Atypical Brucella Research Articles

A Survey for Atypical Brucella Species in Wild-Caught Marine Toads (Rhinella marina) Using Qualitative PCR: A Pilot Study

Abstract The genus Brucella is a group of intracellular bacterial pathogens documented to cause brucellosis, a zoonotic disease of worldwide concern. In the past decade, reported cases of atypical Brucella species in amphibians have increased. These species are considered atypical based on their tropism, phenotypic characteristics, and phylogeny differences. Atypical Brucella species can be misdiagnosed due to their similarity to classical Brucella species and closely related genera. Recent literature has provided evidence for the zoonotic potential of atypical Brucella linked to amphibians. Therefore, understanding the prevalence of these atypical Brucella species in captive amphibian populations is significant for amphibian health, conservation, and public health. This pilot study evaluated the prevalence of atypical Brucella species in a population of marine toads (Rhinella marina) using a validated pan-Brucella qualitative PCR. Samples were analyzed from 56 asymptomatic wild-caught marine toads from Miami, FL, USA, using skin swab samples. Brucella DNA was not detected in any of the skin swab samples analyzed. This pilot study represents a viable non-invasive sampling technique and PCR assay for rapidly screening populations of amphibians for the presence of atypical Brucella species.

Investigation of the presence of Brucella spp. in amphibians collected from some regions in Türkiye

Brucellosis is a zoonotic disease that causes economic losses in many countries worldwide, especially in livestock. With the development of molecular techniques, the diversity of Brucella species has been increasing in recent years. Recently, atypical Brucella spp. have been isolated mainly from cold-blooded animals. Firstly, the African bullfrog and then B. inopinata and B. microti-like strains have been identified in various frog species worldwide. This study aimed to determine the presence of Brucella in amphibian frogs for the first time in Türkiye by bacteriological and molecular methods. Internal organ samples (spleen, liver, lung, kidney, etc.) of 150 frogs from different regions of Türkiye (Edirne/Ipsala (53 pieces), Adıyaman/Kâhta (97 pieces)) were used for the isolation of Brucella spp. Simultaneously, DNA extraction was performed using a commercial isolation kit from tissue homogenates and samples cultured in media and left to incubation. As a result of Brucella genus-specific PCR (Polymerase Chain Reaction) and Multiplex PCR (Bruce-Ladder) methods performed with these extracts, no positivity was detected in the frog samples taken from Edirne province, while in 4 of the frog samples taken from Adıyaman province, bands of approximately 250, 600, 700, 1000, 1500 and 3000 bp were observed in Multiplex PCR. Sphingomonas paucimobilis (S. paucimobilis) was identified by analysing the culture obtained from these samples with VITEK®2:Healtcare. As a result, the presence of Brucella spp. could not be detected both bacteriologically and molecularly in the study samples. However, observing similar multiple bands in multiplex PCR suggested that this bacterium and Brucella species are phylogenetically close. At the same time, it was determined that this opportunistic pathogen isolated from various human infections can colonise amphibians. Since S. paucimobilis, which is found together with Brucella species under the class Alphaproteobacteria, causes opportunistic infections, especially in immunocompromised humans, it was concluded that this bacterium may pose a risk for those working in farms where frogs are raised for human consumption, and it is thought that it may be essential to take the necessary preventive measures.

Brucella spp. are facultative anaerobic bacteria under denitrifying conditions.

