Evaluation of Multiplex Type-Specific Real-Time PCR Assays Using the LightCycler and Joint Biological Agent Identification and Diagnostic System Platforms for Detection and Quantitation of Adult Human Respiratory Adenoviruses

Every year, thousands of basic military trainees in each service of the U.S. Armed Forces experience acute respiratory disease. The majority of this disease burden results from infection with human adenoviruses. We designed single- and multiplex assays that detect and discriminate adenovirus types B3, E4, B7, B11, B14, and B21. A total of 116 oropharyngeal swab specimens obtained from patients at the Naval Health Research Center were used to validate the new assays. Type-specific singleplex assays were designed and used independently to successfully identify 94 representative patient specimens. The lower limits of detection for our singleplex real-time PCR assays were calculated to be 50, 500, 500, 50, 50, and 50 genomic copies per reaction for human adenovirus type B3 (HAdV-B3), HAdV-E4, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21, respectively. These were then multiplexed to increase efficiency and tested against singleplex assays using titrated controls. The HAdV-B3/B11 and HAdV-E4/B7 multiplex assays were as sensitive and specific as they were individually. The HAdV-B14/B21 multiplex assay was not as efficient at detecting HAdV-B14 as the singleplex assay. Interestingly, a statistically significant difference was found between the viral loads of HAdV-B14 and those of HAdV-B3, -E4, -B7, and -B21 (P < 0.001). The assays did not cross-react with other adenoviruses, influenza virus, respiratory syncytial virus, or respiratory disease-causing bacteria. These assays have the potential to be useful as clinical diagnostic tools for the detection of HAdV infection in adult populations.Human adenoviruses (HAdVs) were first associated with clinical illness among military trainees with respiratory disease in the early 1950s (6, 17). HAdVs were the first respiratory viruses to be isolated and characterized. Epidemiological studies showed that adenoviruses are a primary cause of acute respiratory disease (ARD) among military recruits (3, 5) and are a common cause of epidemic respiratory illness in crowded adult civilian populations (18, 21). The diverse human pathogens in the genus Mastadenovirus are categorized into 54 types of adenoviruses. The 54 known types have been grouped into seven species (A to G), based on their immunochemical responses, nucleic acid characteristics, hexon and fiber protein characteristics, biological properties, and phylogenetic analysis (7, 24). They are associated with a broad range of symptoms, including those associated with ARD, conjunctivitis, genitourinary infections, and gastroenteritis, and specific types of adenovirus are associated with specific types of disease (18, 21).Adenoviruses of species B, C, and E are associated with ARD. Species C types, including HAdV type C1 (HAdV-C1), -C2, -C5, and -C6, are common causes of endemic respiratory illness in pediatric populations, and the majority of adults have acquired immunity to these types (8, 16). These may generate asymptomatic carrier-state infections that last into young adulthood, resulting in viral shedding detectable by PCR. Diverse species B types, including HAdV-B3, -B7, -B11, -B14, and -B21, and the sole species E type, HAdV-E4, cause epidemic outbreaks of ARD and conjunctivitis among adults and children (14, 22). These are also associated with essentially continuous outbreaks among unvaccinated military recruits (14, 23). Symptoms range from those associated with mild ARD to severe pneumonia, occasionally resulting in death, even among these otherwise healthy young adults (1, 4, 11, 19). Among U.S. military trainees alone, HAdVs are estimated to cause 22,000 cases of ARD that is severe enough to require medical attention, delaying training schedules, decreasing the quality of life of trainees, and costing the U.S. government $40 million annually (19).The impact and distribution of different types varies over time and can be drastically altered through use of type-specific vaccines (14, 22, 23). An effective vaccine for HAdV-B7 and HAdV-E4 was used in the United States between 1971 and 1999, when production ceased and it became unavailable. Currently, a replacement vaccine is in clinical trials and may soon be available (19, 22), but this vaccine will be limited to use for types 4 and 7. The recent emergence in the United States of a new type, HAdV-B14a (which had not previously been seen in the Western Hemisphere), caused severe outbreaks among both military trainees and civilians and was the subject of several specific outbreak investigations (2, 12, 22). Similarly, in other regions and other circumstances, the severity, attack rates, specific symptoms, and relative risk of fatality may vary greatly between types. It is clearly of significant public health interest to track adult respiratory adenoviruses in a type-specific manner.Historically, adenoviruses were detected by tissue culture methods and discriminated by type-specific serum neutralization methods (13). However, traditional (probeless) PCR assays have since replaced these methods, owing to their greater speed, their significantly lower cost, and the decline in availability of type-specific antisera (15). A variety of reliable PCR assays have been developed and used, including species-specific (26) and type-specific (25) tests. Universal PCR assays paired with sequence analysis have been used to provide a truly comprehensive detection and discrimination method for all HAdV types (20). Real-time (probe-based) PCR platforms now offer even greater efficiency, improved sensitivity and specificity, and the added information value resulting from quantitative analysis of viral titers (9).In this study, we developed a series of single- and multiplexed real-time PCR assays for both the LightCycler and the military Joint Biological Agent Identification and Diagnostic System (JBAIDS) platforms which can detect and discriminate all HAdVs implicated in adult epidemic ARD in the United States, including HAdV-B3, -E4, -B7, -B11, -B14, and -B21. Combined, these tools offer a rapid, high-throughput method for universal detection, discrimination, and quantitation of HAdVs in uncultured throat swab specimens. These assays will allow for much more rapid outbreak assessment and, if validated as in-house diagnostic assays, more rapid individual and public health responses.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Computational Analysis Identifies Human Adenovirus Type 55 as a Re-Emergent Acute Respiratory Disease Pathogen

