In this context, Kim and coworkers 73 demonstrated the expression

of TLR2 and TLR4 in skin samples obtained from preterm delivered babies by immunohistochemistry. As Saracatinib manufacturer for function of TLR in fetus, studies of mouse and human fetal cells show stimulation of fetal intestinal cells or fetal monocyte with LPS results in production of chemokines and cytokines.74,75 These findings indicate that fetal cells are also capable of recognizing microbial products and participate in innate immune defense in the case of microbial invasion of the amniotic cavity; although the expressions of other PRRs in various fetal tissues/organs still need to be elucidated. Recent studies from our laboratory have shown that viral infection of the mouse placenta, which does not induce preterm labor, has a detrimental effect on fetal development.59 A striking finding was the observation of a general inflammatory fetal condition, very similar to those

observed in the human condition known as fetal inflammatory response syndrome (FIRS).76 This inflammatory condition was present in the fetus in spite of undetectable buy Tanespimycin viral titers. Morphologic examination of the fetus reveled changes in the brain, heart and lungs. This data suggests that although the virus may not reach the fetus, an inflammatory process at the placenta will affect the normal development of the fetus, with potential after birth severe consequences. Recent clinical studies have linked TLRs to pregnancy disorders. In the following section, we will discuss some of the most relevant observations. Intrauterine infection and subsequent chorioaminionitis (CAM) are known to be among the most important causes of preterm delivery.1 We evaluated the expression of TLR2 and TLR4 in chorioamniotic membranes in spontaneous labor at term and in preterm parturition that are associated with CAM. TLR2 and TLR4 mRNA expression were significantly higher in membranes from women at

term with spontaneous labor than women not in labor. TLR2 MycoClean Mycoplasma Removal Kit expression in chorioamniotic membranes was significantly higher in patients with CAM than those without CAM. The expression of TLR2 was also restricted to the basal surface of amniotic epithelial cells in non-CAM preterm, labor whereas in CAM cases, diffuse and strong positive staining for the entire cytoplasm of epithelium was observed.39 On the other hand, Rindsjo et al.77 demonstrated that TLR2 expression in trophoblast was decreased in patients with CAM compared to those without CAM. These findings suggest that the response to infection varies in the different parts of the maternal–fetal interface. However, we have to take into consideration the possibility that these variations might be the result of technical variations among study groups. As for TLR4, Kumazaki et al.

e., Allen & Miller, 1999) as both were lower for /puk/ than /buk/. F0 was the only cue near significance INCB018424 supplier for distinguishing between /buk/ and /puk/. Phonetic data suggest that F0 should be lower for /b/ than /p/, and at voicing onset, /buk/’s F0 was indeed 27 msec lower than /puk/’s; this was not even marginally significant, t(106) = 1.59, p = .11. However, it seems unlikely that F0 could serve even to augment the noncontrastive variability in Experiment 3 as 28 /buk/s had F0 values less than the median, compared with 26 /puk/s. Although there was an almost marginal effect in the right direction, there were not

enough tokens showing this relationship to make F0 a worthwhile cue. Moreover, Experiment 2 ruled out that F0 in the absence of noncontrastive variability drives this effect. As a result, the cue that came closest to distinguishing the words does not appear to have much utility as a constrastive cue in this particular set of

stimuli. These experiments investigated the role of contrastive and noncontrastive phonetic variability in infants’ word learning in the switch-task procedure. Experiments 1 and 2 examined whether variability in a contrastive cue was necessary for LY2157299 chemical structure minimal-pair learning in the switch task. Our initial hypothesis was that the switch task requires children to determine that a given exemplar is not a member of the /buk/ (or /puk/) category, and as a result, some estimate of the extent of a category along the contrastive dimension may be needed to make this determination. However, this was not the case: across both experiments there was no evidence for learning, even when three cues to voicing varied simultaneously. Indirectly, this provides evidence that the kind of statistical learning first reported by Maye et al. (2002, 2008) (see also Kuhl et al., 2007; McMurray et al., 2009; Vallabha et al., 2007) can not account why for learning in Rost and McMurray (2009) as variability along the contrastive dimension of voicing alone is not sufficient to support learning. We do not

