Capsule impairs efficient adherence of Streptococcus agalactiae to intestinal epithelium in tilapias Oreochromis sp.

Streptococcosis caused by Streptococcus agalactiae is one of the most important diseases in the tilapia aquaculture industry. The role of the capsule of Streptococcus agalactiae in adherence to fish surfaces has not been evaluated and the mechanism of capsular regulation during adhesion has not been described. The aim of this study was to evaluate the role of the capsule of S. agalactiae during adhesion to intestinal epithelium of tilapia (Oreochromis sp.) in an ex vivo infection model. We show that the capsule impairs the adhesion of bacteria to host intestinal epithelium. Wild type (WT) strain SaTiBe08-18 (S. agalactiae recovered from tilapia) had reduced adhesion (P < 0.0001) in comparison with its unencapsulated mutant of SaTiBe08-18 (Δcps). When WT was treated with sterile saline solution (pH 5) before infection of intestine explants, the adhesion was reached. The results suggest that the capsule impairs the adhesion of S. agalactiae to tilapia intestine and that the acidic milieu could regulate adherence of encapsulated strains. We found GlcNAc on the surface of adherent Δcps but not over the capsule in WT. This difference could be explained by the GlcNAc composition of Lancefield group B antigen and the peptidoglycan in GBS (Group B Streptococcus) and also may be related with better exposure of glycosylated adhesins in unencapsulated fish GBS. Understanding capsular regulation during adhesion of S. agalactiae may provide new leads to find a successful anti-adherence therapy to prevent streptococcosis in tilapia.

Experimental early pathogenesis of Streptococcus agalactiae infection in red tilapia Oreochromis spp.

Streptococcus agalactiae causes a severe systemic disease in fish, and the routes of entry are still ill-defined. To address this issue, two groups of 33 red tilapia Oreochromis spp. each of 10 g were orally infected with S. agalactiae (n = 30), and by immersion (n = 30), six individuals were control-uninfected fish. Three tilapias were killed at each time point from 30 min to 96 h post-inoculation (pi); controls were killed at 96 h. Samples from most tissues were examined by haematoxylin-eosin (H&E), indirect immunoperoxidase (IPI) and periodic acid-Schiff; only intestine from fish infected by gavage was evaluated by transmission electron microscopy. The results of both experiments suggest that the main entry site of S. agalactiae in tilapia is the gastrointestinal epithelium; mucus seems to play an important defensive role, and environmental conditions may be an important predisposing factor for the infection.

Mestizos with systemic lupus erythematosus develop renal disease early while antimalarials retard its appearance: data from a Latin American cohort.

OBJECTIVES: The objective of this paper is to assess the predictors of time-to-lupus renal disease in Latin American patients. METHODS: Systemic lupus erythematosus (SLE) patients (n = 1480) from Grupo Latino Americano De Estudio de Lupus (GLADEL's) longitudinal inception cohort were studied. Endpoint was ACR renal criterion development after SLE diagnosis (prevalent cases excluded). Renal disease predictors were examined by univariable and multivariable Cox proportional hazards regression analyses. Antimalarials were considered time dependent in alternative analyses. RESULTS: Of the entire cohort, 265 patients (17.9%) developed renal disease after entering the cohort. Of them, 88 (33.2%) developed persistent proteinuria, 44 (16.6%) cellular casts and 133 (50.2%) both; 233 patients (87.9%) were women; mean (± SD) age at diagnosis was 28.0 (11.9) years; 12.2% were African-Latin Americans, 42.5% Mestizos, and 45.3% Caucasians (p = 0.0016). Mestizo ethnicity (HR 1.61, 95% CI 1.19-2.17), hypertension (HR 3.99, 95% CI 3.02-5.26) and SLEDAI at diagnosis (HR 1.04, 95% CI 1.01-1.06) were associated with a shorter time-to-renal disease occurrence; antimalarial use (HR 0.57, 95% CI 0.43-0.77), older age at onset (HR 0.90, 95% CI 0.85-0.95, for every five years) and photosensitivity (HR 0.74, 95% CI 0.56-0.98) were associated with a longer time. Alternative model results were consistent with the antimalarial protective effect (HR 0.70, 95% CI 0.50-0.99). CONCLUSIONS: Our data strongly support the fact that Mestizo patients are at increased risk of developing renal disease early while antimalarials seem to delay the appearance of this SLE manifestation. These data have important implications for the treatment of these patients regardless of their geographic location.