Novel human adenoviruses (HAdVs) arise from genome recombination. Analysis of HAdV type 55 from an outbreak in China shows a hexon recombination between HAdV-B11 and HAdV-B14, resulting in a genome that is 97.4% HAdV-B14. Sporadic appearances as a re-emergent pathogen and misidentification as “HAdV-B11a” are due to this partial hexon.Human adenoviruses (HAdVs) were first identified as respiratory pathogens (7, 18) but are now recognized as causing a range of diseases, including those that are ocular, gastrointestinal, and metabolic (4). There are 51 “serotypes” defined by using biological characteristics, including immunochemical methods, e.g., serum neutralization and hemagglutination (4). A novel HAdV was characterized using nonimmunochemical methods, e.g., genomics and bioinformatics, and was named HAdV-G52 (9). Recently, genomics and bioinformatics data have defined two emergent, pathogenic, and recombinant HAdVs (D53 and D54) (8, 21). All three are characterized by genomics and therefore should be termed appropriately as “type” rather than “serotype,” as discussed at the 9th International Adenovirus Meeting (Dobogókő, Hungary, April 2009) (our unpublished data). Described here is a re-emergent respiratory pathogen, HAdV-B55, which contains a partial hexon recombination conferring a change in serotype and, hence, an escape from immune reactivity against HAdV-B14. It was isolated recently from an acute respiratory disease (ARD) outbreak in China and incorrectly termed “HAdV-B11-like” (22, 23).Genomes of “HAdV-B11 strain QS-DLL,” noted here as HAdV-B55 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ643676","term_id":"256028986","term_text":"FJ643676"}}FJ643676), HAdV-11p (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF532578","term_id":"31339691","term_text":"AF532578"}}AF532578), and HAdV-B14p (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY803294","term_id":"57115621","term_text":"AY803294"}}AY803294), were obtained from GenBank. Computational analysis included sequence comparisons with zPicture (15), restriction enzyme pattern analysis (http://www.acaclone.com/), phylogeny analysis using MAVID (1), and recombination analysis with SimPlot and Bootscan (12). Genome percent identities were determined using zPicture, MAFFT (10), and Chimera (16).The genome “chassis” of HAdV-B55 is HAdV-B14 (19), an ARD pathogen, with a partial HAdV-B11 hexon providing the misleading serum neutralization result and incorrect, incomplete original identification, as well as an incorrect clinical diagnosis. HAdV-B55 hexon contains 907 nucleotides from HAdV-B11 out of 2,841 nucleotides (31.9%), embedded in the 34,755 base pair genome (2.6%) (Fig. 1A and B). Sequence comparisons reflect the following: HAdV-B55 versus HAdV-B14 at 98.86%; HAdV-B55 versus HAdV-B11 at 97.64%; and HAdV-B11 versus HAdV-B14 at 97.21% (Fig. ​(Fig.1C).1C). HAdV-B55 appears to have evolved from a recombination between HAdV-B14 and -B11 and should be noted as a new type, in the context of both the recombinant HAdVs that are accepted as novel types (8, 21) and its change in serotype (22). In phylogeny analyses, HAdV-B55 groups into a subclade with HAdV-B14 and HAdV-B14a, whereas hexon (loops 1 and 2) groups into a subclade with HAdV-B11. As “HAdV-B11a” did not cross-react with HAdV-B14 antisera, it is not a simple variant of HAdV-B14. Detailed restriction enzyme (RE) digestion pattern analysis, seemingly anachronistic but highly effective visually, confirms this, showing HAdV-B55 as closer to the HAdV-B14 genome than to the HAdV-B11 genome (data not shown), and also matching the original RE analysis for HAdV-B11a (11).Open in a separate windowFIG. 1.(A) Bootscan recombination analysis of the HAdV-B55 genome. The dark green reflects HAdV-B14, and the red indicates HAdV-B11 sequences. Parameters are as follows: 1,000-bp window; 200-bp step; 100 repetitions; neighbor-joining algorithm. The HAdV types analyzed and the colors are the same for both panels A and B, in descending order: HAdV-B14 (top; dark green), B11, B35, B34, B3, B7, B16, B21, B50, simian adenovirus (SAdV)-B21, A12, F40, F41, D53, D9, SAdV-E25, E4, C5, C2, G52, and SAdV-G1 (bottom; light green). (B) Fine-resolution analysis of the partial hexon gene recombination. Bootscan analysis of the hexon gene shows that the proximal region, containing HVR1-6 (“Loop 1”; 407 to 1,034 nucleotide gene location) and HVR7 (“Loop 2”; 1,363 to 1,484 nucleotide gene location), derives from HAdV-B11. The distal portion derives from HAdV-B14, as does the rest of the genome. Parameters are as follows: 200-bp window; 20-bp step; 100 repetitions; neighbor-joining algorithm. (C) Comparative genomics. zPicture compares the nucleotide sequences of pairs of genomes by using a moving overlapping window and scoring percent identities. The y axis is set between 90 and 100%, indicating a very high level of identity.The divergent hexon comprises two regions of identity indicating a recombination event within the gene (Fig. ​(Fig.1B).1B). Analysis of an earlier described “HAdV-B11a” hexon (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY972815","term_id":"62836655","term_text":"AY972815"}}AY972815) (2) shows it is identical to the one embedded in the “QS” genome (23), suggesting that “HAdV-B11a” was incorrectly characterized earlier as a variant of HAdV-B11 (2, 6, 11, 22) by the use of “then-available” assays. Serum neutralization has the hexon as its target, as does limited molecular typing (3, 13). Since genome rearrangements are missed by serological and molecular assays against a limited repertoire, it is problematic to rely solely on these methods to characterize a virus thoroughly. Thus, due to the limitations of these assays, the original “HAdV-B11a” isolates have been misnamed as well (2, 6, 11).As “HAdV-B11a,” this virus represented a paradox. HAdV-B11 is a member of subspecies B2 and was not originally associated with respiratory disease (14); only HAdV-B14 in this group is associated with respiratory disease (20). A clue as to the incorrect association