argue that infants ignore variability along dimensions, such as VOT. Indeed, it is likely to be important in establishing the location of categories within a dimension. However, it seems that this is not the information that they must glean to succeed here by this more advanced age. This suggests that the perceptual development that supports learning on this task is not simply locating categories within a dimension. Rather, some other component of perceptual development must be occurring. By contrast, Experiment 3 suggests that variability along noncontrastive acoustic dimensions supports minimal-pair learning in the switch task, even when contrastive variability is minimized. Before reaching this conclusion, however, it is important to assess several alternatives. One possible explanation for this is that the stimuli presented in Experiment 3 are more natural than those in Experiments 1 and 2.

, 2004; Kuula et al., 2009). The findings presented in this paper support the therapeutic

usefulness of the nonantibiotic properties of doxycycline in the treatment of chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease, where suppression of interstitial collagenase and 92-kDa gelatinase (gelatinase B) may be beneficial to reduce pathologically excessive degradation of the ECM. It is noteworthy, as shown in this and previous studies (Hanemaaijer et al., 1997), that the inhibition/reduction of MMP-8 and -9 expression and activities by doxycycline and CMTs is not complete, thus allowing these MMPs to carry out the protective actions (McMillan et al., 2004; Sorsa & Golub, 2005; Kuula et al., 2009). Both doxycyclines and chemically modified tetracyclines, when used in conjunction with other chemotherapy agents, CHIR-99021 in vivo may not only lead to more successful periodontal treatments but may reduce the risks for other significant medical conditions including diabetes, heart attack, stroke and other CVDs (Golub et al., 2009; Payne et al., 2009). This study was supported by grant no. A43273 from the New York State Office of Science, Technology and Academic Research

(NYSTAR), through NYSTAR’s Center of Advanced Technology, Stony Brook University. The authors would like to acknowledge Dr Mary Truhlar, Chair of Department of General Dentistry, Stony Brook University, for her support and encouragement of this project. “
“The complement system is regulated

by inhibitors such as factor see more I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms Dipeptidyl peptidase in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complementfactor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. In conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS. Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure 1.

Plasmid curing was used to examine the function of plasmids. Five plasmids of A. AUY-922 datasheet baumannii A3 were cured but no differences were observed between wild-type and plasmid-cured strains with respect to the biofilm formation capabilities. The prevalence of A. baumannii strains with biofilm mode of growth could explain their ability to persist in clinical environments and their role in device-related infections. The genus Acinetobacter includes a group of bacteria that are nonmotile, Gram-negative coccobacilli, displaying strict aerobic metabolism.

Acinetobacter spp. have evolved as important nosocomial pathogens. They are found in diverse environments such as soil, water, food products and are often isolated from medical devices (Bergogne-Bérézin & Towner, 1996). They cause severe infections in immune-compromised patients by colonizing on different medical

devices and surviving on these surfaces (Tomaras et al., 2003). A large number of reports describe the outbreaks of Acinetobacter-associated nosocomial infections such as secondary meningitis, pneumonia, wound, burn and urinary tract infections (UTI) (Bergogne-Berenzin et al., 1993; Patwardhan et al., 2008). Biofilm formation www.selleckchem.com/PARP.html is an important feature of most clinical isolates of Acinetobacter spp. Biofilms are assemblages of surface microbial cells that are enclosed in an extracellular polymeric matrix (Donlan, 2002). It is clear from the epidemiologic evidence that Acinetobacter biofilms play a role in infectious diseases such ADAMTS5 as cystic fibrosis, periodontitis, in bloodstream and UTI because of their ability to indwell