Bacteria of the genus Brucella are responsible for human and animal brucellosis worldwide and comprise the well-characterized, so-called classical species belonging to the core clade, with a host range limited to mammals, and the novel atypical strains mainly considered as non-zoonotic to date, but able to colonize non-traditional hosts such as amphibians. Despite marked phenotypical differences between the two groups, Brucella species display a high degree of genome identity, including genes encoding reductases involved in the denitrification pathway, a respiratory process able to reduce nitrate to nitrogen under anoxic conditions. Brucella spp. have always been considered as strictly aerobic pathogens. Our study, however, describes for the first time that atypical Brucella spp. grow well under anoxia, but in a strictly nitrate-dependent manner, whereas classical species show a heterogeneous behavior. The rate of anaerobic growth of the atypical species Brucella microti was dependent on nitrate concentration, highlighting a more efficient denitrification pathway with rapid consumption of intermediate nitrite, whereas the classical species Brucella suis exhibited lower, nitrate concentration-independent growth coupled with nitrite accumulation. Transcriptional studies performed under anoxic conditions revealed a higher expression of genes participating in the last three denitrification steps in B. microti, as compared to B. suis, which correlates with the rapid and efficient consumption of nitrite in this atypical species and its rapid growth.IMPORTANCERespiration is a fundamental and complex process that bacteria use to produce energy. Despite aerobic respiration being the most common, some bacteria make use of a mode of respiration in the absence of oxygen, called anaerobic respiration, which can yield advantages in adaptation to various environmental conditions. Denitrification is part of this respiratory process ensuring higher respiratory flexibility under oxygen depletion. Here, we report for the first time the evidence of anaerobic growth of Brucella spp. under denitrifying conditions, which implies that this genus should be reconsidered as facultative anaerobic. Our study further describes that efficient denitrification is not equally found within the Brucella genus, with atypical species showing a greater ability to denitrify, correlated with higher expression of the genes involved, as compared to classical species.

Isolation of Brucella inopinata from a White's tree frog (Litoria caerulea): pose exotic frogs a potential risk to human health?

Cold-blooded hosts, particularly exotic frogs, have become a newly recognized reservoir for atypical Brucella species and strains worldwide, but their pathogenicity to humans remains largely unknown. Here we report the isolation and molecular characterization of a B. inopinata strain (FO700662) cultured from clinical samples taken from a captive diseased White's Tree Frog (Litoria caerulea) in Switzerland. The isolation of B. inopinata from a frog along with other reports of human infection by atypical Brucella raises the question of whether atypical Brucella could pose a risk to human health and deserves further attention. The investigations included histopathological analysis of the frog, bacterial culture and in-depth molecular characterization of strain FO700662 based on genome sequencing data. Originally identified as Ochrobactrum based on its rapid growth and biochemical profile, strain FO700622 was positive for the Brucella- specific markers bcsp31 and IS711. It showed the specific banding pattern of B. inopinata in conventional Bruce-ladder multiplex PCR and also had identical 16S rRNA and recA gene sequences as B. inopinata. Subsequent genome sequencing followed by core genome-based MLST (cgMLST) analysis using 2704 targets (74% of the total chromosome) revealed only 173 allelic differences compared to the type strain of B. inopinata BO1T, while previously considered the closest related strain BO2 differed in 2046 alleles. The overall average nucleotide identity (ANI) between the type strain BO1T and FO700622 was 99,89%, confirming that both strains were almost identical. In silico MLST-21 and MLVA-16 also identified strain FO700662 as B. inopinata. The nucleotide and amino acid-based phylogenetic reconstruction and comparative genome analysis again placed the isolate together with B. inopinata with 100% support. In conclusion, our data unequivocally classified strain FO700622, isolated from an exotic frog, as belonging to B. inopinata.

The Retrospective on Atypical Brucella Species Leads to Novel Definitions.

The genus Brucella currently comprises twelve species of facultative intracellular bacteria with variable zoonotic potential. Six of them have been considered as classical, causing brucellosis in terrestrial mammalian hosts, with two species originated from marine mammals. In the past fifteen years, field research as well as improved pathogen detection and typing have allowed the identification of four new species, namely Brucella microti, Brucella inopinata, Brucella papionis, Brucella vulpis, and of numerous strains, isolated from a wide range of hosts, including for the first time cold-blooded animals. While their genome sequences are still highly similar to those of classical strains, some of them are characterized by atypical phenotypes such as higher growth rate, increased resistance to acid stress, motility, and lethality in the murine infection model. In our review, we provide an overview of state-of-the-art knowledge about these novel Brucella sp., with emphasis on their phylogenetic positions in the genus, their metabolic characteristics, acid stress resistance mechanisms, and their behavior in well-established in cellulo and in vivo infection models. Comparison of phylogenetic classification and phenotypical properties between classical and novel Brucella species and strains finally lead us to propose a more adapted terminology, distinguishing between core and non-core, and typical versus atypical brucellae, respectively.