of HAdV-B11 as a respiratory pathogen lies in the reports that “HAdV-B11a” was unusual in having cellular tropism to respiratory epithelial cells rather than renal cells, as observed for original and subsequent isolates of HAdV-B11 (11, 23). In addition, the original “HAdV-B11a” did not agglutinate monkey erythrocytes, unlike the true HAdV-B11 field strains and the prototype Slobitski strain (5). If HAdV-B55 is truly a variant of HAdV-B11 and an ARD pathogen, it presents a drastic change in cell and tissue tropism—from renal and urinary tract to lung, with high morbidity and some mortality (11, 23). This paradox is resolved by the analysis of its genome, that it is HAdV-B14-like rather than HAdV-B11-like. By inference, the cell recognition epitope is either the distal 68.1% of the HAdV-B14 hexon or the fiber, rather than the proximal 31.9% encompassing “loops 1 and 2” of the HAdV-B11 hexon.Although originally identified in sporadic and infrequent historical ARD outbreaks by serum neutralization as “HAdV-B11-like” and recently recharacterized by immunochemistry (serum neutralization and enzyme-linked immunosorbent assay [ELISA]) and limited molecular typing (PCR and hexon sequencing) also as “HAdV-B11-like” (2, 6, 11, 22), genomics and bioinformatics demonstrate that it is a novel type. All of these older techniques, assaying only the hexon gene, simply reconfirmed a HAdV-B11-like hexon epitope. Therefore, it is incorrectly named and noted in GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ643676","term_id":"256028986","term_text":"FJ643676"}}FJ643676) as “HAdV-B11a” or “HAdV-B11 strain QS-DLL” (23). Based on the reported and additional computational analyses and within the context of recent reports of HAdV molecular evolution based on recombination (8, 17, 21), this pathogen is a novel adenovirus type that should be named HAdV-B55.A tsunami of genome sequence information from both newly isolated and presciently archived HAdV strains and their accompanying bioinformatics are leading to an in-depth understanding of the biology of HAdVs. Correct identification and nomenclature of viruses are critical for defining and understanding them as well as their pathogenic variants, particularly in GenBank. The correction of the name of this re-emergent type is urgent, as research groups characterizing other ARD outbreaks by similar or the same HAdV pathogens are on the verge of reporting their findings, adding to the confusion. Genome recombination plays an important role in the molecular evolution of HAdVs, with consequences of newly emerging strains and new understandings of re-emerging pathogens that have tropism change or that become more virulent. An intriguing thought for HAdV-B55 is that perhaps this region of the hexon is a recombination hot spot, driving the evolution, selection, and appearance of a recurrent re-emergent pathogen by altering its serotype and giving it an advantage in a population with seroprevalence against HAdV-B14. As more HAdV genomes are elucidated, they will lead to better understandings of other pathogens that may utilize similar evolution pathways in appearing as emergent and re-emergent pathogens.     

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated,Fibronectin-Binding Protein of Trichomonas vaginalis

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.Trichomonas vaginalis, an extracellular protozoan parasite, is the cause of trichomonosis, the most prevalent nonviral sexually transmitted disease (47). In women, vaginitis due to T. vaginalis clinically manifests with symptoms of vaginal itching, odor, and discharge. Adverse health outcomes for women with this sexually transmitted disease include cervical cancer (46) and preterm delivery and low-birth-weight infants (25). There is a relationship between seropositivity to T. vaginalis and prostate cancer (43). This disease is significant due to its association with human immunodeficiency virus (33, 45). More recently, persistent, undetected T. vaginalis infections associated with asymptomatic carriage were found among women (40).T. vaginalis penetration of the mucous layer (28), followed by adherence to vaginal epithelial cells (VECs), is preparatory for colonization (9, 10). VEC adherence by parasites is mediated by numerous distinct trichomonad surface adhesins (5, 10, 18). Brief contact of T. vaginalis with VECs and fibronectin (FN) elicited dramatic changes in parasite morphology, suggesting a host-specific signaling of parasites (8, 9). Importantly, iron and cell contact by parasites each upregulated the expression of adhesins in a coordinated fashion via distinct mechanisms (2, 4, 6, 21, 29). Genetic approaches using antisense (AS) inhibition of synthesis (36, 37) and heterologous expression in Tritrichomonas foetus (26, 36) have reaffirmed the role of these T. vaginalis proteins as adhesins. T. vaginalis organisms secrete or release numerous metabolic enzymes, including adhesin AP65 (decarboxylating malic enzyme), α-enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during growth and multiplication (27). AP65 and α-enolase were found to reassociate with the parasite surface for the expression of adhesin function (19) and binding to plasminogen (35), respectively.There is an increased awareness of the existence of metabolic enzymes on the surfaces of bacterial pathogens, yeast, and parasites (12, 24, 35). These surface-associated enzymes appear to be novel virulence factors (17, 22, 38, 39). The anchorless glycolytic enzymes GAPDH (13, 31, 38) and α-enolase (39) are present on the surface of group A streptococcus. The surface-associated GAPDH of Candida albicans binds with strong affinity to FN and laminin (22). In enterohemorrhagic Escherichia coli and enteropathogenic E. coli, GAPDH is an extracellular protein that binds human plasminogen and fibrinogen and also interacts with intestinal epithelial cells (17).We demonstrate that GAPDH is another enzyme on the surface of T. vaginalis. A monoclonal antibody (MAb) that inhibited parasite associations with FN was immunoreactive with GAPDH. Importantly, iron was found to regulate gene expression and synthesis and surface placement of GAPDH. Both low-iron-grown trichomonads and AS-transfected parasites with decreased amounts of GAPDH had smaller amounts of surface GAPDH and corresponding lower levels of binding to FN. GAPDH was not involved in adherence of trichomonads to immortalized VECs. Interestingly, as with other microbial pathogens, T. vaginalis GAPDH also bound plasminogen and collagen but not laminin (17, 22).     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Involvement of SOCS3 in Regulation of CD11c+ Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