Functional characterization of pheromone receptors in the tobacco budworm Heliothis virescens.

Functional analyses of candidate Heliothis virescens pheromone odorant receptors (HvORs) were conducted using heterologous expression in Xenopus oocytes. HvOR6 was found to be highly tuned to Z9-14:Ald, while HvOR13, HvOR14 and HvOR16 showed specificity for Z11-16:Ald, Z11-16:OAc and Z11-16:OH, respectively. HvOR15, which had been considered a candidate receptor for Z9-14:Ald did not respond to any of the pheromone compounds tested, nor to 50 other general odorants. Thus, while HvOR15 is specifically expressed in H. virescens male antennae, its role in pheromone reception remains unknown. Based on our results and previous research we can now assign pheromone receptors in H. virescens males to each of the critical H. virescens agonistic pheromone compounds and two antagonistic compounds produced by heterospecific females.

Differential expression of odorant receptor genes involved in the sexual isolation of two Heliothis moths.

Moth sexual communication systems are highly diverse, but the mechanisms underlying their evolutionary diversification remain unclear. Recently, genes coding for odorant receptors (ORs) OR6, OR14, OR15 and OR16 have been genetically associated with species-specific male response to female pheromone blends in Heliothis virescens (Hv) and Heliothis subflexa (Hs). Quantitative real-time PCR analysis indicates that expression of HvOR6, HsOR6, HvOR14, HsOR14, HvOR15 and HsOR15 is male biased, which supports the hypothesis that they have a role in mediating female sex pheromone detection. The genes HvOR14, HvOR15 and HvOR16 are expressed at higher levels than their corresponding orthologues HsOR14, HsOR15 and HsOR16 in male antennae, while HvOR6 and HsOR6 transcripts are equally abundant in male antennae. The lack of higher expression of any of the receptor genes in H. subflexa antennae suggests that interspecific sequence differences, rather than gene regulation differences, underly the species-specific male response to pheromone components.

Spatial relationships between nitrogen status and pitch canker disease in slash pine planted adjacent to a poultry operation.

Pitch canker disease (Fusarium circinatum Nirenberg & O'Donnell) causes serious shoot dieback, reduced growth and mortality in pines found in the southern and western USA, and has been linked to nutrient imbalances. Poultry houses with forced-air ventilation systems produce nitrogen (N) emissions. This study analyzed spatial correlations between pitch canker disease and foliar, forest floor, soil, and throughfall N in a slash pine (Pinus elliottii var. elliottii Engelm.) plantation adjacent to a poultry operation in north Florida, USA. Tissue and throughfall N concentrations were highest near the poultry houses and remained elevated for 400 m. Disease incidence ranged from 57-71% near the poultry houses and was spatially correlated with N levels. Similarly, stem mortality ranged from 41-53% in the most heavily impacted area, and declined to 0-9% at distances greater than 400 m. These results suggest that nutritional processes exacerbate changes in disease susceptibility and expression in slash pine.

Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins.

Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2'-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4(+) target cells and mediated virus-induced, CD4-dependent "fusion from without" comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

Administration of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) for prevention of perinatal simian immunodeficiency virus infection in rhesus macaques.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model to explore novel strategies to reduce perinatal human immunodeficiency virus (HIV) infection. The availability of two easily distinguishable virus isolates, SIVmac251 and the simian/human immunodeficiency virus chimera SHIV-SF33, allows tracing the source of infection following inoculation with both viruses by different routes. In the present study, we evaluated the efficacy of pre- and postinoculation treatment regimens with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) to protect newborn macaques against simultaneous oral SIVmac251 and intravenous SHIV-SF33 inoculation. Untreated newborns became persistently infected following virus inoculation. When three pregnant macaques were given a single subcutaneous dose of PMPA 2 hr before cesarean section, their newborns became SIV-infected following SIV and SHIV inoculation shortly after birth. In contrast, when four newborn macaques were inoculated simultaneously with SIV and SHIV, and started immediately on PMPA treatment for 2 weeks, only one animal became persistently SIV-infected; the remaining three PMPA-treated newborns, however, had some evidence of an initial transient virus infection but were seronegative and healthy at 8 months of age. Our data demonstrate that PMPA treatment can reduce perinatal SIV infection and suggest that similar strategies may also be effective against HIV.

Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction.

Internally controlled RT-PCR methods (QC-RT-PCR) for quantification of SIV RNA are effective, but are relatively cumbersome, expensive, and time and labor intensive. For greater throughput and efficiency, we have developed a method for quantification of plasma SIV RNA levels by real-time RT-PCR using the Applied Biosystems Prism 7700 sequence detection system. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential phase of amplification, regardless of the input template copy number. Simultaneous amplification and analysis eliminates any requirement for handling amplified products, increasing throughput and eliminating a potential source of assay contamination. The assay we have developed for quantification of SIV RNA has a nominal threshold sensitivity of 300 copy Eq/ml of plasma, although as little as 10 copy Eq/reaction of SIV RNA template can be detected. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 25% (coefficient of variation), based on studies of extraction and analysis of replicate aliquots of the same plasma specimens. The combination of sensitivity, precision, and broad dynamic range allows reliable quantification of viral load even during dynamic phases of SIV infection, such as through the onset and resolution of primary infection, or during treatment with antiretroviral agents. The primer-probe combinations we have developed allow quantification of SIV isolates most commonly used for experimental studies. Availability of this assay should greatly facilitate studies of basic pathogenesis and evaluation of therapeutic and prophylactic approaches in the SIV-infected macaque.

Viral dynamics of primary viremia and antiretroviral therapy in simian immunodeficiency virus infection.

Mathematical modeling of viral replication dynamics, based on sequential measurements of levels of virion-associated RNA in plasma during antiretroviral treatment, has led to fundamental new insights into human immunodeficiency virus type 1 pathogenesis. We took advantage of the simian immunodeficiency virus (SIV)-infected macaque model to perform detailed measurements and mathematical modeling during primary infection and during treatment of established infection with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). The calculated clearance half-life for productively infected cells during resolution of the peak viremia of primary infection was on the order of 1 day, with slightly shorter clearance half-lives calculated during PMPA treatment. Viral reproduction rates upon discontinuation of PMPA treatment after 2 weeks were approximately twofold greater than those obtained just prior to initiation of treatment in the same animals, likely reflecting accumulation of susceptible target cells during treatment. The basic reproductive ratio (R0) for the spread of SIV infection in vivo, which represents the number of productively infected cells derived from each productively infected cell at the beginning of infection, was also estimated. This parameter quantifies the extent to which antiviral therapy or vaccination must limit the initial spread of virus to prevent establishment of chronic disseminated infection. The results thus provide an important guide for efforts to develop vaccines against SIV and, by extension, human immunodeficiency virus.

Morphogenesis of Stigmatella aurantiaca fruiting bodies.

Scanning electron micrographs of intermediate stages of fruiting body formation in the myxobacterium Stigmatella aurantiaca suggest that fruiting body formation can be divided into several stages distinguishable on the basis of the motile behavior of the cells. Aggregates formed at sites where cells glide as groups in circles or spirals. Thus, each aggregate was surrounded by a wide band of cells. Several streams of cells were pointed toward and connected to the wide band of cells at the base of the aggregate, suggesting directed cell movement toward the aggregate. The pattern of cells at the base of taller, more mature aggregates suggested that groups of cells enter the aggregate from the surrounding band of cells by changing the pitch of their movement, thus creating an ascending spiral. Stalk formation was characterized by a distinctly different pattern, which suggested that single cells emerge from the band of cells and move toward the aggregate, under it, and then vertically to create the stalk. At this stage, the aggregate appeared to be torn from the substrate as it was lifted off the surface. The cells in the completed stalks were well separated, and most had their long axes pointed in a vertical direction. A great deal of the stalk material appeared to be slime in which the cells were embedded and through which they were presumably moving in the live material. Some suggestions regarding factors that may direct the observed morphogenetic movements are discussed.