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Background: Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) have been associated with aggressive (AgP) and chronic periodontitis.

Objective: The aim of this study was to evaluate the levels of Aa and Pg in gingival crevicular fluid (GCF) of patients with AgP and its relation with clinical parameters.

Design: Sixteen females and fourteen males with clinical diagnosis of AgP aged 17-23 years and their match's controls, were included in this study. Clinical recording concerning probing pocket depth, clinical attachment level, plaque index and gingival bleeding index were performed at baseline, 30 and 60 days after baseline. After clinical examination GCF samples were analyzed for Aa and Pg with a real-time polymerase chain reaction technique. Patients group was treated with a combined of mechanical and oral antibiotic therapy (doxycycline 100 mg/day, during 21 days). A multivariate analysis was used to determine the relationship between Aa and Pg counts with clinical parameters.

Results: GCF from all subjects was positive for Aa and PG. In controls Pg concentration was higher than Aa (Pg: 42,420 ± 3,034 copies/ml; Aa: 66.6 ± 5.4 copies/ml p < 0.001) while in patients both microbes showed the same concentration (Aa: 559,878 ± 39,698 Pg: 572,321 ± 58,752). A significant and positive correlation was observed between counts of Aa and Pg (R square: 0.7965, p < 0.0001). Female showed more counts/ml. Aa might be closely associated with clinical parameters while Pg did not. At 30 and 60 days Aa counts in patients were similar to controls while Pg counts were equal to baseline. However, in spite of Pg presence a clinical improvement was observed in all patients.

Conclusions: In our population the presence of Aa may be associated with AgP while Pg may be in GCF as an opportunistic pathogen which might caused disease when the ecological balance was favorable.

Owing to genotype-specific neutralizing antibodies, analyzing differences in the immunogenic variation among dengue virus (DENV) genotypes is central to effective vaccine development. Herein, we characterized the viral kinetics and antibody response induced by DENV type 2 Asian I (AI) and Asian/American (AA) genotypes using marmosets (Callithrix jacchus) as models. Two groups of marmosets were inoculated with AI and AA genotypes, and serial plasma samples were collected. Viremia levels were determined using quantitative reverse transcription-PCR, plaque assays, and antigen enzyme-linked immunosorbent assay (ELISA). Anti-DENV immunoglobulin M and G antibodies, neutralizing antibody titer, and antibody-dependent enhancement (ADE) activity were determined using ELISA, plaque reduction neutralization test, and ADE assay, respectively. The AI genotype induced viremia for a longer duration, but the AA genotype induced higher levels of viremia. After four months, the neutralizing antibody titer induced by the AA genotype remained high, but that induced by the AI genotype waned. ADE activity toward Cosmopolitan genotypes was detected in marmosets inoculated with the AI genotype. These findings indicate discrepancies between heterologous genotypes that influence neutralizing antibodies and viremia in marmosets, a critical issue in vaccine development.

We previously reported that heterogeneity and homogeneity of the infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced after primary, secondary, and tertiary infections [28]. Defining the variation in viremia induction, antibody response pattern, and antibody-dependent enhancement (ADE) activity in different genotypes of DENV is essential for understanding dengue pathogenesis and developing effective vaccines. Common marmosets (Callithrix jacchus) are useful animal models of DENV infection [29,30]. Thus, this study aimed to characterize in detail the differences between DENV2 genotypes in terms of viremia levels, neutralizing antibody levels, and ADE activity using common marmosets that were inoculated in a two-dose series at four-month intervals (at months zero and four) with a homological genotype of DENV2. Elucidating the immunological ability, induced by two homological genotype infections in a four-month interval, toward the different genotypes of DENV2 could clarify the important immunological characterization of each genotype for vaccine development.

DENV is transmitted by Aedes mosquitoes and the virus infects up to 390 million people per year, of which 96 million infections demonstrate a spectrum of clinical severity [13]. DENV type 2 (DENV2) is the most frequent cause of dengue epidemics worldwide [14,15]. Phylogenetic analysis of the E protein revealed six genotypes of DENV2: sylvatic, Cosmopolitan (CM), Asian I (AI), Asian II, Asian/American (AA), and American [15,16]. Although genotype differences in genome sequences are approximately 3% to 6%, they differ in virulence, incidence, and vector competence [17,18,19,20,21]. In-depth analyses of the immunogenicity of each genotype are central to the development of an effective dengue vaccine. Sequence variations within each genotype strain affects the ability of a vaccine to induce antibodies to neutralize all strains with the homologous serotype because neutralizing antibodies are highly genotype-specific [22]. Due to these serotype-specific neutralizing antibody properties, cross-reactive neutralization ability against heterologous DENV serotypes wanes after a few months [2]. Nonetheless, these subneutralizing antibodies facilitate virus entry into the cells via Fc gamma receptor (FcγR) and subsequently enhance the viral infection and lead to an increase in viral load [23,24,25]. Dengvaxia is a tetravalent live-attenuated chimeric DENV vaccine and is implemented in a three-dose series at six-month intervals (at months 0, 6, and 12). However, its efficacy against all DENV serotypes ranges from 44.6% to 65.6% [26,27]. Notably, the efficacy of this vaccine against DENV2 is the lowest compared to that against other serotypes. It has been hypothesized that low efficacy is probably associated with strain variation among DENV genotypes [22,27].