To investigate the role of suppressor of cytokine signaling (SOCS) molecules in periodontal immunity and RANKL-mediated dendritic cell (DC)-associated osteoclastogenesis, we analyzed SOCS expression profiles in CD4⁺ T cells and the effect of SOCS3 expression in CD11c⁺ DCs during periodontal inflammation-induced osteoclastogenesis and bone loss in nonobese diabetic (NOD) versus humanized NOD/SCID mice. Our results of ex vivo and in vitro analyses showed that (i) there is significantly higher SOCS3 expression associated with RANKL⁺ T-cell-mediated bone loss in correlation with increased CD11c⁺ DC-mediated osteoclastogenesis; (ii) the transfection of CD11c⁺ DC using an adenoviral vector carrying a dominant negative SOCS3 gene significantly abrogates TRAP and bone-resorptive activity; and (iii) inflammation-induced TRAP expression, bone resorption, and SOCS3 activity are not associated with any detectable change in the expression levels of TRAF6 and mitogen-activated protein kinase signaling adaptors (i.e., Erk, Jnk, p38, and Akt) in RANKL⁺ T cells. We conclude that SOCS3 plays a critical role in modulating cytokine signaling involved in RANKL-mediated DC-derived osteoclastogenesis during immune interactions with T cells and diabetes-associated severe inflammation-induced alveolar bone loss. Therefore, the development of SOCS3 inhibitors may have therapeutic potential as the target to halt inflammation-induced bone loss under pathological conditions in vivo.Inflammatory bone diseases affect a large portion of the skeletal system, particularly those portions underlying mucosal surfaces, where inflammation-induced bone loss is closely associated with elevated osteoclast (OC) activity (15, 48). Inflammation-induced bone loss is readily manifested in periodontal disease (i.e., periodontitis), resulting in attachments loss, including periodontal connective tissues and supporting alveolar bone, as a consequence of the interplays between the specific subgingival biofilm and the host''s immune/inflammatory responses (36, 37). The tumor necrosis factor (TNF) family member receptor activator of NF-κB ligand (RANKL), its receptor, RANK, and the natural antagonist, osteoprotegrin (OPG), have been shown to be the key regulators of the differentiation, activation, and survival of OCs and OC. precursors (5, 16-18, 23-25, 44, 55, 58). In addition, it is now clear that the host responses, especially T-cell immunity, play a pivotal role in regulating osteoclastogenesis and homeostasis of the bone tissues (termed osteoimmunology [5]). Activated CD4⁺ T cells express RANKL, which can directly trigger osteoclastogenesis and bone loss, and they can also negatively regulate RANKL activity via OPG production. For instance, it has been shown that OPG treatment results in significantly reduced bone loss in arthritis, osteoporosis, and cancer-related bone metastasis (16-18, 24, 45, 48, 55, 58). In murine models of periodontitis, OPG injections yield a robust inhibition of alveolar bone loss of up to 80 to 100%, suggesting that the RANKL-RANK/OPG axis is the key pathway controlling periodontal osteoclastogenesis (6, 27, 46, 49, 53). In addition to its importance in regulating bone remodelling, RANKL-RANK signaling is also critical for lymph node formation and organogenesis and is involved with dendritic cell (DC) survival and DC-T-cell interactions (23, 55, 59) in which modulation of a complex cytokine network in osteoclastogenesis in vivo has been suggested and shown (45, 52, 53).DCs are efficient antigen-presenting cells (APCs) necessary for regulating T-cell immunity (9). They are detected in diseased tissues of periodontitis and arthritic joints, where they form aggregates with T-cell infiltrates in the inflammatory foci (9, 40, 53) and are believed to contribute indirectly to inflammation-induced bone loss. Recent studies showed that Flt3⁺ monocyte/macrophage (Mo/MQ) precursors may differentiate to OCs, microglia, or DCs, thereby suggesting that these cell types may share common progenitors (2, 9, 26, 32). We recently reported the development of functional OCs from a CD11c⁺ DC subset(s) capable of inducing bone resorption in vitro and post-adoptive transfer in vivo (called DC-derived OCs [DDOC]) (1-4). Moreover, under arthritic conditions, human Mo-derived DCs have been shown to trans-differentiate into OCs in vitro, collectively suggesting that DCs may contribute directly to pathological bone loss (1, 2, 38). Thus, further understanding of the molecular mechanisms during DC-T-cell interactions driving disease pathogenesis and the resulting bone loss is imperative to facilitate the development of novel therapeutics for future treatments.Suppressors of cytokine signaling (SOCS) family and cytokine-inducible SH2 domain-containing protein (CIS) are cytoplasmic adaptor proteins that regulate various cytokine responses in leukocytes via negative feedback loops to inhibit inflammatory stimuli (62). There are eight members, including SOCS 1 to 7 and CIS, and each contains an N terminus, a central SH2 domain, and a C-terminal SOCS box. SOCS inhibits cytokine signaling by binding to phosphorylated JAKs and cytokine receptors and interacting with E3 ubiquitin ligases to polyubiquitylate JAKs for degradation (62). Several studies have investigated the roles of SOCS family members in regulating the development and function of immune cells. For instance, SOCS1 and -3 contain a kinase inhibitory region (KIR) in the N terminus, which inhibits JAK tyrosine kinase activity (35, 60, 61). As cytokine signaling activates JAK/STAT pathways, yielding activated SOCS molecules, SOCS1 is known to bind to JAKs with inhibitory catalytic activity and SOCS3 binds to its proximal sites on cytokine receptors, thereby directly inhibiting JAK signaling (62). These studies established the involvement of SOCS1 and SOCS3 molecules in regulating APCs (i.e., DCs) and T-cell functions during both innate and adaptive immunity (61, 62). Meanwhile, researchers have just begun to elucidate the effects of