medical devices (Struelens et al., 1993; Donlan & Costerton, 2002; Gaddy et al., 2009). Acinetobacter is known to show resistance to a majority of commercially available antibiotics (penicillins, aminoglycosides, cephalosporins, quinolones) and therefore raises an important therapeutic problem (Smolyakov et al., 2004; Shin et al., 2009). A control of the spread of these infections thus demands the removal of Acinetobacter spp. from medical settings (Zavascki et al., 2010). Antibiotic resistance markers are often plasmid borne and plasmids present in Acinetobacter strains can be transferred to other pathogenic bacteria (Chopade et al., 1985; Patwardhan et al., 2008). The ability of Acinetobacter species to adhere to the surfaces, form biofilms, display antibiotic resistance and gene transfer means that there is an urgent need to study the factors responsible for their spread. In the present study, biofilm formation on different abiotic surfaces by six clinical isolates of Acinetobacter baumannii obtained from UTI, as well as catheter surfaces, and the effects of physical parameters (temperature, pH and NaCl) on biofilm formation, was investigated. Factors such as cell surface hydrophobicity (CSH) and production of lectins, important in biofilm formation, were also evaluated.

1 before and during infection resulted in decreased morbidity and mortality compared to control influenza-infected mice. Not only did a portion of treated animals survive, those surviving animals also experienced rapid recovery to a normal weight range. These findings implicate NK cells in contributing to or exacerbating pathology arising from influenza infection. This contrasts with the described essential function of NK cells in protecting mice from influenza infection, selleck kinase inhibitor as evidenced by increased morbidity and mortality when NK cells are depleted or rendered less responsive [24-26]. Previous studies have generally used low doses of influenza virus to study NK cell functions and in this case NK

cells may contribute significantly to limiting the early propagation of virus. In comparing our experiments with those in previous reports, it appears that virus dose plays a role in determining the overall influence of NK cells in host morbidity and mortality as a consequence of influenza infection. Here, we clarify this issue by showing that increasing the influenza dose from medium to high switches the contribution of NK cells from reducing to enhancing morbidity and mortality. Our results with high-dose influenza infection confirm recent findings by Abdul-Careem et al. [36], where they observe NK cells contributing to pathology during influenza infection. Unlike our study, Abdul-Careem et

al. did neither examine virus dose vis-à-vis the NK-cell contribution to pathology during Ceritinib mouse influenza infection, nor define the importance of this factor, however, the single dose of virus they used may be similar to the high-dose virus level we used in this study, since they obtained similar outcomes [36]. Interestingly, for LCMV Teicoplanin infection in mice, it has been demonstrated that the dose of virus greatly affects the influence of NK cells in the immune response to the virus and host outcome. A low dose of LCMV results in viral clearance; a medium dose results in a deleterious

NK-cell dependent alteration of T-cell responses, immunopathology, and virus persistence; while with a high dose of virus, NK cells are beneficial by suppressing T cells that would otherwise mediate severe pathology and mortality [13]. It is conceivable that at high influenza dose, the outcome we observed is similar to that seen with infection of mice with a medium dose of LCMV, where there is NK-cell dependent pathology. The age of mice is another factor affecting host pathology and mortality in the context of influenza infection. This can be seen in comparing the survival curves of the unmanipulated influenza infected control groups in Figures 3 and 5. None of the influenza infected control mice in Figure 3 survived, while approximately 30% of the mice used in Figure 5 did survive the same dose of influenza infection. The mice used in Figures 3 were 4 months old, while those used in Figure 5 were 2 months old.

heilmannii infection was investigated (Fig. 4). Regarding the expression of cytokines, the TNF-α mRNA level in the H. heilmannii-infected gastric mucosa of the WT and PP null mice 1 month after infection

was significantly higher than that in uninfected mice, and its expression level was similar between H. heilmannii-infected WT mice and PP null mice. Helicobacter heilmannii infection led to an Ruxolitinib solubility dmso increase in the IFN-γ level without a significant difference in the WT mice and PP null mice 1 month after infection, and the IFN-γ level in the infected WT mice tended to be higher than that in the infected PP null mice. Three months after infection, the expression levels of TNF-α and IFN-γ tended to be decreased in comparison with 1 month after infection, and no significant difference in these expression levels was observed between both groups. Regarding chemokines, 1 month after infection, the mRNA expression of CCL2, which is known to be involved in the chemoattraction of monocytes and the attraction, activation, and differentiation of T cells (Luther & Cyster, 2001), was significantly upregulated in both the infected WT and PP null mice compared with that in the uninfected mice, and the CCL2 level in the infected WT mice was higher than that in the infected PP null mice. In the H. heilmannii-infected check details WT mice, the mRNA expression level of CXCL13,

