy of genome reduction suggesting a specialization process. Considering its relatedness with Brucella, this specialization might be a commitment toward pathogenic lifestyle that could be driven by technological selective pressure related medical and industrial technologies.
Assays for Identification and Differentiation of Brucella Species: A Review
Microorganisms
Brucellosis is one of the most important and widespread bacterial zoonoses worldwide. Cases are reported annually across the range of known infectious species of the genus Brucella. Globally, Brucella melitensis, primarily hosted by domestic sheep and goats, affects large proportions of livestock herds, and frequently spills over into humans. While some species, such as Brucella abortus, are well controlled in livestock in areas of North America, the Greater Yellowstone Ecosystem supports the species in native wild ungulates with occasional spillover to livestock. Elsewhere in North America, other Brucella species still infect domestic dogs and feral swine, with some associated human cases. Brucella spp. patterns vary across space globally with B. abortus and B. melitensis the most important for livestock control. A myriad of other species within the genus infect a wide range of marine mammals, wildlife, rodents, and even frogs. Infection in humans from these others varies with geog...
Uncovering the Hidden Credentials of Brucella Virulence
Microbiology and Molecular Biology Reviews, 2021
Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world’s most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals.
Frontiers in Microbiology, 2016
An extended multilocus sequence analysis (MLSA) scheme applicable to the Brucella, an expanding genus that includes zoonotic pathogens that severely impact animal and human health across large parts of the globe, was developed. The scheme, which extends a previously described nine locus scheme by examining sequences at 21 independent genetic loci in order to increase discriminatory power, was applied to a globally and temporally diverse collection of over 500 isolates representing all 12 known Brucella species providing an expanded and detailed understanding of the population genetic structure of the group. Over 100 sequence types (STs) were identified and analysis of data provided insights into both the global evolutionary history of the genus, suggesting that early emerging Brucella abortus lineages might be confined to Africa while some later lineages have spread worldwide, and further evidence of the existence of lineages with restricted host or geographical ranges. The relationship between biovar, long used as a crude epidemiological marker, and genotype was also examined and showed decreasing congruence in the order Brucella suis > B. abortus > Brucella melitensis. Both the previously described nine locus scheme and the extended 21 locus scheme have been made available at http://pubmlst.org/brucella/ to allow the community to interrogate existing data and compare with newly generated data.
Omp2b Porin Alteration in the Course of Evolution of Brucella spp
Frontiers in Microbiology, 2020
The genus Brucella comprises major pathogenic species causing disease in livestock and humans, e.g. B. melitensis. In the past few years, the genus has been significantly expanded by the discovery of phylogenetically more distant lineages comprising strains from diverse wildlife animal species, including amphibians and fish. The strains represent several potential new species, with B. inopinata as solely named representative. Being genetically more distant between each other, relative to the "classical" Brucella species, they present distinct atypical phenotypes and surface antigens. Among surface protein antigens, the Omp2a and Omp2b porins display the highest diversity in the classical Brucella species. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. They share between 85 and 100% sequence identity depending on the Brucella species, biovar, or genotype. Only the omp2b gene copy has been shown to be expressed and genetic variation is extensively generated by gene conversion between the two copies. In this study, we analyzed the omp2 loci of the non-classical Brucella spp. Starting from two distinct ancestral genes, represented by Australian rodent strains and B. inopinata, a stepwise nucleotide reduction was observed in the omp2b gene copy. It consisted of a first reduction affecting the region encoding the surface L5 loop of the porin, previously shown to be critical in sugar permeability, followed by a nucleotide reduction in the surface L8 loopencoding region. It resulted in a final omp2b gene size shared between two distinct clades of non-classical Brucella spp. (African bullfrog isolates) and the group of classical Brucella species. Further evolution led to complete homogenization of both omp2 gene copies in some Brucella species such as B. vulpis or B. papionis. The stepwise omp2b deletions seemed to be generated through recombination with the respective omp2a gene copy, presenting a conserved size among Brucella spp., and may involve short direct DNA repeats. Successive Omp2b porin alteration correlated with increasing porin permeability in the course of evolution of Brucella spp. They possibly have adapted their porin to survive environmental conditions encountered and to reach their final status as intracellular pathogen.
Brucellosis in wildlife in Africa: a systematic review and meta-analysis
Scientific Reports, 2021
This study aimed to consolidate current knowledge of wildlife brucellosis in Africa and to analyse available predictors of infection. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. Information on species, test used, test results, area, rainfall, livestock and wildlife contact and year of study were extracted. This systematic review revealed 42 prevalence studies, nine disease control articles and six articles on epidemiology. Brucella abortus, Brucella melitensis, Brucella inopinata and Brucella suis were reported in wildlife. The prevalence studies revealed serological evidence of brucellosis in buffalo, antelope (positive in 14/28 species), carnivores (4/12) and other species (7/20) over the last five decades. Buffalo populations were more likely to be infected and had a higher seroprevalence than other species; the pooled seroprevalence was 13.7% (95% CI 10.3-17.3%) in buffalo, 7.1% (95% CI 1.1-15.5%) in carnivores and 2.1% (95% CI 0.1-4.9%) in antelope. Wildlife in high rainfall areas (≥ 800 mm) were more likely to be infected, and infected populations showed higher seroprevalence in high rainfall areas and in studies published after 2000. Domestic animal contact was associated with increased seroprevalence in antelope and carnivore species, but not in buffalo, supporting the hypothesis that buffalo may be a reservoir species. Brucellosis caused by Brucella spp. is a disease of significant economic, public health and veterinary importance. Since its identification over 120 years ago in humans it has been isolated in wide variety of animals and found to have a global distribution. The main aetiological agents in humans are Brucella abortus and Brucella melitensis, which are predominantly carried by large and small ruminants respectively. Brucellosis in wild animals in Africa has been documented in a variety of countries since the early 1960′s with serological studies and some Brucella isolations in many wildlife species 1 . Most studies have been serological surveys to try to better understand the epidemiological situation in wildlife, with an assumption that wildlife infected with Brucella spp. may have implications for domestic animals and humans. There has been no previous systematic review and meta-analysis of brucellosis in wildlife in Africa of this nature. The objectives of this systematic review were to update answers to the following questions: I. Which wildlife species have been exposed to brucellosis and where are they found? II. Which Brucella species are known to infect wildlife species? III. Which wildlife species are able to sustain Brucella infections? IV. Are wildlife species a brucellosis risk to domestic animals and vice-versa? V. What is known about effective control and prevention methods in wildlife? VI. What factors are associated with brucellosis infection in wildlife?
Frontiers in Cellular and Infection Microbiology, 2016
Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.