SOCS expression on the development of OCs and OC precursors, whereby specific SOCS molecules regulating osteoclastogenesis via cytokine signaling have been shown (35, 62). To date, however, the role(s) of SOCS family molecules and their impact on modulating RANKL expression and alveolar bone destruction in periodontal lesions remain unclear. Of particular interest to us is SOCS3, as it has been previously shown to be involved during inflammation-induced osteoclastogenesis and its activity is closely associated with key osteotropic cytokines, such as interleukin-1 (IL-1), TNF-α, transforming growth factor β, IL-6, IL-17, etc. (8, 10, 11, 35, 43, 60).We and others have established the NOD and humanized NOD/SCID mouse models to study periodontal inflammation and immunity and discovered that diabetic NOD mice manifest enhanced alveolar bone loss associated with increases in (i) T-cell proliferation and RANKL expression postinfection by anaerobic Aggregatibactor actinomycetemcomitans compared to prediabetic and nondiabetic NOD mice, and (ii) the Th1-inducing capability of the CD11c⁺ DC subset (14, 27-29). The enhanced alveolar bone loss in diabetic NOD and the physiological relevance of humanized NOD/SCID to periodontitis (14, 27, 49, 63) provide robust systems to study disease pathogenesis during the host-microbe interactions and alveolar bone loss. Herein, we employed these established animal models to assess the role of key SOCS molecules during periodontal immunity and RANKL-mediated alveolar bone loss by studying SOCS expression profiles in pathogen-reactive CD4⁺ T cells and the impact of halting functional SOCS3 on the development and activity of CD11c⁺ DDOC.     

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids,but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H₂O₂) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam₃CSK₄ and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam₃CSK₄ and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam₃CSK₄ or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam₃CSK₄ and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H₂O₂ for 6 h at 37°C inactivates Pam₃CSK₄, FSL-1, and LTA by more than 80%. Although HF, LPL, and H₂O₂ treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.Bacterial sepsis is a leading cause of death within intensive care units (43). Although bacterial sepsis was traditionally associated with gram-negative (Gr−) bacteria, recently, the prevalence of sepsis caused by gram-positive (Gr+) bacteria has rapidly increased (2, 3, 38). In fact, in 2000, Gr+ bacteria accounted for 52% of sepsis cases whereas Gr− bacteria accounted for only 37.6% (7, 31, 38). In bacterial sepsis, the innate immune system provides both the initial immune responses and the early inflammatory responses (1, 8, 12). Early responses to infections with Gr+ and Gr− bacteria have been shown in previous studies to involve different cytokine profiles (9, 16, 25, 51, 54). Other studies have found that infections with Gr− bacteria activate Toll-like receptor 4 (TLR4) primarily with lipopolysaccharide (LPS), a membrane component of Gr− bacteria (26, 27, 44, 53). In contrast, infections with Gr+ bacteria involve TLR2, but the nature of the key TLR2 ligand is still controversial (34, 52, 56).Two components of the cell walls of Gr+ bacteria have been proposed to be TLR2 ligands. One group of studies suggests that lipoteichoic acid (LTA) is the key ligand (10, 46, 49, 57). LTA is a polyphosphate attached to the cell membrane via a diacyl glycolipid and is an abundant component of the envelopes of Gr+ bacteria (47). Highly purified LTA, as well as its synthetic analogs, has been shown to trigger TLR2-mediated inflammatory responses (10, 15, 20, 35). However, the biological role of the LTA is unclear because it is difficult to purify natural LTA without introducing contaminants or damaging the structure of the LTA (41). Another group proposes bacterial lipoproteins as the critical ligand (22). Lipoproteins are a functionally diverse class of bacterial membrane proteins characterized by an N-terminal lipid moiety (4) and are TLR2 ligands (22-24). Although synthetic analogs of lipoproteins were found to be potent TLR2 ligands (5, 6, 42), natural lipoproteins are difficult to purify, and their properties are poorly understood.To avoid the technical difficulties involved in purification, a different investigational approach was developed. This approach uses methods to selectively inactivate either LTA or lipoproteins in bacterial culture supernatants or crude bacterial cell wall extracts (22-24, 49). LTA inactivation is usually performed with hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) (23, 48, 49), which, respectively, hydrolyzes the phosphodiester bonds in the LTA or deacylates one of its acyl chains (17, 28, 36, 55). Lipoprotein inactivation is commonly achieved by deacylation with a lipoprotein lipase (LPL) or by oxidation with hydrogen peroxide (H₂O₂) (22, 24, 62). Despite their wide use, the reaction selectivities of these methods have not been evaluated. Thus, we investigated the reaction specificities of these methods by studying the impacts of these four reactions on the biological properties as well as the chemical structures of LTA and lipoprotein analogs.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10⁵ to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r² = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.Since the discovery of human T-lymphotropic virus type 1 (HTLV-1) in 1980 (16, 40), three other genotypes and 10 subtypes have been recognized. The precise geographical distribution and the clinical consequences of these infections are still a matter of debate. This can be attributed at least in part to the fact that there are insufficient accurate tools for HTLV diagnosis, genotyping, and measurement of viral burden.HTLV-1 is endemic in several geographical areas, including sub-Saharan Africa, South America, the Caribbean Islands, Japan, and Melanesia. It has been estimated that worldwide 10 to 25 million people are infected with this retrovirus (41, 53). Most HTLV-1-infected individuals remain asymptomatic throughout their lifetimes. However, 5 to 10% of infected people develop clinical complications, among which adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are the most severe. Other