Dengue virus (DENV) belongs to the genus Flavivirus in the family Flaviviridae. DENV is a small (~11 kb) enveloped virus that contains a single-stranded, positive-sense RNA genome [1,2,3]. The RNA genome of DENV is formed by a single open reading frame that encodes three structural proteins (capsid [C], pre-membrane [prM], and envelope [E]) and seven non-structural proteins (NS, NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) [3,4,5]. There are four antigenically distinct serotypes of DENV, referred to as DENV1–4, with the identity among serotypes being less than 80% at their E protein amino acid level [5,6,7]. Nonetheless, infection with any serotype of DENV causes similar clinical symptoms ranging from mild febrile illness, dengue without warning signs, dengue with warning signs, severe dengue, and occasionally dengue-related death. Symptoms in patients diagnosed as dengue with warning signs include abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, liver enlargement, increased hematocrit with a decrease in platelets [8]. In 2009, the World Health Organization classified dengue cases as severe dengue case symptoms, including severe plasma leakage, with or without severe hemorrhagic, and severe organ impairment including myocarditis, hepatitis, and encephalitis [9,10,11,12].

Animal studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the NIID, Tokyo, Japan. The study was approved by the Institutional Animal Care and Use Committee of NIID (approval nos. 613006 and 516010) on 29 January and 7 March 2013, respectively. All animal and infection experiments were performed in accordance with NIID Institutional.

Dengue NS1 antigen levels were determined using a Platelia™ Dengue NS1 Ag ELISA kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Plasma samples from three DENV-naive marmosets were used as negative controls. A positive: negative ratio was calculated using the following formula: (index value of sample/index value of negative controls). A positive: negative ratio (P: N ratio) ≥2 was considered positive, and all tests were performed in duplicates.

Baby hamster kidney cell line-21 (BHK-21) was seeded in 12-well plates in MEM (Merck Millipore) supplemented with 10% Hi-FBS (Thermo Fisher Scientific) and then incubated at 37 °C overnight until the cells reached approximately 70% confluency. The plasma samples were serially diluted 10-fold from 1:10 to 1:106. A total of 50 µL of diluted plasma was inoculated onto the cell monolayer. After incubation for 60 min, 1 mL of maintenance medium containing MEM and 1% methylcellulose supplemented with 2% Hi-FBS was added. The plates were incubated at 37 °C in 5% CO2 until visible plaques were observed (5–7 days of incubation). Plaques were fixed with 10% formaldehyde, stained with methylene blue, washed with water, and counted. All tests were conducted in duplicate. Viral titers were expressed as plaque-forming units per milliliter (PFU/mL) using the following formula: (average number of plaques observed × sample dilution)/inoculum volume, µL).

The presence of enhancing activity was determined in plasma samples collected at 140 days after the first virus inoculation using an ADE assay, as described previously [33]. Plasma samples were heat-inactivated at 56 °C for 30 min before use. The heat-inactivated plasma samples were serially diluted 10-fold, starting from 1:10 to 1:106 in MEM supplemented with 10% Hi-FBS. The fold-enhancement was calculated using the following formula: virus titers in plasma sample/virus titers in the absence of plasma samples (negative control). To assess the contribution of the virus-immune complex to virus titers (infection-enhancement), the ratios of fold-enhancement were calculated using the following formula: average fold-enhancement assessed with FcγR-expressing BHK cells (VN)/average virus fold-enhancement assessed with BHK cells (VO) [34].

BHK cells were seeded in 12-well plates in Minimum Essential Medium (MEM) (Merck Millipore, Burlington, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Hi-FBS) (Thermo Fisher Scientific, Waltham, MA, USA) and then incubated at 37 °C overnight until the cell monolayer reached 70%–80% confluency. Plasma samples were heat-inactivated at 56 °C for 30 min before use. The heat-inactivated plasma samples were serially diluted two-fold, starting from 1:2.5 to 1:20,480 in MEM supplemented with 10% Hi-FBS. The virus-antibody complexes were prepared by mixing 25 μL of DENV at titers of 5000 PFU/mL with 25 μL serially diluted plasma samples to make a final dilution series from 1:5 to 1:40,960. The control samples were prepared by mixing 25 μL of DENV at 5000 PFU/mL with 25 μL of MEM supplemented with 10% Hi-FBS. Next, the virus-antibody complexes were incubated at 37 °C for 60 min. A total of 50 μL of the mixture was then inoculated onto BHK cell monolayers in 12-well plates. After incubation for 60 min, 1 mL of maintenance medium containing MEM and 1% methylcellulose supplemented with 2% Hi-FBS was added. The plates were incubated at 37 °C in 5% CO2 until visible plaques were observed (5–7 days of incubation). Cells were fixed with 10% formaldehyde, stained with methylene blue, and washed with tap water, after which the number of plaques was counted. All tests were conducted in duplicate. The neutralizing antibody titer was expressed as the maximum dilution of the plasma sample, which yielded a ≥50% plaque reduction in the virus inoculum compared to that in the control virus samples. Results are shown as PRNT50 values, expressed as the reciprocal of the highest plasma dilution (end-point titer), resulting in ≤50% of the input plaque count. Differences in the neutralizing antibody titers were determined by comparing the geometric mean (GMT) of genotype-specific neutralizing antibody titers of DENV2 AI, CM, and AA genotypes at each time point.

