Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host

Brucella species are Gram-negative bacteria which belong to α-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified. © 2006 Elsevier Masson SAS. All rights reserved

Evaluating the virulence of a Brucella melitensis hemagglutinin gene in the caprine model

With the completion of the genomic sequence of Brucella melitensis 16M, a putative hemagglutinin gene was identified which is present in 16M and absent in Brucella abortus. The possibility of this hemagglutinin being a potential virulence factor was evaluated via gene replacement in B. melitensis yielding 16MΔE and expression in trans in B. abortus 2308-QAE. Utilizing the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate these strains. B. melitensis 16M hemagglutinin gene expression in trans in 2308-QAE revealed a significant (p≤ 0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking B. melitensis 16M virulence in pregnant goats. The B. melitensis disruption mutant\u27s colonization and abortion rates demonstrated no attenuation in colonization but displayed a 28% reduction in abortions when compared to parental B. melitensis 16M. © 2010 Elsevier Ltd

Characterization of a putative hemagglutinin gene in the caprine model for brucellosis

With the completion of the genomic sequences of Brucella melitensis 16M and B. abortus 2308 and the vaccine strain RB51, a putative hemagglutinin gene was identified that is present in 16M and absent in B. abortus. The possibility of this hemagglutinin being a potential host specificity factor was evaluated via expression in trans in B. abortus 2308-QAE and RB51-QAE. Using the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate the strains. © 2010 Springer Science+Business Media B.V

Tick-Borne Encephalitis Virus, Coxiella burnetii & Brucella spp. in Milk, Kazakhstan

Raw milk was collected from cows in western Kazakhstan in winter 2014-2015. Samples were defatted and frozen at -20C, then tested as follows. For tick-borne encephalitis virus, 65 samples were tested using the VectorBest TBEV antigen capture kit, with 9% positive. For Coxiella burnetii, 50 samples were assayed using a species-specific qPCR assay and all were negative, though positive controls were consistently positive. For Brucella spp., PCR, ELISA and FPA testing is ongoing, with some positive results. These data suggest that consumption of raw cow's milk in western Kazakhstan is a risk factor for tick-borne encephalitis and brucellosis. The risk for Q fever appears to be small during winter, but may be present at other times of the year

Tick-Borne Encephalitis Virus, Coxiella burnetii & Brucella spp. in Milk, Kazakhstan

Raw milk was collected from cows in western Kazakhstan in winter 2014-2015. Samples were defatted and frozen at -20C, then tested as follows. For tick-borne encephalitis virus, 65 samples were tested using the VectorBest TBEV antigen capture kit, with 9% positive. For Coxiella burnetii, 50 samples were assayed using a species-specific qPCR assay and all were negative, though positive controls were consistently positive. For Brucella spp., PCR, ELISA and FPA testing is ongoing, with some positive results. These data suggest that consumption of raw cow's milk in western Kazakhstan is a risk factor for tick-borne encephalitis and brucellosis. The risk for Q fever appears to be small during winter, but may be present at other times of the year

Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle

PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis. This report describes the results of studies conducted with this mutant in the natural bovine host. Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1x1010 colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1. At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308. In a second experiment, eight heifers in mid-gestation were infected with 1x107CFU of either strain via the conjunctival sac. The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves. Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain. These studies demonstrate that the B. abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308. Copyright (C) 2000 Elsevier Science B.V

\u3ci\u3eBrucella\u3c/i\u3e Species Survey in Polar Bears (\u3ci\u3eUrsus maritimus\u3c/i\u3e) of Northern Alaska