manifestations of HTLV-1 infection include infective dermatitis (25), uveitis (34), arthritis (38), and Strongyloides stercoralis infection (53). Some of these manifestations could accelerate disease development and/or progression (12, 16). For HTLV-1, a distinction is made between seven subtypes: the worldwide, cosmopolitan subtype HTLV-1a; the Central African subtypes HTLV-1b, -d, -e, -f, and -g; and the Australo-Melanesic subtype HTLV-1c (8, 23, 41, 52).HTLV-2 was discovered in 1982. This retrovirus is endemic in Amerindian and pygmy populations and epidemic in intravenous drug users (16, 49). In contrast to the case for HTLV-1, convincing epidemiological demonstrations of a definitive etiological role of HTLV-2 in human disease are limited. Nevertheless, HTLV-2 has been linked with the development of neurological disorders similar to HAM/TSP, with arthritis, with pulmonary disorders, and with increased mortality (2, 16, 42). HTLV-2 is divided into three subtypes, namely, HTLV-2a and HTLV-2b, mostly found on the American continent, and HTLV-2d, mostly found in Africa (10, 41, 44, 52).In 2005, two more genotypes, HTLV-3 and -4, were discovered in asymptomatic individuals from Cameroon (6, 7, 47, 56). To date, no diseases have been reported in association with HTLV-3 or -4. Further research is needed to determine the distribution and prevalence as well as the pathogenicity of these two new genotypes.The routine diagnosis of HTLV infections is based on conventional serological techniques such as enzyme-linked immunosorbent assay and Western blotting. However, among samples infected with HTLV-1 or HTLV-2, the proportion of seroindeterminate results is high (20, 21, 28, 57). Moreover, in the cases of HTLV-3 and HTLV-4, an indeterminate Western blot pattern appears to be the rule rather than the exception (6, 29). To confirm and/or support serological assays, diagnostic HTLV PCR techniques were created (51, 54). In the next phase, real-time or quantitative PCR (qPCR) assays were developed that confirm the diagnosis and at the same time quantify the HTLV proviral load (PVL). The majority of the published HTLV qPCR assays are singleplex assays, which detect one HTLV genotype per amplification reaction and hereby were developed for the most prevalent variant of HTLV-1, the cosmopolitan HTLV-1a, or for HTLV-2 infection (11, 22, 26, 32, 55). Multiplex qPCR allows the simultaneous detection and amplification of two or more target DNA sequences in only one amplification reaction. To our knowledge, one specific and one generic biplex qPCR for HTLV-1 and -2 (13, 26) and, just recently, one triplex qPCR for HTLV-1, -2, and -3 have been described (3).To address the current problems with HTLV diagnosis and quantitation, taking into account the diversity in HTLV genotypes and subtypes, we developed a novel triplex qPCR assay for simultaneous detection, genotyping, and quantification of PVL of HTLV-1, -2, and -3 infections. In the future, HTLV-4 can be incorporated into our qPCR technique, provided that viral cell culture is possible. Furthermore, considering the increasing number of HTLV qPCR techniques available at present, together with the lack of validation, we performed the first comparative analysis between two qPCR assays developed at different institutions.     

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb₃) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb₃ and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.Shiga toxin-producing Escherichia coli (STEC) is the principal etiologic agent of diarrhea-associated hemolytic-uremic syndrome (HUS) (42, 60, 66). Renal disease is thought to be due to the combined action of Shiga toxins (Shiga toxin 1 [Stx1] and Stx2), the primary virulence factors of STEC, and bacterial lipopolysaccharide (LPS) on the renal glomeruli and tubules (6, 42, 60, 66). Of these, Stx2 is most frequently associated with the development of HUS (45). Shiga toxin enters susceptible cell types after binding to the cell surface receptor glycosphingolipid globotriaosylceramide (Gb₃) and specifically depurinates the 28S rRNA, thereby inhibiting protein synthesis (42, 60, 66). The damage initiates a ribotoxic stress response consisting of mitogen-activated protein (MAP) kinase activation, and this response can be associated with cytokine release and cell death (21, 22, 25-27, 61, 69, 73). This cell death is often caspase-dependent apoptosis (18, 61). Gb₃ is expressed by human glomerular endothelial cells, podocytes, and multiple tubular epithelial cell types, and damage markers for these cells can be detected in urine samples from HUS patients (10-12, 15, 49, 73). Shiga toxin binds to these cells in renal sections from HUS patients, and along with the typical fibrin-rich glomerular microangiopathy, biopsy sections demonstrate apoptosis of both glomerular and tubular cell types (9, 29, 31).Concomitant development of the most prominent features of HUS: anemia, thrombocytopenia, and renal failure, requires both Shiga toxin and LPS in the murine model (30, 33). Nevertheless, our previous work demonstrated that renal failure is mediated exclusively by Stx2 (33). While it is established that Gb₃ is the unique Shiga toxin receptor (46), the current literature regarding the mechanism by which Shiga toxin causes renal dysfunction in mice is inconsistent. Even though Gb₃ has been localized to some murine renal tubules and tubular damage has been observed (19, 23, 46, 53, 65, 68, 72, 74), the specific types of tubules affected have been incompletely characterized. Although multiple groups have been unable to locate the Shiga toxin receptor Gb₃ in glomeruli in murine renal sections (19, 53), one group has reported that murine glomerular podocytes possess Gb₃ and respond to Stx2 in vitro (40), and another group has reported that renal tubular capillaries express the Gb₃ receptor (46). Furthermore, murine glomerular abnormalities, including platelet and fibrin deposition, occur in some murine HUS models (28, 30, 33, 46, 59, 63). We demonstrate here that murine glomerular endothelial cells and podocytes are unresponsive to Stx2 because they do not produce the glycosphingolipid receptor Gb₃ in vitro or in vivo. Further, murine renal tubules, including collecting ducts, express Gb₃ and undergo Stx2-induced apoptosis, resulting in dysfunctional urine production and dehydration.     