Levels of anti-DENV IgM antibody against all DENV serotypes were determined using a Focus Dengue Fever IgM capture ELISA kit (Focus Diagnostic, Cypress, CA, USA). Anti-DENV IgG antibodies against all serotypes of DENV were determined using a Panbio Dengue IgG Indirect ELISA kit (Abbott Laboratories, Abbott Park, IL, USA). Plasma samples from three DENV-naive marmosets were used as negative controls. A positive: negative ratio was calculated using the following formula: (index value of sample/index value of negative controls). A positive: negative ratio (P: N ratio) ≥2 was considered positive. Index values were calculated according to the manufacturer’s instructions.

Common marmosets (Callithrix jacchus) were obtained from CLEA Japan, Inc. (Tokyo, Japan) and maintained in a specific pathogen-free area at the National Institute of Infectious Diseases (NIID, Tokyo, Japan). The NIID Animal Study Review Board approved this study (approval nos. 613006 and 516010). Each marmoset was inoculated with 1 × 106 plaque-forming units (PFU/dose of DENV). Marmosets M1, M2, and M3 were inoculated with the DENV2 00-43 strain ({"type":"entrez-nucleotide","attrs":{"text":"AB111452","term_id":"31376313","term_text":"AB111452"}}AB111452), and Marmosets M4, M5, and M6 were inoculated with the DENV2 08-77 strain ({"type":"entrez-nucleotide","attrs":{"text":"AB545874","term_id":"308152592","term_text":"AB545874"}}AB545874). The interval between DENV inoculations was 140 days. In total, 1 mL of whole blood was collected in EDTA tubes from each marmoset on day 0 (before virus inoculation), 2, 4, 7, 10, 14, 114, 128, and 140 post-inoculation (p.i.) following the first virus inoculation, and on days 2, 4, 7, 10, 14, and 148 p.i. following the second virus inoculation. The blood samples were centrifuged at 2000 rpm for 10 min at 4 °C to collect the plasma. The plasma samples were stored at −80 °C before use.

DENV type 1 (DENV1) Tokyo-Yoyogi (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"LC006123","term_id":"698162713","term_text":"LC006123"}}LC006123), DENV2 DHF0663 ({"type":"entrez-nucleotide","attrs":{"text":"AB189122","term_id":"51850374","term_text":"AB189122"}}AB189122), DENV2 00-43 ({"type":"entrez-nucleotide","attrs":{"text":"AB111452","term_id":"31376313","term_text":"AB111452"}}AB111452), DENV2 08-77 ({"type":"entrez-nucleotide","attrs":{"text":"AB545874","term_id":"308152592","term_text":"AB545874"}}AB545874), DENV type 3 (DENV3) NRT 09-34, DENV type 4 (DENV4) TVP360, Zika virus (ZIKV) PRVABC59 ({"type":"entrez-nucleotide","attrs":{"text":"KX377337","term_id":"1036637434","term_text":"KX377337"}}KX377337), and Japanese encephalitis virus (JEV) Mie/41/2002 ({"type":"entrez-nucleotide","attrs":{"text":"AB241119","term_id":"81687252","term_text":"AB241119"}}AB241119) strains were used. The DENV2 00-43 strain was isolated from imported dengue fever cases from Indonesia and belonged to the AI genotype. The DENV2 08-77 strain was isolated from an imported dengue fever case from the Maldives and belonged to the AA genotype. DENV2 DHF006 was isolated from a dengue hemorrhagic fever case that belonged to the Cosmopolitan genotype. All virus strains were first isolated using C6/36 cells and passaged in baby hamster kidney (BHK) cells. Virus stocks were used within four culture passages in BHK cells. Culture supernatants collected from infected BHK cells were centrifuged at 3000 rpm for 5 min to remove cell debris. The virus stock was stored at −80 °C before use. The virus RNA was purified and sequenced as described previously [31]. A phylogenetic tree was constructed using the nucleotide sequence of the complete E protein region and obtained using the neighbor-joining method in MEGA software [32].

Levels of cross-reactivity antibodies against ZIKV and JEV 4 months post-inoculation were determined using a PRNT50 assay (). Cross-reactive neutralizing antibodies against ZIKV and JEV were absent in marmosets inoculated with the DENV2 AI and AA genotypes. However, a low neutralizing antibody against JEV was detected in one marmoset inoculated with the DENV2 AA genotype.

In the marmosets inoculated with the DENV2 AI and AA genotypes, the ratio of infection-enhancement to DENV1 was less than 4-fold, suggesting that ADE activity to DENV1 was absent (a). Interestingly, in the marmosets inoculated with the DENV2 AI genotype, the mean fold ratio of infection-enhancement to DENV2 CM genotype at plasma dilutions of 1:10 and 1:100 was 27- and 4-fold, respectively, suggesting the presence of an infection-enhancement antibody to the DENV2 CM genotype (b). In the marmosets inoculated with the DENV2 AA genotype, the ratio of infection-enhancement to DENV2 CM was less than 4-fold, suggesting that ADE activity to DENV2 was absent (b). In the marmosets inoculated with the DENV2 AI and AA genotypes, the ratio of infection-enhancement to DENV3 was less than 4-fold, suggesting that ADE activity to DENV3 was absent (c).

