Since August 2017, CDC has confirmed three cases of brucellosis attributed to Brucella abortus cattle vaccine strain RB51 (RB51). Each case was associated with consumption of domestically acquired unpasteurized (raw) milk products (1). Patient symptoms varied and included fever, headache, overall malaise, and respiratory symptoms. In total, at least eight persons met the probable case definition of a clinically compatible illness epidemiologically linked to a shared contaminated source (2). In addition, hundreds of persons, from dozens of states, were potentially exposed to the contaminated raw milk products (3).
A number of nucleic acid sequences have been targeted for the development of Brucella genus-specific PCR assays, including 16S rRNA, the 16S-23S intergenic spacer region, omp2, and bcsp31 (1, 9, 11, 12). The most frequently described PCR target for the diagnosis of human brucellosis is the bcsp31 gene encoding a 31-kDa antigen conserved among Brucella spp. (8, 9, 15). PCR identification of Brucella strains at the species or biovar level has been more challenging. Recently, Redkar et al. (10) described real-time PCR assays for the detection of B. abortus, B. melitensis, and B. suis biovar 1. These PCR assays target the specific integration of IS711 elements within the genome of the respective Brucella species or biovar. The assays, however, were designed to be tested in separate PCRs. Using similar PCR targets, but in a multiplex format, we have developed a real-time triplex assay that permits rapid confirmation of Brucella spp., B. abortus, and B. melitensis isolates in a single test.
The detection limit of each primer-probe set in the multiplex format was also tested (data not shown). Serial 10-fold dilutions of a known amount of B. abortus or B. melitensis purified DNA were tested in triplicate using the multiplex format. The detection limit for each set of primers and probe was 150 fg of purified DNA. No fluorescence was detected at the level of 15 fg of purified DNA. The analytical sensitivity of this assay suggests its usefulness for the direct detection of Brucella in clinical specimens.
Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.
A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.
Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-β, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-β is also important for the UPR induction during B. abortus infection. Indeed, IFN-β shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-β favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-β signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.
Chlamydia abortus (C. abortus) is a non-motile obligate intracellular Gram-negative pathogenic bacterium, belonging to the Chlamydiales family. C. abortus infects mainly ruminants, especially sheep and goats, and less frequently cattle, pigs and horses; however, it can also affect humans, being of particular concern in pregnant women [1, 2]. C. abortus is known as the causative agent of enzootic abortion of ewes (EAE) or ovine enzootic abortion (OEA) which represents one of the most common causes of ovine and caprine infectious abortion worldwide, along with other infectious agents such as Campylobacter sp, Toxoplasma sp, Listeria sp, Salmonella sp, Border disease virus and Cache Valley virus [3, 4]. Abortion occurs in the later stages of pregnancy, as C. abortus is able to progressively colonize the placenta, causing damage and affecting the fetus(es) to varies degrees . The infection can result in foetal loss (abortion), the birth of stillborn or weak lambs or, in some cases of unaffected animals; presence of a weak lamb with a healthy tween is also not uncommon . Breeders can incur great economic losses if numerous cases occur in a farm (abortion storm), usually when the infection first affects a naïve flock . Another important aspect is the spread of this enzootic infection to humans, which can develop as severe disease, especially in pregnant women , generally affecting female farmers, abattoir workers and veterinarians. However, environmental contamination with the bacteria released by abortion products or infected animals may also play a crucial role in disease spread, interspecies cross-over and adaptation . Indeed, abortion products, in particular vaginal fluids, placentas, dead/aborted lambs, fleeces and still born/infected lambs are all characterized by a high bacterial load and represent a significant risk, both for naïve animals and for humans . Different types of flock management are also involved in the extent of environmental contamination and the spread of the pathogen: in intensively managed flocks, where the animals are kept in smaller enclosures, there is a higher incidence of C. abortus, as the environmental contamination is concentrated in small spaces; conversely in extensively managed flocks, where animals are kept within larger areas, a lower incidence of the pathogen is observed, linked to the fact that animals are less likely to come into contact with a contaminated area . In addition, C. abortus can survive in the environment even in unfavourable conditions from a few days to a few months, thanks to the presence of a spore-like cell wall, which gives it considerable resilience . This resistance seems to be directly connected to the greater possibility for the bacterium to come into contact and infect many animal species, farmed or wild ones, and consequently to spread more easily to humans . Specific aspects of this will be discussed later.
The specific interactions between C. abortus and its respective hosts have been investigated for some but not all its host species; however, some of the general mechanisms can be extrapolated from work carried out on other Chlamydia species .
RBs can also enter a state of quiescence (persistent state) if exposed to critical environmental conditions, for example the depletion of essential growth substances, or the presence of antibiotics. C. abortus, lacking the tryptophan biosynthetic operon (trp), enters into a persistent state if in presence of IFN-γ: this cytokine induces the expression of indoleamine-2,3-dioxygenase (IDO), which in turns degrades the host tryptophan, necessary for bacterial growth. Although C. abortus lacks the tryptophan biosynthetic pathway, it can still grow and multiply into the placenta probably through the rescue of tryptophan from maternal-to-foetal nutrients . IDO is known to have multiple functions related to protective immunity (IFN-γ linked), tolerance (by suppression of T cells) and physiology, therefore the animal physiological, nutritional or immune status could influence induction of persistence [20, 21]. 041b061a72