which is known to be involved in the organogenesis of lymphatic tissues including MALT (Mebius, 2003), was significantly higher than that in the uninfected mice, and no significant increase was observed in the infected oxyclozanide PP null mice 1 month after infection. Three months after infection, the expression

level of these chemokines was drastically increased both in infected WT and in PP null mice. These results raise the possibility that H. heilmannii induces the expression of cytokines and chemokines related to inflammation and infiltration of lymphatic cells in the gastric mucosa in the absence of PP, although increases in the expression of some of these cytokines and chemokines were relatively low 1 month after infection in PP null mice. In this study, the roles of PP in H. heilmannii-induced immune responses and the development of gastric lymphoid follicles in the gastric mucosa were examined using PP null mice because PP enhances antigen-specific immune responses at the infected site in the gut, and it was also reported that PP play important roles in acquired immunity against Helicobacter bacteria including H. pylori and H. felis (Kiriya et al., 2007; Nagai et al., 2007). The most interesting finding of this study is that PP are not essential for the formation and development of gastric lymphoid follicles induced by H. heilmannii infection (Fig. 2). In previous studies, it was reported that no gastritis was observed in H. pylori-infected mice lacking PP 2 months after infection (Nagai et al., 2007), and 3 months after H. felis infection, PP null mice did not develop H.

To examine the effect of OX40 and 4-1BB activation on FoxP3 expression, CD4+FoxP3/gfp+ Tregs were cultured in vitro with IL-2, or TNF/IL-2 with or without agonistic Abs for OX40 or 4-1BB. After 3-day culture, the levels of FoxP3 expression on a per cell basis (MFI) on

Tregs was increased by ∼two-fold after TNF/IL-2 treatment, as compared with IL-2 treatment alone (p<0.001, Fig. 4D). Importantly, the TNF/IL-2-induced enhancement of FoxP3 expression in Tregs was preserved and even modestly increased by treatment with the 4-1BB agonistic Ab (p<0.05, Fig. 4D). However, in our experimental system, the agonistic Abs for OX40 and 4-1BB did not further enhance TNFR2 expression on Tregs (data PF-01367338 in vitro not shown), suggesting that the effect of TNF on the up-regulation of co-stimulatory TNFRSFs was unidirectional. Next, the suppressive capability of Tregs expanded by the combination of TNF and anti-4-1BB Ab or anti-OX40 Ab was investigated. Consistent with our previous report 3, the suppressive activity of Tregs pre-treated with TNF/IL-2 on the proliferation by Teffs was markedly enhanced (Fig. 4E). Moreover, Tregs pre-treated with TNF/IL-2 in combination with anti-4-1BB Ab or anti-OX40 Ab retained and, in the case of anti-4-1BB Ab, could enhance their potent suppressive potential, as compared with Tregs pre-treated with TNF/IL-2 (medium) alone (p<0.05, Fig. 4F and G). Our data therefore indicate that up-regulation of 4-1BB and OX40 by

TNF/IL-2 on Tregs could further promote their proliferation, Selleckchem Proteasome inhibitor while preserving or even enhancing their potent suppressive activity. It has been reported that LPS was able to activate and expand Tregs by interacting with TLR4 expressed on their surface 23. Since LPS is a potent inducer of TNF 24, we hypothesized that TNF produced in response to LPS challenge may also contribute to the LPS-induced expansion of Tregs. The results showed that in vivo injection of LPS resulted in ∼two-fold and >three-fold increase in the proportion of FoxP3+ cells in the splenic CD4+

subsets by 24 and 72 h after injection, respectively (Fig. 5A). Similarly, the proportion of FoxP3+ cells present in the draining mesenteric LN CD4+ subset following intraperitoneal LPS injection was Selleckchem Nutlin-3 also increased from 8.54% in control mice to 14.24% (Fig. 5B). The expansion of Tregs in the CD4+ subset persisted until day 5 (data not shown). Moreover, the surface expression levels of TNFR2, 4-1BB and OX40 were markedly preferentially increased by 6 h on Tregs (Fig. 5C). The up-regulation of these TNFRSF members on Tregs was transient, with a peak expression at 24 h for both TNFR2 and OX40, and 6 h for 4-1BB respectively (Fig. 5C). Thus, our data show that in vivo administration of LPS also results in the activation and proliferation of Tregs. To confirm the role of TNF in the expansion of splenic Tregs, a neutralizing Ab against mouse TNF was injected 24 h and 1 h before LPS challenge.