The virus titers of DENV1 were higher with the mean infection-enhancement of 2.3-fold in the presence of 1:10 diluted plasma from the marmosets inoculated with DENV2 AI genotype than in the absence of plasma in FcγR-expressing BHK cells (a). In marmosets inoculated with the DENV2 AA genotype, the infection-enhancement was 2-fold higher in FcγR-expressing BHK cells (b). In marmosets inoculated with DENV2 AI genotype, the virus titers of the DENV2 CM genotype were higher with a mean infection-enhancement of 2.7-fold in the presence of 1:100 diluted plasma than in the absence of plasma when inoculated in the FcγR-expressing BHK cells (c). In marmosets inoculated with DENV2 AA genotype, the titer of DENV2 was higher with a mean infection-enhancement of 2-fold in the presence of 1:1000 diluted plasma than in the absence of plasma when inoculated in the FcγR-expressing BHK cells (d). Interestingly, in the BHK cells, the virus titers of the DENV2 CM genotype were lower in the presence of plasma than in the absence of plasma from the marmosets inoculated with DENV2 AI and AA genotypes. In marmosets inoculated with DENV2 AI genotype, the virus titers of DENV3 were higher, with a mean infection-enhancement 2- and 2.7-fold in the presence of 1:10 diluted plasma than in the absence of plasma in BHK-and FcγR-expressing BHK cells, respectively (e). In marmosets inoculated with DENV2 AA genotype, the virus titers of DENV3 were higher with a mean infection-enhancement 2-fold in the presence of 1:100 diluted plasma than in the absence of plasma in BHK cells (f).

Levels of genotype and serotype cross-reactive neutralizing antibodies to DENV1, DENV2 AI, DENV2 CM, DENV2 AA, DENV3, and DENV4 plasma at 4 months after the first virus inoculation were determined using a PRNT50 assay (). In the marmosets inoculated with the DENV2 AI genotype, serotype cross-reactive neutralizing antibodies against DENV1, DENV3, and DENV4 were below the detection level. Conversely, low-genotype cross-reactive neutralizing antibodies against the DENV2 CM and AA genotypes were detected (GMTCM < 5, GMTAA = 28). In the marmosets inoculated with the DENV2 AA genotype, low serotype cross-reactive neutralizing antibodies against DENV1, DENV3, and DENV4 were detected (GMTDENV1 = 4, GMTDENV3 = 8, GMTDENV4 = 6), and high levels of genotype cross-reactive neutralizing antibodies to the DENV2 AI and CM genotypes were detected (GMTAI = 160, GMTCM = 253).

In the marmosets inoculated with the DENV2 AA genotype, neutralizing antibodies to the DENV2 AI, CM, and AA genotypes were detected on day 14 p.i. (GMTAI = 50, GMTCM = 14, GMTAA = 63) (b). Four months after the first virus inoculation, levels of neutralizing antibodies to the DENV2 AI, CM, and AA genotypes continued to increase and remained high (GMTAI = 80, GMTCM = 160, GMTAA = 403). Following the second virus inoculation, levels of neutralizing antibodies to the DENV2 AI, CM, and AA genotypes increased up to 10-fold compared to those on day 14 p.i. following the first virus inoculation (GMTAI = 640, GMTCM = 508, GMTAA = 4063). After four months, although the levels of neutralizing antibodies decreased, the antibodies remained detectable (GMTAI = 28, GMTCM = 80, GMTAA = 320).

In the marmosets inoculated with the DENV2 AI genotype, neutralizing antibodies to the DENV2 AI, CM, and AA genotypes were detected on day 14 p.i. (GMTCM = 40, GMTAI = 80, GMTAA = 160) (a). Four months after the first virus inoculation, the levels of neutralizing antibodies to the DENV2 AI, CM, and AA genotypes waned (GMTCM < 20, GMTAI < 20, GMTAA = 25). Following the second virus inoculation with the DENV2 AI genotype, neutralizing antibody levels in the marmosets increased rapidly on day 7 p.i. Levels of neutralizing antibodies against the DENV2 AA genotype (GMTAA = 320) were higher than those of neutralizing antibodies to the DENV2 CM (GMTCM = 57) and DENV2 AI genotypes (GMTAI = 113). On day 14 p.i., levels of neutralizing antibodies to the DENV2 AI, CM, and AA genotypes increased approximately 4- to 8-fold compared to those during primary virus inoculation, and levels of neutralizing antibodies to the DENV2 AA genotype were higher than those of neutralizing antibodies to the DENV2 CM and AI genotypes (GMTAI = 453, GMTCM = 320, GMTAA = 1280). Four months after the second virus inoculation, neutralizing antibodies to the DENV2 AI, CM, and AA genotypes waned (GMTAI = 28, GMTCM = 20, GMTAA = 160) but remained detectable.

Levels of genotype-specific and genotype cross-reactive neutralizing antibodies to three genotypes of DENV2 (AI, CM, and AA) were determined using a PRNT50 on day 0 (before virus inoculation), 4, 7, 14, 114, 128, and 140 p.i. following the first virus inoculation and on days 2, 4, 7, 14, and 148 p.i. following the second virus inoculation ().

In marmosets inoculated with the DENV2 AA genotype, anti-DENV IgM and IgG antibodies were first detected at day 7 and 10 p.i., respectively (b). Before secondary virus inoculation, high anti-DENV IgG antibody levels were still detected in all marmosets, but anti-DENV IgM antibodies were below the detection level. Following the second virus inoculation, anti-DENV IgM antibody levels slightly increased, but the level of anti-DENV IgG continued to increase. Both the DENV2 AI and AA genotypes induced similar antibody responses in marmosets. High levels of anti-DENV IgG antibodies were induced in marmosets inoculated with DENV2 AI and AA genotypes following the second virus inoculation.

