Determination and Analysis of Actinomyces israelii Serotypes by Fluorescent-Antibody Procedures

This study was undertaken to determine the antigenic relationships between serotypes of Actinomyces israelii with fluorescent-antibody (FA) procedures. In addition, the antigenic relationships between A. israelii and other members of the genus Actinomyces were studied by the same methods. Seven FA conjugates were used to determine the serological characteristics of 28 isolates believed to represent A. israelii, serotypes 1 and 2. The results showed that the lower dilutions of serotype 1 conjugates stained serotype 2 antigens; however, serotype 2 conjugates did not stain serotype 1 antigens. Serotype 1 conjugate could be made specific by adsorption. A. israelii serotype 1 conjugate cross-reacted also with A. naeslundii, but this cross-reaction could be eliminated by adsorption or dilution. Serotype 2 conjugate appeared to be specific for A. israelii serotype 2. Adsorption studies revealed antigenic variants among the various A. israelii serotype 1 and 2 isolates. However, all isolates could be identified by direct FA staining with appropriate conjugates. One isolate previously identified as A. israelii was shown, on the basis of FA studies, to be an A. naeslundii. A polyvalent diagnostic reagent was prepared which was specific for A. israelii serotypes 1 and 2. This reagent should find application in diagnostic and reference laboratories.     

Characterization of monoclonal antibodies for rapid identification of Actinomyces naeslundii in clinical samples

The purpose of this study was to generate highly specific serological reagents for the quantitative identification of Actinomyces naeslundii in clinical samples, in particular dental plaque. Balb/c mice were immunized with pasteurized human A. naeslundii strains representing different genospecies and serotypes. Ten hybrid cell lines secreting monoclonal antibodies reactive with A. naeslundii were isolated and characterized. Antibody specificity was determined by indirect immunofluorescence and enzyme-linked immunosorbent assay using strains from 59 species and by immunofluorescence analyses of supragingival plaque from 10 gingivitis patients. Nine monoclonal antibodies reacted selectively with A. naeslundii, whereas one additionally bound to Actinomyces israelii. They recognized at least nine different epitopes with characteristic expression patterns among the test strains. Six clusters of antigenically unique or closely related strains could be distinguished. Clusters 1, 4, and 5 represented by 12, 18, and 5 strains, respectively, comprised over 80% of the A. naeslundii strains tested. All reference strains for genospecies 1 grouped with cluster 1. Strains associated with genospecies 2 fell into clusters 4 and 5. Tests with mutant strains indicated that three monoclonal antibodies recognize type 2 and one type 1 fimbriae of genospecies 2. Only four isolates grouped with clusters 2 and 3 characterized by the expression of cluster-specific antigens. Interestingly, cluster 2 and 3 bacteria were markedly more abundant in vivo than indicated by their sparse representation in our strain collection. Overall, all but one of the new monoclonal antibodies should prove of value for the serological classification and rapid quantitative determination of A. naeslundii in clinical samples.     

Application of MLST and pilus gene sequence comparisons to investigate the population structures of Actinomyces naeslundii and Actinomyces oris

Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n = 37) and A. oris (n = 68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established.     

Effect of the Environment on Genotypic Diversity of Actinomyces naeslundii and Streptococcus oralis in the Oral Biofilm

The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR. The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A. naeslundii organisms than the plaque sampled from the caries-free subjects. These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values. We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable. We found that the diversity of A. naeslundii strains did not change (χ2 = 0.68; P = 0.41) although the proportional representation of A. naeslundii was significantly reduced (P < 0.05). Conversely, the diversity of the S. oralis strains increased (χ2 = 11.71; P = 0.0006) and the proportional representation of S. oralis did not change. We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S. oralis increased, resulting in the increased genotypic diversity of this species. Apparently, A. naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation. These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community.     

Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.     

