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The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi -infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease.

Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm−2) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3 %) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility).

Simian immunodeficiency viruses (SIVs) share a number of biological and structural features with human immunodeficiency virus (HIV), making SIVs powerful tools for studying HIV infection. SIVs are the sources of the HIV pandemic (71), and SIV infection of nonhuman primates (NHPs) is at the origin of animal models of AIDS research (16, 28, 70, 74). Rhesus macaques (RMs; Macaca mulatta) infected with SIVmac, SIVsmm, and several chimeric simian-human immunodeficiency viruses (SHIVs) are the most widely used animal models of AIDS and provide essential insights into HIV/SIV immunopathogenesis and vaccine research (43, 65). The Indian RM (IndRM) is the best-characterized and most commonly studied model; however, significant expansion of AIDS research in recent years has resulted in limited availability of IndRM and a renewed interest in alternative animal models for pathogenic SIV infection (15, 47), such as Chinese RMs, cynomolgus macaques (Macaca fascicularis), and pigtailed macaques (PTMs; Macaca nemestrina) (15, 65). Chinese RMs and cynomolgus macaques show somewhat less pathogenic courses of SIVmac239, SIVmac251, and SHIV89.6P infections than IndRMs (36, 61). SIV and SHIV strains, as well as HIV type 2 (HIV-2), have also been used to infect PTMs (2, 12, 38, 48). Attempts have been also made to infect PTMs with HIV-1, but no persistent infection resulted (1, 18, 19). Several groups now primarily study PTMs as an animal model for HIV (28), and there are growing data on the common major histocompatibility complex molecules expressed by PTMs and the SIV epitopes they present (2, 12, 38, 48), thus enabling detailed immunopathogenic studies in PTMs similar to those originally pioneered with IndRMs. One of the most important applications of the use of PTMs as animal models for AIDS research is the study of cross-species SIV transmission from the natural hosts. It is widely acknowledged that African NHPs that are natural hosts of SIV generally do not progress to AIDS when infected with their species-specific virus despite high levels of viral replication (51, 54, 55, 62). In striking contrast to the pacific SIV-host interactions observed in natural hosts, HIV-1 and HIV-2 infections in humans are highly pathogenic, being characterized by progression to AIDS in a variable time frame (27). Both HIV-1 and HIV-2 originated by cross-species transmission of the SIVcpz from chimpanzees (Pan troglodytes troglodytes) and SIVsmm from sooty mangabeys (Cercocebus atys), respectively (71). SIVs have a high propensity for cross-species transmission, which is not a rare event, having been documented in the wild (8, 31, 72). However, unlike cross-species transmission of SIVs to humans and macaques that resulted in the emergence of highly pathogenic viruses, cross-species transmission of SIVs to new African NHP hosts generally does not result in increases in SIV pathogenicity (71). Therefore, understanding the mechanisms behind the spectacular increase in pathogenicity of SIVs transmitted across species to humans is a high priority in the field, especially considering the high exposure of humans to a plethora of viruses naturally infecting African NHP hosts in sub-Saharan Africa (59). A systematic approach of the events driving the increase in pathogenicity upon SIV cross-species transmission will need appropriate NHP models. For these types of studies RMs are probably not an appropriate host as studies carried out thus far demonstrated that RMs can control the majority of cross-species-transmitted SIV infections. Thus, SIVmnd-2 from mandrills (Mandrillus sphinx) (68), SIVrcm from red-capped mangabeys (RCMs; Cercocebus torquatus torquatus) (37, 64), SIVagm from African green monkeys (genus Chlorocebus) (52), SIVsyk from Syke's monkey (Cercopithecus albogularis) (26), and SIVtal from talapoins (Myopithecus talapoin) (49) are minimally pathogenic or controlled when transmitted to RMs. The only exception is the cross-species transmission of SIVsmm from sooty mangabeys (Cercocebus