Following primary virus inoculation, anti-DENV IgM antibody was first detected after day 4 p.i. and earlier than anti-DENV IgG antibody in the marmosets inoculated with DENV AI genotype (a). Anti-DENV IgG antibody levels were first detected after day 10 p.i. After three months, anti-DENV IgM antibody levels waned below detection, but anti-DENV IgG antibody levels continued to increase. Following the second virus inoculation with the DENV2 AI genotype, anti-DENV IgM levels remained below the detection level, but high levels of anti-DENV IgG antibody were detected in the marmosets.

All marmosets were inoculated with the homologous genotype of DENV2 during the secondary infection to determine the antibody pattern following infection. The levels of anti-DENV IgM and IgG antibodies were determined on days 0 (before the first virus inoculation), 2, 4, 7, 10, 14, 114, 128, and 140 p.i. following the first virus inoculation, and on days 2, 4, 7, 10, and 14 following the second virus inoculation (). Samples could not be obtained from marmoset M3 owing to non-study-related death at day 128 following virus inoculation; marmoset M3 demonstrated no symptoms related to infectious diseases, and the autopsy remained inconclusive regarding the reason for death.

Viral RNA was detected on day 2 p.i. in all marmosets inoculated with the DENV2 AI and AA genotypes after the first virus inoculation (c). In the marmosets inoculated with the DENV2 AI genotype, the average detection period of viral RNA was 3.6 ± 2.9 days, with average peak levels of 3.82 ± 0.9 log10 genome copies/mL. In the marmosets inoculated with the DENV2 AA genotype, the average detection period of viral RNA was 7.3 ± 1.2 days, with average peak levels of 3.97 ± 0.2 log10 genome copies/mL. Interestingly, the levels of viral RNA in marmosets inoculated with the AA genotype were significantly higher than those in marmosets inoculated with the A1 genotype at day 7 p.i. (p = 0.009) and day 10 p.i. (p = 0.001).

In the marmosets inoculated with the DENV2 AI genotype, the average detection period of the infectious virus after the first virus inoculation was 3.0 ± 1.7 days, with average peak levels of (3.4 ± 2.1) × 103 PFU/mL (b). In the marmosets inoculated with the DENV2 AA genotype, the average detection period of the infectious virus was 2.0 ± 0.0 days, with average peak levels of (8.2 ± 5.0) × 103 PFU/mL. Nonetheless, we found no significant differences in infectious virus titers between marmosets inoculated with the DENV2 AI and AA genotypes.

Six marmosets were used in the study. Marmosets numbered 1 to 3 (M1, M2, and M3) were inoculated with the DENV2 Asian I (AI) genotype. Marmosets 4 to 6 (M4, M5, and M6) were inoculated with the DENV2 Asian/American (AA) genotype. NS1 levels, infectious virus titers, and viral RNA copy numbers were determined on days 0, 2, 4, 7, 10, and 14 p.i. (). The mean duration of the NS1 antigen detected in the marmosets inoculated with the DENV2 AI genotype was longer (average detection period = 7.0 ± 1.7 days) than that in marmosets inoculated with the DENV2 AA genotype (average detection period = 6.0 ± 0.0 days) (a). After the second virus inoculation, the levels of NS1 antigen were below the detection level due to the presence of antibodies specific to NS1 antigen. Nonetheless, there were no significant differences in the levels of NS1 antigen between marmosets inoculated with DENV2 AI and AA genotypes in the first and second virus inoculation.

In a previous study, we reported that varying levels of neutralizing antibodies were induced against different DENV2 genotypes after primary, secondary, and tertiary infections and that the neutralizing antibody titers to some heterologous genotypes were higher than those of homologous genotypes within a serotype [29]. Thus, this study analyzed the differences in the immunogenic variation among genotype-specific neutralizing antibodies against DENV by characterizing the viral kinetics and antibody response induced by DENV2 AI and AA genotypes using marmosets as the animal model.

Here, we found that the DENV2 AI and AA genotypes induced different levels of viremia and neutralizing antibody titers in marmosets. Inoculation with the DENV2 AI and AA genotypes induced different levels of genotype-specific neutralizing antibodies against the DENV2 AI, CM, and AA genotypes. On day 14 p.i., following the first virus inoculation, higher levels of genotype-specific neutralizing antibodies against the DENV2 AI, CM, and AA genotypes were detected in marmosets inoculated with the DENV2 AI genotype than in those inoculated with the DENV2 AA genotype. However, at four months p.i., neutralizing antibodies in the marmosets inoculated with the DENV2 AA genotype remained high and detectable compared to those in the marmosets inoculated with the DENV2 AI genotype, which decreased. Neutralizing antibodies in marmosets inoculated with the DENV2 AA genotype had a higher affinity for the homologous genotype. However, neutralizing antibodies in marmosets inoculated with the DENV2 AI genotype had a higher affinity for heterologous genotypes (CM and AA genotypes).

Almost 5 months (~140 days) after the first virus inoculation, ADE activity to DENV1 and DENV3 and serotype cross-reactive neutralizing antibodies against DENV1, DENV3, and DENV4 were not detected in the marmosets inoculated with the DENV2 AI genotype. However, in marmosets inoculated with the DENV2 AA genotype, low serotype cross-reactive neutralizing antibodies against DENV1, DENV3, and DENV4 were detected to enhance DENV1 and DENV3. Although the cross-reactive antibodies retained low levels of virus-neutralizing activity (≤5), the ADE activity levels were below the detection limits of the assay.