Infections ostéo-articulaires à bactéries rares ou atypiques

Microbiological identification is essential for the management of bone and joint infections. However, the detection of the pathogen is sometimes difficult. In case of a negative sample and/or depending on the context, the presence of so-called atypical bacteria is to be mentioned and looked for. This article reports the main characteristics of osteoarticular infections caused by atypical mycobacteria, Brucella sp., Pasteurella sp., Bartonella henselae and Kingella kingae. Tuberculosis infections and Whipple's disease will be discussed in other articles in the journal.

Atypical Brucella inopinata–Like Species in 2 Marine Toads

We describe the isolation of atypical Brucella inopinata–like species and unique clinicopathologic findings in 2 adult marine toads (Rhinella marina), including oophoritis in 1 toad. These findings represent a novel emerging disease in toads and a possible zoonotic pathogen.

First Case of Brucellosis Caused by an Amphibian-type Brucella.

We report the first case of brucellosis caused by an isolate whose genome is identical that of a frog isolate from Texas, demonstrating the zoonotic potential of amphibian-type Brucella. Importantly, with such atypical Brucella, correct diagnosis cannot be performed using routine serological tests or identification methods.

Application of Whole Genome Sequencing and Pan-Family Multi-Locus Sequence Analysis to Characterize Relationships Within the Family Brucellaceae.

The bacterial family Brucellaceae is currently composed of seven genera, including species of the genus Brucella, a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenic Brucella spp. may be hindered by their close phenotypic similarity to other members of the Brucellaceae, particularly of the genus Ochrobactrum. Additionally, a number of novel atypical Brucella taxa have recently been identified, which exhibit greater genetic diversity than observed within the previously described species, and which share genomic features with organisms outside of the genus. Furthermore, previous work has indicated that the genus Ochrobactrum is polyphyletic, raising further questions regarding the relationship between the genus Brucella and wider Brucellaceae. We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel of Brucellaceae type strains, in order to characterize relationships within the family. Phylogenies based on WGS core genome alignments were able to resolve phylogenetic relationships of 31 non-Brucella spp. type strains from within the family, alongside type strains of twelve Brucella species. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypical Brucella isolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genus Ochrobactrum and wider family. Both WGS and MLSA methods closely grouped Brucella spp. with a sub-set of Ochrobactrum species. However, results also confirmed that the genus Ochrobactrum is polyphyletic, with seven species forming a separate grouping. The pan-family MLSA scheme was subsequently applied to a panel of 50 field strains of the family Brucellaceae, isolated from a wide variety of sources. This analysis confirmed the utility of the pan-Brucellaceae MLSA scheme in placing field isolates in relation to recognized type strains. However, a significant number of these isolates did not cluster with currently identified type strains, suggesting the existence of additional taxonomic diversity within some members of the Brucellaceae. The WGS and pan-family MLSA approaches applied here provide valuable tools for resolving the identity and phylogenetic relationships of isolates from an expanding bacterial family containing a number of important pathogens.

Brucella microti-like prevalence in French farms producing frogs.

In the last 10years, many atypical novel members of Brucella species have been reported, including several Brucella inopinata-like strains in wild-caught and "exotic" amphibians from various continents. In 2017, a strain of Brucella was isolated for the first time in animals from a French farm producing frogs-Pelophylax ridibundus-for human consumption and identified as B. microti-like. Following this first isolation, investigations were performed in this farm as well as in the farm of the research unit that provided the domestic frog strain to estimate the prevalence of B. microti-like infection and its presence in the surrounding environment. Farming practices were investigated and samples including frogs at different development stages, surface tank swabs, water, feed and soil were analysed by real-time PCR and bacteriological methods. High B. microti-like prevalence values (higher than 90%) were obtained in frog samples in the commercial farm, and its presence was highlighted in the environmental samples except feed. In the research unit farm, B. microti-like species was also isolated and detected in frog and environmental samples. These results show that B. microti-like organisms are able to colonize amphibians and persist in their environment. Its presence could constitute a possible risk for consumers and workers proving the importance of assessing the zoonotic and pathogenic potentials of these new and atypical Brucella species.

A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae.

