Expansion of brucellosis detection in the country of Georgia by screening household members of cases and neighboring community members.

BACKGROUND: Brucellosis is considered as endemic zoonotic disease in the country of Georgia. However, the burden of the disease on a household level is not known. Therefore, this study sought to determine the benefits of active surveillance coupled to serological screening for the early detection of brucellosis among close contacts of brucellosis cases. METHODS: We used an active surveillance approach to estimate the rate of seropositivity among household family members and neighboring community members of brucellosis index cases. All participants were screened using the serum tube agglutination test (SAT). Blood cultures were performed, obtained isolates were identified by a bacteriological algorithm, and confirmed as Brucella spp. using real-time PCR. Further confirmation of Brucella species was done using the AMOS PCR assay. RESULTS: A total of 141 participants enrolled. Of these, 27 were brucellosis index cases, 86 were household family members, and 28 were neighboring community members. The serological evidence of brucellosis in the household member group was 7% and the rate at the household level was 21%. No screened community members were Brucella seropositive. Majority of brucellosis cases were caused by B. melitensis; only one index case was linked to B. abortus. CONCLUSION: We found evidence of brucellosis infection among household family members of brucellosis index cases. B. melitensis was the most common species obtained. Findings of this active surveillance study highlight the importance of screening household family members of brucellosis cases and of the use of culture methods to identify Brucella species in the country of Georgia.

Spiroplasma spp. biofilm formation is instrumental for their role in the pathogenesis of plant, insect and animal diseases.

Spiroplasma spp. are important phyto and insect pathogens, and candidate causal agent/s of transmissible spongiform encephalopathies (TSE) in man and animals. These filterable wall-less bacteria are widely distributed in nature with an unspecified environmental reservoir. In this study we showed by scanning electron microscopy that spiroplasma form biofilm on an assortment of hard surfaces including mica, nickel and stainless steel. Spiroplasma were stuck to the surfaces by fibrillar threads consistent with curli fibers (an amyloid protein found in bacterial biofilms). After a lengthy time in cultures (6 weeks), spiroplasma in biofilm bound to mica disks lost their spiral shapes and formed coccoid forms interconnected by long (>2 µm) branched membranous nanotubules, therein representing direct conjugate connections between the cells. The affinity of spiroplasma biofilms for mica and nickel, and the membrane communications suggest that soil could be a reservoir for these bacteria. The persistence of clay bound spiroplasma in soil could serve as the mechanism of lateral spread of TSEs by ingestion of soil by ruminants. Spiroplasma binding to stainless steel wire supports bacterial contamination of surgical instruments following surgery on dementia patients as a mechanism of iatrogenic transmission of TSEs, especially with resistance of spiroplasma in biofilms to drying or exposure to 50% glutaraldehyde. The discovery of biofilm formation by spiroplasma addresses questions regarding environmental persistence of these organisms in nature and suggests novel mechanisms of intercellular communication and transmission.

B cell-deficient mice display markedly enhanced resistance to the intracellular bacterium Brucella abortus.

BACKGROUND: Brucella species are facultative intracellular bacteria that cause lifelong infections in humans and livestock. METHODS: Here we evaluated the contribution of B cells in control of murine brucellosis in the more susceptible BALB/c and the more resistant C57BL/6 mice by infecting B cell-deficient mice. RESULTS: Strikingly, in the absence of B cells in both C57BL/6 and BALB/c mice, 99% and 99.5% of the infection found in wild type mice was cleared, respectively. This augmented clearance was not reversed in either strain by passive transfer of immune serum. In C57BL/6 mice, the clearance of infection coincided with an increase in interferon γ (IFN-γ)-producing CD4 and CD8 T cells and a reduction in interleukin 10 (IL-10)-producing cells. In BALB/c mice, this clearance was IFN-γ-dependent, as B cell/IFN-γ dual knockout mice were unable to clear the infection, and was inversely related to the levels of transforming growth factor ß (TGF-ß). Furthermore, B cells were found to produce TGF-ß and IL-10 during early stages of infection in BALB/c wild-type and C57BL/6 wild-type mice, respectively. CONCLUSIONS: Thus, we demonstrate that the establishment of the high plateau phase of infection is dependent on non-antibody-mediated B cell effector mechanisms, including B regulatory functions, during murine brucellosis.

Spiroplasma found in the eyes of scrapie affected sheep.

OBJECTIVE: Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model. PROCEDURE: The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE). RESULTS: The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie-affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA. CONCLUSION: These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.

Spatio-temporal thermal kinetics of in situ MWCNT heating in biological tissues under NIR laser irradiation.