Quantitative Analysis of Actinomyces Cell Walls

Quantitative data on the amino acid composition of cell walls of five species of Actinomyces were obtained by using a Beckman-Spinco amino acid analyzer. The major amino acids in A. israelii, A. naeslundii, A. eriksonii, and A. bovis species included alanine, glutamic acid, lysine, aspartic acid, and ornithine, as reported by previous workers, whereas A. propionicus contained diaminopimelic acid. Other amino acids, including glycine, valine, leucine, proline, isoleucine, and threonine, were present in at least some of the walls in quantities equal to or slightly less than that of lysine. This raised the question of whether these may represent cross-links in the peptidoglycan or other cell wall structural components or whether the wall preparations contained nonpeptidoglycan material despite the use of electron microscopy as a standard of purity; further work is required to supply the answer. The quantitative data furnish relative molar concentrations of amino acids, which can provide definitive identification of some of the species and differentiation of Actinomyces from other members of the Actinomycetales and from morphologically similar genera such as Corynebacterium and Propionibacterium.     

Mixed infection involving Actinomyces,Aggregatibacter, and Fusobacterium species presenting as perispinal tumor

A representative case in which a polymicrobial infection involving Fusobacterium nucleatum, Actinomyces israelii and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans was initially diagnosed as malignancy in an edentulous patient. Additional history obtained after the nature of the syndrome was elucidated revealed that he had had his two remaining teeth extracted four months prior to this episode.     

Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.     

Phenotypic and Genotypic Selection of Microbiota Surviving under Dental Restorations

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, β-N-acetylglucosaminidase, and β-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P = 0.002 and P = 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.     

Effect of saliva viscosity on the co-aggregation between oral streptococci and Actinomyces naeslundii

doi: 10.1111/j.1741‐2358.2011.00595.x Effect of saliva viscosity on the co‐aggregation between oral streptococci and Actinomyces naeslundii Background: The co‐aggregation of oral bacteria leads to their clearance from the oral cavity. Poor oral hygiene and high saliva viscosity are common amongst the elderly; thus, they frequently suffer from pneumonia caused by the aspiration of oral microorganisms. Objectives: To examine the direct effect of saliva viscosity on the co‐aggregation of oral streptococci with actinomyces. Materials and methods: Fifteen oral streptococcal and a single actinomyces strain were used. Co‐aggregation was assessed by a visual assay in phosphate buffer and a spectrophotometric assay in the same buffer containing 0–60% glycerol or whole saliva. Results: Nine oral streptococci co‐aggregated with Actinomyces naeslundii ATCC12104 in the visual assay and were subsequently used for the spectrophotometric analysis. All tested strains displayed a decrease in co‐aggregation with increasing amounts of glycerol in the buffer. The co‐aggregation of Streptococcus oralis with A. naeslundii recovered to baseline level following the removal of glycerol. The per cent co‐aggregation of S. oralis with A. naeslundii was significantly correlated with the viscosity in unstimulated and stimulated whole saliva samples (correlation coefficients: ?0.52 and ?0.48, respectively). Conclusion: This study suggests that saliva viscosity affects the co‐aggregation of oral streptococci with actinomyces and that bacterial co‐aggregation decreases with increasing saliva viscosity.     

BALB/c mice infected with antimony treatment refractory isolate of Leishmania braziliensis present severe lesions due to IL-4 production

Background

Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis–infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult.

Methodology/Principal Findings

Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S).

Conclusion/Significance

We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.     