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.

The New Strains Brucella inopinata BO1 and Brucella Species 83-210 Behave Biologically Like Classic Infectious Brucella Species and Cause Death in Murine Models of Infection

Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species.

Natural IS711 insertion causing omp31 gene inactivation in Brucella ovis

The present report describes an atypical Brucella ovis strain (Bo10) isolated from the epididymis and testis of an infected ram. Macroscopic and microscopic lesions characteristic for the infection, including positive Brucella immunostaining, were observed within lesions in the genital organs. Compared to other isolates, strain Bo10 required an additional day (a total of 96 hr) of incubation to form visible colonies, showed a distinct carbon source utilization profile, agglutinated only weakly with rough (R) serum, but showed a high capacity for autoagglutination. Isolate Bo10 failed to produce the 1,071-bp fragment in the outer membrane protein (omp) 31 gene-based part of the "Bruce-ladder" multiplex polymerase chain reaction system but did produce a 1,915-bp amplicon, thus presenting a profile similar to Brucella abortus. Sequence analysis of the 1,915-bp fragment revealed an 842-bp long insertion sequence (IS)711 transposon element inserted into the promoter region of the omp31 gene, immediately upstream from the ribosome binding site (-10 box/Pribnow box). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a whole-cell lysate showed the absence in Bo10 of the approximately 31-kDa protein fragment associated with omp31. The results demonstrate a natural inactivation of omp31 and, consequently, the absence of the Omp31 protein in this B. ovis isolate. The novel location of IS711 within the genome of a naturally occurring B. ovis strain supports the hypothesis that IS711 could be an active transposon in this Brucella species.

A potential novel Brucella species isolated from mandibular lymph nodes of red foxes in Austria

The wild red fox ( Vulpes vulpes) is a known indicator species for natural foci of brucellosis. Here, we describe phenotypic and molecular characteristics of two atypical Brucella strains isolated from two foxes hunted 2008 in Eastern Austria. Both strains agglutinated with monospecific anti- Brucella A serum and were positive in ELISA with monoclonal antibodies directed against various Brucella lipopolysaccharide epitopes. However, negative nitrate reductase- and negative oxidase-reaction were atypical traits. Affiliation to the genus Brucella was confirmed by 16S rRNA gene sequencing and by detection of the Brucella specific insertion element IS 711 and gene bcsp31 using real-time PCR. Both fox strains showed identical IS 711 Southern blot profiles but were distinct from known brucellae. The number of IS 711 copies detected was as high as found in B. ovis or marine mammal Brucella strains. Molecular analyses of the recA and omp2a/b genes suggest that both strains possibly represent a novel Brucella species.

Atypical isolates of Brucella abortus from Canada and the United States characterized as dye sensitive with M antigen dominant

A total of 41 Brucella isolates, examined by standard biotyping procedures, were found to be similar to Brucella abortus biovar 2 in dye sensitivity but had a dominant M antigen. Oxidative metabolic tests performed on 39 of the isolates confirmed them as B. abortus. Additional biochemical and bacteriophage susceptibility studies were performed on 35 of the isolates. The isolates had identical reactions in the various tests, except for one isolate which was resistant to lysis by all phage strains used. Two isolates were injected into guinea pigs and shown to be virulent. The isolates described in this study appear similar to atypical Brucella isolates previously reported in the United Kingdom and the United States and may form the basis of a new biovar, B. abortus biovar 10.

Properties of a cell-wall-defective variant of Brucella abortus of bovine origin.

The properties of an atypical Brucella strain isolated from lymph node tissue of a cow slaughtered as a brucellosis reactor were examined. The organism was Gram negative and highly pleomorphic, existing as cocci, coccobacilli, rods, branched and irregular forms which stained with fluorescent antibody conjugates prepared against rough and smooth Brucella abortus strains. It produced lecithinase and required at least 15% v/v equine or bovine serum for growth. It did not need supplementary CO2 for growth, produced H2S and was inhibited by brucella dyes and partially by i-erythritol. Growth inhibition or lysis was produced by brucella-phages. The organism was not pathogenic for guinea-pigs or mice but evoked antibodies mainly to rough Brucella antigens.