atys), which is pathogenic in RMs upon direct cross-species transmission (41, 62). Note that the emergence of SIVmac and SIVsmm reference strains currently used for pathogenesis and vaccine studies occurred through accidental transmission of SIVsmm from sooty mangabeys to different species of macaques in primate centers in the United States (4, 5, 34, 44) and that the high pathogenicity of these reference strains might have resulted as an effect of serial passages (5, 24). This conclusion is also supported by the recent observation that the intrinsic pathogenicity of primary SIVsmm isolates is significantly lower than previously believed (C. Apetrei, unpublished observations). In contrast to RMs, PTMs appear to be more susceptible to cross-species-transmitted SIV, with cases of AIDS reported to occur in PTMs after experimental infection with SIVsmm (17), SIVagm from different species of African green monkeys (SIVagm.ver and SIVagm.sab) (22, 25) (I. Pandrea, unpublished observations), SIVlhoest from l'hoest monkeys (Cercopithecus lhoesti) (6), and SIVsun from sun-tailed monkeys (Cercopithecus solatus) (6). This higher susceptibility of PTMs to cross-species-transmitted viruses may be explained by peculiarities of the TRIM5 genes in PTMs. The TRIM5 gene encodes Trim5α, a protein that blocks infection of the cell by retroviruses through the inhibition of reverse transcription immediately after viral entry into the cell (67). An aberrant splicing of TRIM5 mRNA resulting in TRIM5α isoform transcripts was described in PTMs (46). These isoforms (TRIM5η or TRIM5θ) are incapable of restricting either HIV-1 or SIV infection (11), thus explaining the high susceptibility of PTMs to different retroviral pathogens. The RCMs are found along the Atlantic forest coastal area of west and central Africa (9) and are naturally infected with a specific lentivirus, SIVrcm (7, 13, 21, 64). Since the RCMs were identified by genus and species previously (two paragraphs above), the identification was deleted from the sentence beginning “The RCMs are found.”− SIVrcm is a unique primate lentivirus in that it uses chemokine receptor CCR2 as a coreceptor for entry (13, 76) in contrast to the majority of HIV and SIV strains that use CCR5 and CXCR4 as the main coreceptors (14, 35). This peculiar coreceptor usage pattern is due to a deletion of 24 bp in the RCM CCR5 (Δ24 CCR5) gene that prevents expression of CCR5 on the surface of CD4+ T cells. This Δ24 CCR5 allele has a high frequency in RCM populations (13). Thus, SIVrcm is an example of a primate lentivirus that has adapted its coreceptor usage to allow productive replication and persistence in its natural host species. Given the unique coreceptor usage, along with the observation that the CCR2 coreceptor is expressed mainly on macrophages and only at very low levels on CD4+ T cells, SIVrcm may be ideal for pathogenesis studies of macrophage-tropic viruses. To date, there is no available data on SIVrcm replication in its natural host, but it is believed that SIVrcm rarely causes disease in RCMs, suggesting that it has been associated with its natural host for a long time. Previous studies on experimental infection of cynomolgus macaques and RMs reported that SIVrcm replicated in both macaque species during acute infection, but the replication was completely controlled by day 60 postinfection (p.i.) (21, 64). This controlled pattern of infection was maintained even after serial passage of SIVrcm in RMs (37). In order to develop an animal model for the in vivo study of SIVrcm pathogenesis and to confirm that this virus can be used as a model of macrophage-tropic SIV infection, we infected PTMs with SIVrcm and monitored the dynamics of viral loads (VLs), changes in lymphocyte subsets, antibody responses, and clinical and pathological features over a period of 27 months. Surprisingly, the patterns of SIVrcm replication and memory CD4+ T-cell depletion in PTMs were similar to those observed in lymphotropic SIV infections. Following inoculation in PTMs, SIVrcm expanded its coreceptor usage, becoming able to use CCR4 in vivo in addition to CCR2. This result indicates that lentiviral adaptation to a new host may occur rapidly through strain selection and involve new pathogenic pathways, pointing to the need for in vivo studies to characterize the pathogenesis of primate lentiviruses.