The presence of ADE activity in heterologous serotypes (DENV1 and DENV3) was not observed in any marmoset. Interestingly, ADE activity to the heterologous genotype of DENV2 (Cosmopolitan genotype) was detected in the plasma of marmosets inoculated with DENV2 AI (at 1:10 and 1:100 dilution). These results suggest that the antibodies induced by the DENV2 AI did not neutralize the heterologous genotype in FcγR-expressing BHK cells and that a low concentration of cross-reactive neutralizing antibodies is correlated with the presence of infection-enhancing activity. In addition, the results indicated that a certain threshold of cross-reactive neutralizing antibodies to heterologous genotypes and serotypes is critical for protection. Additionally, determining the spectrum of neutralizing antibodies induced by each selected DENV strain associated with protection and ADE activity is crucial for dengue vaccine development.

This study revealed significant differences in viral growth patterns between the DENV2 AI and AA genotypes in the marmoset model. Infection with the DENV2 AI genotype induced a longer detectable duration of viremia than infection with the DENV2 AA genotype in the marmoset model. Interestingly, infection with the DENV2 AA genotype induced higher levels of viremia than the DENV2 AI genotype. A study on pediatric dengue cases in Vietnam found that infection with the DENV2 AI genotype induced higher levels of viremia in children than those infected with the DENV2 AA genotype [20]. However, in this marmoset model, we found that inoculation with the DENV2 AA genotype induced higher levels of viremia than inoculation with the DENV2 AI genotype. A plausible explanation for these differences is that the DENV2 strains used in this study differed from the circulating strains in Vietnam, indicating possible discrepancies in phenotypes and pathogenicity. In pediatric dengue cases in Vietnam, clinical background, infection history, and host factors, including immune responses and genetic background, are associated with overall clinical outcomes, thus contributing to differential outcomes between human and marmoset models. Overall, our results confirm differential infectivity between genotypes, and this may, in turn, lead to distinct immune responses upon infection with different genotypes and viral strains.

This study highlights the discrepancies between genotype strains in inducing DENV-neutralizing antibodies and viremia in marmosets. The results indicate that antigenic differences between genotypes may influence the levels of serotype-specific and cross-reactive neutralizing antibodies during primary and secondary infections. Interestingly, low levels of cross-reactive neutralizing did not thoroughly neutralize the heterologous genotype in FcγR-expressing BHK cells, and ADE activity was confirmed in these samples. The results indicate the importance of optimizing the selection of DENV strains that possess the capability to neutralize homologous and heterologous genotypes. Additionally, the results indicate that neutralizing activity titers, as determined by the FcγR-expressing BHK cells, detect both infection-enhancing and neutralizing activity as neutralizing titers may better reflect the biological properties of DENV antibodies in vivo.

NUESTRO CARISMA

Imitar la vida de Hostia y de Inmolacion que llevo nuestro Senor Jesucristo desde su encarnacion hasta su Ascension triunfante y que continua aun en la tierra, en el adorable Sacramento del Altar, en el cielo a la diestra del Eterno Padre. Carisma recibido del Espiritu Santo a traves del fundador Padre Jose Julio Maria Matovelle.

LEMA DE LA CONGREGACION

Esta expresado en las siglas O.A.D. “Todo por amor de Dios” debiendo las hermanas realizar todas sus acciones movidas por este amor.

NUESTRA ESPIRITUALIDAD

Es Eucaristica, el Espiritu de la Congregacion es el de Inmolacion por amor, en union con Jesucristo. Cada uno de sus miembros debe considerarse como Victimas de caridad ofrecidas en union con nuestro Senor Jesucristo, por los mismos fines con que se ofrecen diariamente el sacrifico del altar, esto es:

1.- Glorificar y ensalzar a Dios, reconociendo su infinita soberania y absoluto senorio sobre todas las criaturas.

2.- Darle gracias por sus incesantes beneficios.

3.- Reparar los ultrajes que el pecado irroga a esta majestad infinita.

4.- Suplicar en su generosidad inefable los bienes y gracias que continuamente necesitamos todas las criaturas.

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Vodafone ha realizado una gran apuesta por el contenido en resolucion 4K. Los mas modernos decodificadores de la operadora ya ofrecen esta resolucion, pero faltaba contenido. Hasta ahora solo podi­amos ver algun partido de futbol y algun documental. Sin embargo, Vodafone ha anunciado un acuerdo con Sony Pictures y Paramount Pictures para incorporar a su Videoclub peli­culas en 4K. La compani­a estrenara semanalmente una peli­cula sin coste adicional y ofrecera peli­culas de estreno por 7 euros cada una.

La resolucion 4K ha llegado para quedarse. Hoy en di­a ya es muy complicado encontrar un televisor moderno que no ofrezca esta resolucion. Sin embargo, el problema viene a la hora de encontrar contenido. Mas alla de las series de Netflix y de Amazon, o las peli­culas Blu-Ray 4K, no encontrabamos demasiadas opciones. Por ello la operadora Vodafone Espana ha llegado a sendos acuerdos con Sony Pictures Television y Paramount Pictures para incluir en su Videoclub peli­culas con resolucion 4K.

El contenido de Sony Pictures se compone de dos tipos de peli­culas. Por un lado tendremos 16 ti­tulos de estreno en VOD con un precio de 7 euros cada uno. Por otro lado, se incluiran 52 peli­culas de libreri­a, que se estrenaran semanalmente en el servicio de Vodafone TV sin coste adicional.

En cuanto a Paramount Pictures, Vodafone TV ofrecera en los proximos meses 11 peli­culas de esta productora. De los once ti­tulos tendremos cuatro estrenos en VOD, con un precio de 7 euros cada uno. Los otros siete ti­tulos se incluiran en la libreri­a y tendran un coste de 5 euros cada uno.