, 2008) and embedded in Epon selleck inhibitor according to standard protocols (Hayat, 2000). Specimens were sputter-coated with gold and imaged with a Quanta 3D FEG (FEI). Features within the FIB–SEM dataset were segmented using Amira (Visage Imaging Inc.), and 3D images were

created. To compare the different microscope techniques, we investigated the biofilm development (day 1 trough 4) of P. aeruginosa PAO1 in once-through flow chambers, perfused with media as described previously (Bjarnsholt et al., 2005). SEMs are used to examine topographies of materials with magnifications that range from that of optical microscopy to the nanoscale. SEM scans the surface of the specimen with a finely focused electron beam to produce an image. SEM micrographs have a large depth of field yielding a three-dimensional Alisertib ic50 appearance, which is useful for understanding the surface structure of the sample. Accordingly, SEM is a good option to visualize the bacteria residing in the biofilms. As shown in Fig. 1, it is possible to obtain high-resolution images of P. aeruginosa aggregating on the glass substratum of a flow cell. As with CLSM, it is possible to see the spatial distribution of bacteria including the so-called mushrooms (for comparison se Fig. 2). It seems that the bacteria are uncovered but interconnected by fiber-like structures. Most biofilm literature agrees that

an alginate- and water-containing matrix, which protects the bacteria against adverse conditions, surrounds the bacteria. We were not able to show or find any evidence of a gel-like matrix covering the bacteria using conventional SEM. This is not surprising because an important step in conventional SEM preparation is dehydration. selleck It is hard to evaluate whether the biofilm structures, including the fibers, that are visualized with this method are influenced by the preparation. We speculate that these structures are condensed matrix

components or are actual polymers found underneath the water-containing matrix. When investigating a biological structure in the electron microscope, the problem of artifact formation because of specimen preparation always needs to be considered and analyzed carefully. It is generally considered that vitrification by ultra fast freezing, for example high-pressure freezing, is the gold standard for nonsolid specimen fixation (Walther & Ziegler, 2002; Hohenberg et al., 2003; Walther, 2003a). The clear advantage of cryo-SEM is the lack of preoperational steps including dehydration and the investigation of time-based specimens ‘frozen in time’. The total preparation occurs within a minute of time, which is significantly less than with conventional SEM that takes days. The sample in the current study was fixed by plunging it into sub-cooled nitrogen (nitrogen slush) close to the freezing point of nitrogen at −210 °C.

However, the proliferation of naïve and memory T cells in lymphodepleted mice is regulated differently; homeostasis of naïve CD8+ T cells is regulated by IL-7 and self-MHC/peptide ligands, whereas homeostasis of memory-like CD8+ T cells

is MHC-independent, and controlled by both IL-7 and IL-15. In addition to lymphopenia-driven proliferation, the co-transfer of a small number of Ag-specific TCR transgenic T cells into irradiated mice following Ag exposure resulted in a dramatic expansion of Ag-specific T cells 12. Our recent published data also demonstrated Ag-induced proliferation of melanoma-specific T cells in lymphodepleted hosts, and FDA-approved Drug Library supplier showed that both Ag-induced expansion and lymphopenia-driven proliferation of non-Ag specific T cells were IL-7 dependent 6. The more rapid expansion of Ag-activated T cells enabled them to outpace the lymphopenia-driven proliferation of non-Ag specific T cells during the first 2 wk of immune reconstitution, but contraction followed. The contraction was presumably due