We investigated the impact of rhesus macaque (RM) B-cell depletion before inoculation with the isolate SIVsmmD215. Seven RMs were treated every 3 weeks with 50 mg/kg of an anti-CD20 antibody (rituximab) starting 7 days before inoculation for 2 (n = 4) and 5 (n = 3) months. Four control animals received no antibody. Three animals were completely depleted of CD20+ B cells, but 4 were only partially depleted of CD20 cells in the LNs and intestine. The decrease in antibody production was consistent with the efficacy of tissue CD20 depletion. Seroconversion and neutralizing antibody production was significantly delayed in animals showing complete tissue CD20 depletion and remained at low titers in all CD20-depleted RMs. Surprisingly, there was no significant difference in acute or chronic viral loads between CD20-depleted and control animal groups. There was a tendency for lower viral set points in CD20-depleted animals. At 6 weeks after inoculation, cellular immune responses were significantly stronger in CD20-depleted animals than in controls. There was no significant difference in survival between CD20-depleted and control animals. Our data suggest that a deficiency of Ab responses did not markedly affect viral replication or disease progression and that they may be compensated by more robust cellular responses.

The mechanisms underlying the lack of disease progression in natural simian immunodeficiency virus (SIV) hosts are still poorly understood. To test the hypothesis that SIV-infected African green monkeys (AGMs) avoid AIDS due to virus replication occurring in long-lived infected cells, we infected six animals with SIVagm and treated them with potent antiretroviral therapy [ART; 9-R-(2-phosphonomethoxypropyl) adenine (tenofovir) and beta-2,3-dideoxy-3-thia-5-fluorocytidine (emtricitabine)]. All AGMs showed a rapid decay of plasma viremia that became undetectable 36 h after ART initiation. A significant decrease of viral load was observed in peripheral blood mononuclear cells and intestine. Mathematical modeling of viremia decay post-ART indicates a half-life of productively infected cells ranging from 4 to 9.5 h, i.e., faster than previously reported for human immunodeficiency virus and SIV. ART induced a slight but significant increase in peripheral CD4+T-cell counts but no significant changes in CD4+T-cell levels in lymph nodes and intestine. Similarly, ART did not significantly change the levels of cell proliferation, activation, and apoptosis, already low in AGMs chronically infected with SIVagm. Collectively, these results indicate that, in SIVagm-infected AGMs, the bulk of virus replication is sustained by short-lived cells; therefore, differences in disease outcome between SIVmac infection of macaques and SIVagm infection of AGMs are unlikely due to intrinsic differences in the in vivo cytopathicities between the two viruses.

Aquifer biofilm-forming isolates from the Edwards Aquifer, Texas, were characterized biochemically and by phylogenetic analyses based on partial 500 bp 16S rRNA sequencing. Five of the 8 strains evaluated were gram-negative and the other 3 were gram-positive. Phylogenetic analyses suggested novelty of 3 isolates, and biochemical analysis showed a range of carbon-source use patterns among the isolates. Such baseline information will be useful for investigating biofilm population interactions, as well as understanding the microbial ecology of unpolluted karst aquifers.

Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.

This chapter reviews many commonly used organisms, experimental approaches, and techniques used for the growth and study of biofilms. Here, the authors focus on commonly used approaches with a greater emphasis on currently used techniques. They have summarized several diverse techniques for growing and evaluating biofilms below. These include examples of biofilm techniques for exploring naturally occurring biofilms, medically important biofilms, industrial applications, laboratory biofilm techniques, imaging and spectroscopy techniques, and broad-based genetic techniques. Overall, the authors concentrate on several techniques that are presently used in current biofilm research. Included in this discussion are biofilm growth strategies, a brief mention of genetic strategies, imaging techniques, and data analysis. The authors hope that this discussion will serve as an informative reference for the biofilm research community. In the field of dental microbiology, the constant depth fermenter is a device in which oral microorganisms are cultured on hydroxyapatite disks that are coated with saliva. Such standardized methods will permit a meaningful and rational comparison of data among individual laboratories. Finally, microbiologists now realize the many diverse environments and situations in which biofilms occur. As a result, there will be a continuing need for innovation in the experimental designs used to study biofilms.

Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of α S1 -casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.

Although biofilm formation is widely documented on Earth, it has not been demonstrated in the absence of gravity. To explore this possibility, Pseudomonas aeruginosa, suspended in sterile buffer, was flown in a commercial payload on space shuttle flight STS-95. During earth orbit, biofilm formation was induced by exposing the bacteria to sterile media through a 0.2-microm (pore size) polycarbonate membrane. Examination of these membranes by confocal microscopy revealed biofilms to be present and that these biofilms could persist in spite of vigorous agitation. These results represent the first report of biofilm formation under microgravity conditions.