No es el primer contenido 4K que incluye Vodafone en su oferta televisiva. El ultimo partido entre Real Madrid y Barcelona se emitio en 4K. Y la compani­a ya ha anunciado que ofrecera la final de la Champions en 4K. Ademas tambien se han incorporado algunos contenidos de plataformas como Netflix.

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Objective: To analyze drug adherence, overall survival (OS) and hospitalization rates of patients with castration-resistant prostate cancer (CRPC) treated with arbiraterone acetate (AA), enzalutamide (ENZ) and their sequence in a real-life setting.

Methods: The database of the largest public insurance company in Austria was analyzed. All CRPC patients with at least one prescription of AA and/or ENZ between September 2013 and August 2016 in the pre-chemotherapy and post-chemotherapy setting were extracted and matched to the Austrian death and hospital admission statistics. Drug adherence was estimated by the medication possession ratio (MPR).

Results: Data of 457 patients (mean age: 74.4 ± 8.5 years) were analyzed. The mean MPR was 90% for AA, 85% for ENZ and 88% for the sequence therapy cohort. The median overall survival (OS) of the entire cohort was 21 months: 15 months for AA, 24 months for ENZ, 26 months for the sequence group, and 10 months for the sequence group after switching. In the post-chemotherapy setting, the median OS was 14 months in AA treatment (mean: 15.8 ± 0.9 months), 19 months in the ENZ treatment (mean: 17.2 ± 1.4 months) and 25 months in the sequence group (mean: 22.7 ± 0.8 months). Median OS in the pre-chemotherapy setting was 25 months (mean: 21.5 ± 1.1 months), 18 months in AA treatment group (mean: 18.9 ± 1.5 months) and 17 months in ENZ treatment group (mean: 18.2 ± 1.9 months). Only 43 (9.4%) patients were not hospitalized during the course of the study. On average patients spent 13% of their remaining life span in hospital care (median 8%, range: 1-34%).

Conclusion: This Austrian prescription database allows some insights into the outcome of CRPC patients treated with AA and ENZ and their sequence in a real-life setting. Drug adherence was satisfactory, OS was shorter for AA and ENZ as compared to the pivotal phase III trials.

There is an active and complex microbial community in the animal's gastrointestinal (GI) tract, which plays key roles in immune system [1,2], nutrition absorption [3], pathogenesis [4], and further in the health and physiological functions of the host [5]; thus, the use of intestinal microflora as probiotic has become one of the hot topics in the international research, especially the use of Lactobacillus [6–8]. Lactobacillus is one of the predominant bacteria in the animal's GI tract [9–11], and it can produce abundant lactic and acetic acids to lower the pH value in the GI tract [12] or compete for the nutrients and epithelial adhesion sites with pathogens to inhibit the growth of pathogens [13], or has the excellent properties of acid and bile salt tolerance as well as powerful capacity of colonization and adhesion, and so on [14]. It has been used as an alternative to antibiotic growth promoters in the poultry industry owing to the hazards posed by the indiscriminate use of antibiotics to human and animal health [15], or as feed supplements for improving animal's production performance [16–18], or as the delivery vector or host to express heterologous genes and antigens [19,20]. However, prior to choosing which species of Lactobacillus to be used as an alternative to antibiotic or feed supplements or the expression host and to explore the probiotic effects of Lactobacilli from the animal's GI tract, more veracious detection technologies are needed to monitor and keep track of them, to know the distribution and quantity in the animal's GI tract, as well as to determine which Lactobacillus is the most predominant one. In addition, the biological characteristics should also be analyzed for the effective use and the development of probiotics.

Generally, the diagnosis of Lactobacilli is carried out by culture-dependent techniques (isolation and culture, microscopic examination, physiological and biochemical events), which is time-consuming and prone to misinterpretation [21]. Compared with the previous method, the conventional polymerase chain reaction (PCR) detection is more prompt, but it is not feasible or accurate to determine complex microbial community due to false positives with low sensitivity and inconveniency [22]. Real-time PCR is an innovative technology based on the conventional PCR with high accuracy and speed, strong specificity and reliability, which can reduce the pollution of false positives to directly measure bacterial DNA in various environments [23].

In the present study, we successfully identified the most predominant Lactobacillus species in the chicken GI tract from crop to rectum by real-time PCR, analyzed the probiotic characteristics, and obtained a desirable Lactobacillus for expressing heterologous genes that can be subsequently used as a vector or host.

The experimental protocol was approved by the Institutional Experimental Animal Care and use Committee of Northwest A & F University. One hundred sixty 1-day-old male Arbor Acres broilers purchased from commercial producer were raised under controlled management similar to commercial practice, and fed with standard commercial diets and water ad libitum without any antibiotics. At 42 days of age, 20 broilers were randomly selected and sacrificed by cutting the carotid arteries. The GI tracts were aseptically removed, and the contents from crop to rectum were collected into sterile tubes and stored immediately at −80°C.

Genomic DNA from crop to rectum's content was isolated using a Stool DNA kit (Omega, Moorpark, USA) according to the manufacturer's instruction, and the DNA concentration and purity were determined by a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Pittsburgh, USA) and then stored at −20°C.