to the suppression mediated by Treg 13–15, or competition with other lymphocyte subsets that undergo delayed proliferation driven by the lymphopenic condition 16. The disruption of T-cell homeostasis leads to profound changes in programs of T-cell activation, differentiation, and survival. Different programming might promote or dampen T-cell reactivity to Ag 17, 18. Thus, it is critically important to determine how Sirolimus mw to set the T-cell regulating programs and determine what underlying mechanisms promote the development of effective antitumor immunity during immune reconstitution in lymphodepleted hosts. Various MYO10 investigators have provided data to suggest that improved activation of T cells may be the result of elimination of Treg, creation of space, or removal of cytokine sinks 7, 19. However, the relative contribution of these mechanisms needs

to be further characterized. In this report, we carefully assessed the effect of lymphopenia-driven proliferation of different subsets of lymphocytes on the concomitant Ag-driven proliferation of melanomas-specific T cells, and the antitumor efficacy of adoptive T-cell therapy in melanoma-bearing mice. We have previously documented that vaccination with peptide-pulsed DC induced a rapid and large expansion of melanoma-specific T cells in lymphodepleted mice that was followed by a delayed lymphopenia-driven proliferation of co-transferred polyclonal naïve spleen cells 6. We hypothesized that the delayed proliferation of co-transferred spleen cells could reduce the maximum expansion of tumor-specific T cells, and thus limit the therapeutic activity of adoptively transferred T cells.

HRP-conjugated goat antirabbit IgG (Dingguo Biotechnology, Beijing, China) diluted by 1:10000 was added and incubated for 1 hr at 37°C. The plates were washed four times with PBS before adding diaminobenzidine substrate (Dingguo Biotechnology), 20 M H2SO4 was added to cease the reaction and the OD490nm was measured. A positive control, a negative control and a blank control were always included on each plate. Six BALB/c mice (6–8 weeks of age) were immunized with the purified recombinant protein. For primary immunization, each mouse was s.c.

injected with 50 μg of antigen (recombinant click here 56-kDa protein) emulsified in Freund’s complete adjuvant. Ten days later, they were given an i.p. booster injection of RXDX-106 in vivo 50 μg antigen emulsified in Freund’s incomplete adjuvant. Control mice were injected similarly with PBS emulsified in Freund’s complete adjuvant or incomplete adjuvant. After that, mice were bled and sera were obtained and stored at −20°C. The animal use was reviewed and approved by the Beijing Administrative Committee for Laboratory Animals and the animal care met the standard of the committee. Bleeding of the mice was performed by tail clip after primary immunization and cardiac puncture after booster immunization. To determine

IgG titers of the sera, an IFA test with antigen slides of O. tsutsugamushi Karp was carried out with fluorescein isothiocyanate-conjugated goat antimouse IgG (Kierkegaard & Perry Laboratories, Gaithersburg, MD,

USA). Meanwhile, an ELISA test was also performed as described above. A fragment of 1107 bp that would yield a 46-kDa His-tagged protein with a deletion of 99 amino acid residues at the N terminal and 64 amino acid residues at the C terminal was amplified by PCR and the product was cloned into pET30a. The resulting recombinant plasmid, designated pET30a-Ot56, was detected by both PCR and restriction enzyme digestion (Fig. 1) and was verified by direct DNA sequencing. Analysis by SDS-PAGE showed that a band approximately at 46 kDa, the expected size those for the truncated protein, was observed in E. coli Rossetta cells transformed with pET30a-Ot56 (Fig. 2a). The purified protein appeared as a single band corresponding to the molecular mass of the recombinant protein on SDS-PAGE (Fig. 2b). The amount of protein after purification was 0.7 mg/mL. Immunoblot assay showed that the protein was recognized by O. tsutsugamushi Karp-immunized rabbit serum (Fig. 2c). The recombinant protein was also validated by MALDI-TOF-MS, which revealed that it had 100% identity to 56-kDa protein of O. tsutsugamushi (Fig. 3). Enzyme-linked immunoassay was performed to assess the extent of cross-reactivity of the recombinant protein with the rabbit polyclonal sera described above. All of the sera detected, except sera against O. tsutsugamushi strains TA763, TH1817, Kato, B quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis were negative (Tables 1,2).