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

Simian immunodeficiency virus (SIV) SIV smm naturally infects sooty mangabeys (SMs) and is the source virus of pathogenic infections with human immunodeficiency virus type 2 (HIV-2) and SIV mac of humans and macaques, respectively. In previous studies we characterized SIV smm diversity in naturally SIV-infected SMs and identified nine different phylogenetic subtypes whose genetic distances are similar to those reported for the different HIV-1 group M subtypes. Here we report that, within the colony of SMs housed at the Yerkes National Primate Research Center, at least four SIV smm subtypes cocirculate, with the vast majority of animals infected with SIV smm subtype 1, 2, or 3, resulting in the emergence of occasional recombinant forms. While SIV smm -infected SMs show a typically nonpathogenic course of infection, we have observed that different SIV smm subtypes are in fact associated with specific immunologic features. Notably, while subtypes 1, 2, and 3 are associated with a very benign course of infection and preservation of normal CD4 + T-cell counts, three out of four SMs infected with subtype 5 show a significant depletion of CD4 + T cells. The fact that virus replication in SMs infected with subtype 5 is similar to that in SMs infected with other SIV smm subtypes suggests that the subtype 5-associated CD4 + T-cell depletion is unlikely to simply reflect higher levels of virus-mediated direct killing of CD4 + T-cells. Taken together, this systematic analysis of the subtype-specific features of SIV smm infection in natural SM hosts identifies subtype-specific differences in the pathogenicity of SIV smm infection.

Five strains of Lactobacillus plantarum and 4 strains of Lactobacillus ssp. isolated from fresh meat contained between 1 and 5 plasmids ranging in Mr from 1.3 to 49 MDa. Plasmid-curing studies suggested that maltose utilization is associated with a 49 MDa plasmid (pML291) in Lactobacillus sp. DB29 and 34.5 MDa plasmids in Lactobacillus ssp. DB27, DB28 and DB31. Restriction digestion of pML291 and a putative plasmid deletion derivative, pML292, isolated from a maltose negative mutant of DB29, generated common restriction fragments. Southern blot DNA-DNA hybridization using pML 291 as a probe indicated that there is strong homology between putative maltose plasmids.

We report in vitro characterization of 11 SIVsmm strains of six lineages co-circulating in naturally infected sooty mangabeys (SMs) from US Primate Centers and showed no major differences in the in vitro replication pattern between different SIVsmm lineages. Primary SIVsmm isolates utilized CCR5 and Bonzo co-receptors in vitro. SIVsmm growth in human T cell lines was isolate-, not lineage-specific, with poor replication on Molt4-Clone8, CEMss and PM1 cells and better replication on MT2, SupT1 and CEMx174 cells. All primary SIVsmm isolates replicated on SM and human PBMCs. In vitro replication in macaques varied widely, with moderate to high replication in pig-tailed macaque PBMCs, enhanced by CD8+ T cell depletion, and highly variable replication on rhesus macaque (Rh) PBMCs. Primary SIVsmm isolates replicated in Rh monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). In vivo, SIVsmm isolates replicated at high levels in all SIVsmm-infected Rh. The poor in vitro replication of primary SIVsmm isolates in Rh cells did not correlate with in vivo replication, emphasizing the value of in vivo studies.

Virus-like particles (VLPs) offer a platform to test the hypothesis that, since antibody binding to native envelope glycoprotein (Env) trimers results in HIV-1 neutralization, that native Env trimers presented in membranes may be useful for inducing neutralizing antibodies (nAbs) in a vaccine setting. So far, VLPs have not fulfilled this potential. Here, using a “shotgun” approach, we evaluated a wide cross-section of variables in a series of VLP immunizations. We identified 3 tentative leads. First, that VLP doses may not have been sufficient for optimal nAb induction. Second, that dampening the antigenicity of non-functional Env (for example uncleaved gp160) using either protease digests or IgG masking may be useful. Third, that guinea pig sera preferentially target non-conserved epitopes and exhibit relatively high background activity, suggesting that rabbits may be preferable as small animal vaccine models. Recent immunogenicity studies in rabbits appear to bear out all 3 of these leads.