Carbon nanotubes have many potential applications in life sciences and engineering as they have very high absorbance in the near-infrared (NIR) spectrum, while biological tissues do not. The purpose of this study was to determine the effect of 1064 nm NIR laser power levels on the spatial temperature distribution and the temperature kinetics in mammalian tissue at both macroscopic and microscopic scales. The model tissue was the 'flat' of a chicken wing (the section containing the radius and ulna), which was injected under the skin in the subcutaneous layer of tissue. Specimens were exposed to laser radiation and an infrared thermography system was used to measure and record the temperature distributions in the specimens at both the macroscopic and microscopic scales. Experimental results concluded that power levels of 1536 mW easily achieved hyperthermic temperatures with localized values as high as 172.7 °C.

Enhanced immune response of red deer (Cervus elaphus) to live rb51 vaccine strain using composite microspheres.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. The occurrence of the infection in humans is largely dependent on the prevalence of brucellosis in animal reservoirs, including wildlife. The current vaccine used for cattle Brucella abortus strain RB51, has proven ineffective in protecting bison (Bison bison) and elk (Cervus nelsoni) from infection and abortion. To test possible improvements in vaccine efficacy, a novel approach of immunization was examined from April 2004 to November 2006 using alginate composite microspheres containing a nonimmunogenic, eggshell-precursor protein of the parasite Fasciola hepatica (Vitelline protein B, VpB) to deliver live vaccine strain RB51. Red deer (Cervus elaphus), used as a model for elk, were vaccinated orally (PO) or subcutaneously (SC) with 1.5x10(10) viable organisms per animal. Humoral responses postvaccination (immunoglobulin G [IgG] levels), assessed at different time points, indicated that capsules containing live RB51 elicited an anti-Brucella specific IgG response. Furthermore, the encapsulated vaccine elicited a cell-mediated response that the nonencapsulated vaccinates failed to produce. Finally, red deer were challenged with B. abortus strain 19 by conjunctival exposure. Only animals that received encapsulated RB51 vaccine by either route exhibited a significant reduction in bacterial counts in their spleens. These data suggest that alginate-VpB microspheres provide a method to enhance the RB51 vaccine performance in elk.

Novel Spiroplasma Spp. Cultured From Brains and Lymph Nodes From Ruminants Affected With Transmissible Spongiform Encephalopathy.

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 dark-field 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.

Spiroplasma spp. from transmissible spongiform encephalopathy brains or ticks induce spongiform encephalopathy in ruminants.

Spiroplasma, small motile wall-less bacteria, are linked by molecular and serological studies to the transmissible spongiform encephalopathies (TSEs), which include scrapie in sheep, chronic wasting disease (CWD) in deer and Creutzfeldt-Jakob disease in humans. In this study, two experiments were undertaken to determine the role of spiroplasma in the pathogenesis of TSE. In experiment 1, Spiroplasma mirum, a rabbit tick isolate that had previously been shown to experimentally induce spongiform encephalopathy in rodents, was inoculated intracranially (IC) into ruminants. S. mirum-inoculated deer manifested clinical signs of TSE after 1.5 to 5.5 months incubation. The deer, as well as sheep and goats, inoculated with S. mirum developed spongiform encephalopathy in a dose-dependent manner. In experiment 2, spiroplasma closely related to S. mirum were isolated from TSE-affected brains via passage in embryonated eggs, and propagated in cell-free M1D media. Spiroplasma spp. isolates from scrapie-affected sheep brain and from CWD-affected deer brain inoculated IC into sheep and goats induced spongiform encephalopathy closely resembling natural TSE in these animals. These data show spiroplasma to be consistently associated with TSE, and able experimentally to cause TSE in ruminant animal models, therein questioning the validity of studies that have concluded the prion, a miss-folded protease-resistant protein that builds up in TSE brains during the course of the disease, to be the sole causal agent. The spiroplasma infection models reported here will be important for investigating factors involved in the pathogenesis of TSE since ruminants are the natural hosts.

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

Brucella species are gram-negative bacteria which belong to alpha-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.

Evaluation of Brucella abortus strain RB51 and strain 19 in pronghorn antelope.

Free-roaming elk and bison in the Greater Yellowstone Area remain the only wildlife reservoirs for Brucella abortus in the United States, and the large number of animals and a lack of holding facilities make it unreasonable to individually vaccinate each animal. Therefore, oral delivery is being proposed as a possible option to vaccinate these wild ungulates. One of the main problems associated with oral vaccination is the potential exposure of nontarget species to the vaccines. The purpose of this study was to determine the effects of two Brucella vaccines, strain 19 (S19) and the rough strain RB51 (SRB51), in pregnant pronghorn antelope. We conclude that S19 and SRB51 rarely colonize maternal and fetal tissues of pregnant pronghorn and were not associated with fetal death. Oral delivery of either vaccine at this dose appears to be nonhazardous to pregnant pronghorn.