Effect of metabolic substances of oral Actinomyces on Candida albicans

Actinomyces naeslundii, Actinomyces viscosus and Candida albicans are associated with root cavity. The aim of this study was to determine, in vitro, the effect produced by the metabolic substances elaborated by Actinomyces naeslundii and Actinomyces viscosus on Candida albicans. The strains were isolated of saliva. There were used the double plaque diffusion method (DPDM) and the method of radial diffusion (MRD). The effect of the time of incubation and of different concentrations of metabolic substances elaborated by Actinomyces naeslundii and Actinomyces viscosus on the kinetics of growth of C. albicans were studied. Later, the nature of the substances produced by the two strains of Actinomyces was determined. It was found that there was no inhibition of the growth of C. albicans by A. naeslundii and A. viscosus in the DPDM and the MRD. There was stimulation of the growth of C. albicans by the two strains of Actinomyces when the DPDM was used. In the MRD the results were negative. Metabolic substances produced by both species stimulated the growth of C. albicans in low concentrations but at high concentrations inhibition was observed. The best concentration of the stimulating factor, a protein substance stable to 70 degrees C, corresponds to a dilution of 1/80. The inhibition of the growth of C. albicans was produced by the decrease of the pH, the higher effect being obtained with the dilution 1/5. The metabolic substances produced by A. naeslundii and A. viscosus can have both inhibitory and stimulant effects on C. albicans, according to their concentration. These metabolic interactions would condition the proportion of C. albicans in the oral microbial ecosystems.     

Effect of Biofilm Growth on Expression of Surface Proteins of Actinomyces naeslundii Genospecies 2

The predominant surface proteins of biofilm and planktonic Actinomyces naeslundii, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly up-regulated relative to planktonic cells.     

Experimental reproduction of the Jorge Lobo's disease in BALB/c mice inoculated with Lacazia loboi obtained from a previously infected mouse

Long-term maintenance of Lacazia loboi in the laboratory has not been reported. We report here the use BALB/c mice to maintain the Lacazia loboi for extended period of time. Eight to ten week-old mice were inoculated intradermally in both hind footpads with a fungal suspension from a macerated footpad obtained from an original mouse previously infected with the fungi and sacrificed 8 months after inoculation. The inoculated animals were sacrificed at different time intervals, footpads were excised, the right one was submitted to histopathological examination and the left one was macerated in sterile saline for fungal count and viability index determination. The inoculated animals presented the histopathological picture identical to the mice previously inoculated with material from human lesion. Granulomatous infiltrates with predominance of macrophages and giant cells were observed. The granulomas evolved progressively as observed in the different times of sacrifice. After 7 months of inoculation, macroscopic lesions were observed, and the number of fungi obtained from macerated footpads was higher than the number of inoculated fungi. The pattern of lesion development was similar to what was observed in animals infected with a fungal suspension obtained from a human lesion. Considering the histopathological findings, the clinical manifestations, and the finding of a higher number of fungi obtained than the inoculated into footpads of each mice, we believe the BALB/c mice strain is as an excellent way to maintain L. loboi in laboratory. Moreover, even after serial passages of the fungi, the granulomatous lesions are reproduced consistently in laboratory conditions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Influence of Fluid Shear and Microbubbles on Bacterial Detachment from a Surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s⁻¹) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

Assessment of crude oil degradation efficiency of newly isolated actinobacteria reveals untapped bioremediation potentials

Bioremediation is gaining favorable attention as a more economical and environmentally friendly technique for the remediation of crude oil hydrocarbons. This makes the search for crude oil–degrading microbes very crucial. In this study, the isolation and identification of actinobacteria in soil samples from a selected crude oil spill site were carried out. Eighteen isolates from different soil depths (20–120 cm) were screened for their ability to grow on crude oil–based medium (COBM). Actinomyces naeslundii, Actinomyces viscosus, Actinomyces israelii, Actinomyces meyeri, and Nocardia formicae from a 20 cm soil depth exhibited higher growth profiles on COBM than on glucose-based medium (GBM). A. viscosus and A. isrealii exhibited 5- and 3-fold increase in growth over GBM and were selected for biodegradation studies. Growth kinetics and residual crude oil were used to measure the degradation efficiency of A. viscosus and A. israeli over varying crude oil concentrations. Surprisingly, A. viscosus and A. isrealii achieved 98% degradation of 10 g/L crude oil in 12 days and 97% degradation of 30, 50, and 75 g/L in 16 and 18 days, respectively. Specific activity of total peroxidase was assayed over the biodegradation period. Peroxidase activity increased with degradation efficiency of A. viscosus and A. isrealii, suggesting that peroxidases play a key role in the crude oil biodegradation process. The unique tolerance exhibited by A. viscosus and A. israelii to crude oil and high crude oil degradation efficiencies indicate their promising potential for bioremediation applications.     