We report on the presence of specific antibodies to Brucella spp. and Yersinia enterocolitica in polar bears (Ursus maritimus) from northern Alaska (southern Beaufort Sea) during 2003–2006. Based on numerous known stressors (e.g., climate change and loss of sea ice habitat, contaminants), there is increased concern regarding the status of polar bears. Considering these changes, it is important to assess exposure to potentially pathogenic organisms and to improve understanding of transmission pathways. Brucella or specific antibodies to Brucella spp. has been reported in marine mammals. Various assays were used to elucidate the pathway or source of exposure (e.g., “marine” vs. “terrestrial” Brucella spp.) of northern Alaska polar bears to Brucella spp. The standard plate test (SPT) and the buffered Brucella antigen card test (BBA) were used for initial screening for antibodies specific to Brucella. We then evaluated positive reactors (presence of serum antibody specific for Brucella spp.) using immunoblots and competitive enzyme-linked immunosorbent assay (cELISA; based on pinniped-derived Brucella spp. antigen). Annual prevalence of antibody (BBA and SPT) for Brucella spp. ranged from 6.8% to 18.5% over 2003–2006, with an overall prevalence of 10.2%. Prevalence of Brucella spp. antibody did vary by age class. Western blot analyses indicated 17 samples were positive for Brucella spp. antibody; of these, 13 were negative by marine (pinniped) derived Brucella antigen cELISA and four were positive by marine cELISA. Of the four samples positive for Brucella antibody by marine cELISA, three cross-reacted with Y. enterocolitica and Brucella spp. (one sample was Brucella negative and Y. enterocolitica positive). It appears the polar bear antibody does not react with the antigens used on the marine cELISA assay, potentially indicating a terrestrial (nonpinniped) source of Brucella spp

Brucella species survey in polar bears (ursus maritimus) of northern Alaska

We report on the presence of specific antibodies to Brucella spp. and Yersinia enterocolitica in polar bears (Ursus maritimus) from northern Alaska (southern Beaufort Sea) during 2003-2006. Based on numerous known stressors (e.g., climate change and loss of sea ice habitat, contaminants), there is increased concern regarding the status of polar bears. Considering these changes, it is important to assess exposure to potentially pathogenic organisms and to improve understanding of transmission pathways. Brucella or specific antibodies to Brucella spp. has been reported in marine mammals. Various assays were used to elucidate the pathway or source of exposure (e.g., marine vs. terrestrial Brucella spp.) of northern Alaska polar bears to Brucella spp. The standard plate test (SPT) and the buffered Brucella antigen card test (BBA) were used for initial screening for antibodies specific to Brucella. We then evaluated positive reactors (presence of serum antibody specific for Brucella spp.) using immunoblots and competitive enzyme-linked immunosorbent assay (cELISA; based on pinniped-derived Brucella spp. antigen). Annual prevalence of antibody (BBA and SPT) for Brucella spp. ranged from 6.8% to 18.5% over 2003-2006, with an overall prevalence of 10.2%. Prevalence of Brucella spp. antibody did vary by age class. Western blot analyses indicated 17 samples were positive for Brucella- spp. antibody; of these, 13 were negative by marine (pinniped) derived Brucella antigen cELISA and four were positive by marine cELISA. Of the four samples positive for Brucella antibody by marine cELISA, three cross-reacted with Y. enterocolitica and Brucella spp. (one sample was Brucella, negative and Y. enterocolitica positive). It appears the polar bear antibody does not react with the antigens used on the marine cELISA assay, potentially indicating a terrestrial (nonpinniped) source of Brucella spp. © Wildlife Disease Association 2010

Novel Spiroplasma Spp. cultured from brains and lymph nodes from ruminants affected with transmissible spongiform encephalopathy

© 2017 American Association of Neuropathologists, Inc. Spiroplasma spp., tiny filterable wall-less bacteria, are consistently associated with the transmissible spongiform encephalopathies (TSE). Spiral forms have been transiently isolated from TSE-affected brain tissues in SP4 growth media designed for isolation of Spiroplasma spp., but the isolate could not be propagated in SP4 media. A bacterium must grow in vitro in cell-free cultures to allow full characterization of a suspect pathogen. Here, a novel Spiroplasma sp. was isolated from scrapie- and chronic wasting disease (CWD)-affected brains and lymph nodes. Filtrates of tissue homogenates inoculated into Brucella media incubated for 14 days at 35 °C resulted in high titers of spiroplasma as shown by darkfield microscopy. A drop assay of infected media on Bacto Schaedler agar showed spiroplasma isolates forming unique subsurface colonies after 21 days incubation. Spiroplasma coils, coccoid forms and clumps of entwined spiroplasma filaments were seen on the agar by scanning electron microscopy. Since Brucella media has a sodium bisulfite additive that lowers oxygen tension, TSE spiroplasma growth requires media with low oxygen tension. Brucella media allows for isolation and propagation of spiroplasma from TSE-affected tissues, which will lead to complete characterization of this TSE pathogen and determine its role as a candidate causative agent of TSE