Mobilization of CD34+ Progenitor Cells in Association with Decreased Proliferation in the Bone Marrow of Macaques after Administration of the Fms-Like Tyrosine Kinase 3 Ligand

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34⁺ progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo. However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34⁺ progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34⁺ progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34⁺ cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34⁺ progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34⁺ cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34⁺ cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.The cytokine receptor Fms-like tyrosine kinase 3 (FLT3) is expressed at high levels on both primitive and early lymphoid/myeloid CD34⁺ progenitor cells (3, 21). Interaction with its cognate ligand (FLT3-L), found in both soluble and membrane-bound isoforms, contributes to the regulation of self-renewal and differentiation potential of these cells (43, 44). However, dysregulation of FLT3/FLT3-L signaling can result in the development of various leukemias (1, 6, 22, 29), and increased serum levels of FLT3-L are often indicative of other hematologic and autoimmune abnormalities (17, 25, 39). Nonetheless, after both murine and human FLT3-L were cloned in the early 1990s (24), this hematopoietic cytokine was used effectively in vitro to expand and maintain CD34⁺ progenitor cells (26, 32, 33) and, in combination with other growth factors, was used to induce differentiation of myeloid lineage cells (4), dendritic cells (2, 15), natural killer (NK) cells (42), erythroid precursors (12), and even endothelial cells (41). In addition, FLT3-L was shown to specifically suppress apoptosis of CD34⁺ progenitor cells (27).Early in vivo studies in mice demonstrated that FLT3-L administration not only mobilized and expanded murine CD34⁺ progenitor cells but also promoted expansion of human CD34⁺ cells transferred into SCID mice (8, 9, 24). In nonhuman primates FLT3-L was used to expand dendritic cell subsets (7, 30, 35), to treat radiation-induced myelosuppression (13, 14, 19), and as an adjuvant for various vaccines (23, 40). Although CD34⁺ cells primarily reside in the bone marrow (BM), examination of mobilization of these cells in vivo in nonhuman primates has been limited and typically restricted to analyses of blood (5, 18, 28). CD34⁺ cell mobilization and hematopoiesis is of particular interest in macaque models of lentivirus infections because, during both HIV and SIV infections, BM damage and reduced hematopoiesis is evident early after infection and is associated with decreased numbers and clonogenic potential of CD34⁺ progenitors, despite low levels of infection and virus replication in these cells (10, 16, 20, 34, 36, 37). Therefore, in the present study we quantified and characterized mobilization of CD34⁺ progenitor cells in BM in relation to that observed in peripheral blood by examining BM aspirates taken at various times during and after FLT3-L administration to naive and SIV- or SHIV-infected macaques.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.     

Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health problem in Central Asia, Southeast Asia, Africa, and Latin America (11, 52, 53). It was estimated that more than 21 million cases of typhoid fever and >200,000 deaths occurred in 2000 (10). The treatment of patients and management of asymptomatic carriers are becoming more difficult due to the worldwide emergence of multidrug-resistant (MDR) strains (2, 15, 29, 42, 43). Vaccination is considered the most promising strategy for the control of typhoid fever in developing countries (11, 19, 52, 53).Typhoid fever in children younger than 5 years old has often been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of less than optimal culture media (35, 46). Several studies have shown that the incidence of typhoid fever among children less than 5 years old is similar to that in school age children and young adults (14, 27, 34, 50, 51).The 3 licensed typhoid vaccines have limited efficacy, and none are suitable for young children under 5 years old. The use of heat-inactivated whole-cell vaccine was suspended in many countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were introduced in the late 1980s; both vaccines are well accepted and confer moderate protection (50 to 70%) in older children and adults. However, neither vaccine is licensed for routine immunization of infants (52).The Vi capsular polysaccharide is both an essential virulence factor and a protective antigen for S. Typhi (36, 38, 39). The concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is not suitable for routine immunization of infants and young children because of its age-related immunogenicity and T-cell independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae (12, 20), Vi covalently bound with protein conferred T-cell dependence and increased immunogenicity (48-50). To date, diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins (CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi polysaccharide in laboratory studies (16, 17, 32, 48-50; personal communications). An improved method was developed (24), utilizing adipic acid dihydrazide (ADH) as the linker and Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier. Clinical trials of Vi-rEPA conjugates conferred 89% protection in Vietnamese children 2 to 5 years old for 46 months (23, 26, 28). The level of serum IgG anti-Vi induced by Vi-rEPA conjugates was correlated with prevention of typhoid fever in these studies (7, 21-23, 26, 28).One limitation of using rEPA as the carrier protein is the lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by pharmaceutical companies in China and India were prepared (24, 48, 49). Modifications of conjugation procedures were made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility of stockpiling in disaster relief.Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule using alternating injections of polysaccharide and conjugate. Priming or boosting effects of polysaccharide on its conjugate vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no consistent conclusion about various types of polysaccharides studied (6, 9, 31, 40, 41). Here, we compared the immune response of Vi polysaccharide injected before or after the administration of Vi-DT with the responses of those receiving 2 injections of Vi-DT. We also investigated the dosage effect for the purpose of better formulation.     