Effect of environment on staphylococcal lesions in mice

The influence of reduced barometric pressure on the development and healing of staphylococcal skin lesions in mice was investigated by exposing groups of animals to the test environment before, after, or before and after subcutaneous inoculation with 3.5 × 10⁸ colony-forming units of a phage type 80 strain of Staphylococcus aureus. Similarly infected control animals were not exposed to the experimental environment. The results indicate that the lesions which developed in mice exposed to the test environment prior to infectious challenge were larger and healed at a slower rate than those in mice maintained at ground level before infection. Exposure after inoculation produced no demonstrable effect in the size or healing rate of the experimental lesions.     

Taenia crassiceps antigens induce a Th2 immune response and attenuate injuries experimentally induced by neurotoxoplasmosis in BALB/c mice

Toxoplasma gondii is a pathogenic agent responsible for causing both systemic and local disease which elicits a typically pro-inflammatory, Th1 immune response. Taenia crassiceps antigen induces a Th2 immune response that immunomodulates Th1 based infections. Therefore the aim of this study was to evaluate whether T. crassiceps cysticerci antigens are able to modulate the inflammatory response triggered in experimental neurotoxoplasmosis (NT). BALB/c mice were inoculated with T. gondii cysts and/or cysticerci antigens and euthanized at 60 and 90 days after inoculation (DAI). The histopathology of the brains and cytokines produced by spleen cells culture were performed. The animals from the NT group, 90DAI (NT90), presented greater intensity of lesions such as vasculitis, meningitis and microgliosis and cytokines from Th1 profile characterized by high levels of IFN-gamma. While in the T. crassiceps antigens group, 60DAI, there were more discrete lesions and high levels of IL-4, a Th2 cytokine. In the NT co-inoculated with cysticerci antigens group the parenchyma lesions were more discrete with lower levels of IFN-gamma and higher levels of IL-4 when compared to NT90. Therefore the inoculation of T. crassiceps antigens attenuated the brain lesions caused by T. gondii inducing a Th2 immune response.     

Identification of Actinomyces israelii and Actinomyces naeslundii by Fluorescent-Antibody and Agar-Gel Diffusion Techniques

This study was an attempt to develop a fluorescent-antibody (FA) test to differentiate Actinomyces israelii and A. naeslundii as an aid in their laboratory identification. Two strains of A. israelii (X522 and A601) and two strains of A. naeslundii (X454 and X600), which had received intensive study by several investigators, were used for the immunization of rabbits. Working titers, based on tests with antigens prepared from the homologous strains and from well-established heterologous strains, were determined for each labeled antibody preparation. These conjugates and their normal serum control conjugates were used separately to stain 85 cultures of Actinomyes species and 23 strains of other species that might be confused with them. Acetone-precipitated soluble antigens from these same strains were tested with different antisera in the agar-gel diffusion test. Results showed that A. israelii (X522 and A601) and A. naeslundii (X454 and X600) labeled antiglobulins, when used at their working titers, stained most strains of their homologous species. Agar-gel diffusion results showed general agreement with those of the FA tests. The two tests appear to be equal in sensitivity, but the FA test is more specific, since several cross-reactions were noted with the agar-gel diffusion test whereas no cross-reactions were obtained with the FA reagents. Agar-gel and FA studies suggest that at least two serotypes of A. israelii may be associated with human disease. Although the majority of strains tested in this study appear to belong to a common serotype, "serotype 1," two strains of an apparent second serotype, "serotype 2," were encountered. FA staining of tissue impression smears from experimentally infected mice was successful when a counterstain, Evans Blue dye, was used.     

Use of a Safe,Reproducible, and Rapid Aerosol Delivery Method to Study Infection by Burkholderia pseudomallei and Burkholderia mallei in Mice

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10², 10³ and 10⁴ organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10³ and 10⁴ B. pseudomallei cells, animals infected with 10² organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those seen in human infections.