Characterization of a Campylobacter jejuni VirK Protein Homolog as a Novel Virulence Determinant

Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.Campylobacter jejuni is a leading cause of infectious diarrhea in industrialized and developing countries (2, 67). Although most often self-limiting, C. jejuni infections can also lead to severe disease and harmful sequelae, such as Guillain-Barré syndrome (4, 55). Despite the significant progress made during the past few years, the mechanisms of C. jejuni pathogenesis remain poorly understood. A number of potential virulence factors have been identified, and in some cases, their role in virulence and/or colonization has been demonstrated in animal models of infection. For example, motility has been shown to be crucial in order for C. jejuni to colonize or cause disease in several animal models of infection (1, 15, 30, 54). A variety of surface structures, such as adhesins (34, 40, 64) and polysaccharides (5, 6), and glycosylation systems (38, 74), which presumably modify some of these surface structures, have also been shown to be important for infection. Additional studies have revealed the importance of specific metabolic pathways in C. jejuni growth both in vitro and within animals (16, 25, 31, 60, 76). The ability of C. jejuni to invade and survive within nonphagocytic cells has also been proposed to be an important virulence determinant (21, 41, 57, 58, 68, 75, 80).The available genome sequences of several C. jejuni strains have provided significant insight into C. jejuni physiology and metabolism (22, 32, 62, 63, 65). Remarkably, however, analysis of these C. jejuni genome sequences has revealed very few homologs of common virulence factors from other pathogens. A notable exception is the toxin CDT (cytolethal distending toxin), which is also encoded by several other important bacterial pathogens (36, 44, 45). In this paper we describe the identification of a transposon insertion mutant in C. jejuni 81-176, which results in increased susceptibility to antimicrobial peptides and a significant defect in the ability of the organism to cause disease in an animal model of infection. The insertion mutant was mapped to the CJJ81176_1087 open reading frame (Cj1069 in the C. jejuni NCT 11168 reference strain), which encodes a protein with very significant amino acid sequence similarity to the VirK (DUF535) family of virulence factors (13, 20, 56).     

Immune Defenses against Batrachochytrium dendrobatidis,a Fungus Linked to Global Amphibian Declines,in the South African Clawed Frog,Xenopus laevis

Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.Batrachochytrium dendrobatidis is a newly described chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians (29). Growing evidence links amphibian declines in Australia, Central America, the western United States, Europe, and Africa to this emerging infectious disease (4, 9, 12, 26, 29, 34-36, 45, 65). B. dendrobatidis colonizes skin cells of adults and the keratinized mouth parts of tadpoles (3, 4, 29, 34) but does not invade other tissues. It is spread by waterborne zoospores that attach to the skin and migrate to the basal layer of the epidermis (3). The pathogen replicates within the epidermal cells and moves to the surface as the cells mature. Emerging zoospores may infect the same host or another nearby host (3, 4, 29, 34). Recent evidence supports the hypothesis that death results from impaired retention of essential ions by the skin resulting in eventual cardiac arrest (63, 64). Some species of amphibians are very resistant to lethal infections of B. dendrobatidis, whereas others are more susceptible (4, 26, 27, 38, 66-68), and the factors that determine resistance or susceptibility are not well understood. Although much is known about amphibian immunity in general (9, 14, 41), there is limited information about specific immune responses against B. dendrobatidis.We hypothesized that resistant species have antimicrobial peptides or antibodies in the mucus that limit initial infections by B. dendrobatidis zoospores and prevent the further colonization of the same host by zoospores emerging from the skin. Previous work has shown that individual purified antimicrobial peptides (11, 44-50, 52, 68) and enriched skin peptides (48, 52, 66-68) from many species can inhibit the growth of B. dendrobatidis zoospores and mature sporangia in vitro. The skin of amphibians is also protected by the adaptive immune system. Antigens in the skin can be transported to the spleen, where an immune response involving both T cells and B cells can occur (9, 14, 41). In mammals and in fish, antibodies are present in mucosal secretions (10, 28, 31, 53), but there have been no previous studies of antibodies in amphibian mucus.X. laevis was chosen as the species to investigate immunity to B. dendrobatidis because this species has been widely used as a model for studies of amphibian immunity since the 1960s (9, 14, 41). X. laevis is quite resistant to the lethal effects of infection with B. dendrobatidis in nature. Infections were detected in archived specimens of X. laevis as early as 1938, and the incidence of infected individuals appears to be constant (∼3%) over the last 60 years (1941 to 2001) (65).We show here that after exposure to B. dendrobatidis, immunocompetent frogs developed a mild infection that is almost completely cleared by 45 days. Antimicrobial skin peptides inhibited B. dendrobatidis growth, were present at effective concentrations in resting frogs, and increased in number when frogs were exposed to an “alarm” stress. Treatment with norepinephrine depleted skin peptide stores and increased host susceptibility to infection. X-irradiation depleted leukocytes in the spleen without altering the capacity to secrete skin peptides, and the infection intensity was significantly greater in the irradiated frogs. Immunization with heat-killed B. dendrobatidis induced significantly elevated pathogen-specific IgM and IgY in the serum detectable for at least 1 month after the last immunization. In addition to antimicrobial peptides, skin mucus samples from frogs exposed to B. dendrobatidis 5 months earlier contained antibodies of all three immunoglobulin classes that bind B. dendrobatidis. Whether the mucosal antibodies are protective will be determined in ongoing studies. Collectively, these data demonstrate that X. laevis is a good model species to study immune defenses against B. dendrobatidis, and both innate and adaptive immune mechanisms appear to be involved in the resistance to lethal infections.