The species-specific primers used to quantify Lactobacillus spp. were listed in Supplementary Table S1, which were adapted from published papers [24–27] and the specificity of these primers was checked by nucleotide–nucleotide BLAST of NCBI database. The specific regions of target bacteria were amplified by conventional PCR using the different species-specific primers. The PCR product was purified with a Gel extraction kit (Omega), then inserted into the pGEM-T easy vector (Promega, Madison, USA) and sequenced by Genscript Bio-Technology Co. (Nanjing, China) to confirm the recombinant plasmids. The concentration and number of copies of the correct recombinant plasmids were determined and calculated, respectively. The standard plasmids were obtained from serially 10-fold diluted (from 10³ to 10⁹) recombinant plasmids, and then real-time quantitative PCR was performed to construct the standard curve with the threshold cycle (C_t) corresponding to the number of copies.

Real-time quantitative PCR was performed with the iCycler iQ real-time detection system (Bio-Rad, Hercules, USA) using 96-well optical-grade PCR plates. The PCR was performed in triplicate in a total volume of 25 μl using SYBRII green PCR master mix (TaKaRa, Dalian, China). Each reaction contained 150 ng of template DNA, 12.5 μl of SYBRII green PCR master mix and 2 mM of each primer. The reaction conditions were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Melt curve: 81 cycles of 55°C for 30 s, temperature change 0.5°C, ending temperature 95°C. Data analysis was carried out with iCycler Optical System Interface software (version 2.3, Bio-Rad).

A total of 20 μl of 4–6 h grown (early log phase) indicator pathogenic organism (purchased from China General Microbiological Culture Collection Center, Beijing, China) was spread on Lysogeny broth (LB) agar plates, which had perforated holes of about 8 mm. The ant-apical half of holes was covered by a little sterile and unset LB agar medium. After solidification, 80 μl of Lactobacillus culture supernatant, pH = 3.5, lactic acid, MRS broth or sterile water were added to each hole and incubated at 37°C for 24 h, and then the diameter of inhibition zone was measured. The antimicrobial compound was further characterized by treating the culture supernatants with lactic acid and acetic acid as well as proteinase K and catalase for 1 h at 37°C.

The isolates were tested for resistance to nine antibiotics produced by Oxoid (Waltham, USA). The result was expressed as sensitive (S) or resistant (R). This testing was performed using the standard disc diffusion method (CLSI 2009) [28].

A 1 ml aliquot of the Lactobacillus cell culture incubated for 16 h was centrifuged at 5000 g for 10 min at 4°C, and the pellets were washed twice and resuspended in sterile phosphate buffered saline. One percent of the mixture was inoculated into 5 ml MRS broth, and the pH was adjusted to 2–6, and then incubated for 24 h at 37°C. Similarly, 1% of the mixture was inoculated into the 5 ml MRS broth containing 0.3 and 0.5% bile salt and incubated for 10 h at 37°C, then the OD₆₀₀ values were determined every 2 h.

The specific regions of target bacteria amplified by conventional PCR using the species-specific primers are shown in Fig. 1. Electrophoresis analysis indicated that all of the amplified products displayed a specific band without primer dimmer and mixed bands. The melting curve analysis also indicated that all primer pairs produced only a single PCR product. The standard curve, PCR efficiency, and linear correlation coefficient (R²) are listed in Supplementary Table S2, where R² of all the standard curves reached >0.998. The PCR efficiency reached 93.8–107.2%.

As shown in Fig. 2, the counts of Lactobacillus spp. were higher in the crop, proventriculus, gizzard, duodenum, jejunum, ileum, and rectum than those in the cecum (P < 0.05). Lactobacilli had the highest diversity in the crop and the lowest one in the cecum. As shown in Table 1, in the crop, the counts of L. reuteri, L. johnsonii, and L. acidophilus were significantly higher than those of L. paracasei, L. brevis, L. plantarum, L. aviarius, L. delbrueckii, and L. gasseri (P < 0.05). The counts of L. reuteri, L. johnsonii, and L. acidophilus had no significant difference (P > 0.05). In the proventriculus and gizzard, the counts of L. paracasei and L. brevis were significantly lower than those of other Lactobacillus spp (P < 0.05). In the duodenum, the counts of L. aviarius, L. reuteri, L. johnsonii, L. crispatus, and L. acidophilus were significantly higher than those of L. brevis, L. plantarum, and L. gasseri (P < 0.05). In the jejunum and ileum, the counts of L. aviarius, L. reuteri, L. johnsonii, L. crispatus, L. acidophilus, L. kefiranofaciens, and L. salivarius L. plantarum were significantly higher than those of L. delbrueckii, L. paracasei, L. brevis, and L. gasseri (P < 0.05). In the cecum, the counts of L. reuteri, L. aviarius, L. johnsonii, L. crispatus, L. acidophilus, L. kefiranofaciens, L. salivarius, and L. delbrueckii were significantly higher than those of L. plantarum, L. paracasei, L. brevis, and L. gasseri (P < 0.05). In the rectum, the counts of L. reuteri, L. aviarius, L. johnsonii, L. acidophilus, L. crispatus, L. kefiranofaciens, and L. salivarius were significantly higher than those of L. plantarum, L. paracasei, L. brevis, and L. gasseri (P < 0.05). In the crop, proventriculus, gizzard, cecum, and rectum, L. reuteri had the highest diversity. In the duodenum, jejunum, and ileum, L. aviarius had the highest diversity. Compared with the lower GI tract, the upper GI tract colonized more Lactobacilli.

Lactobacillus reuteri has been used to express heterologous genes as the delivery vector or host in previous studies [19,20], and its genome-wide sequence has also been reported by Morita et al. [29]. Therefore, we analyzed the probiotic properties of six Lactobacillus reuteri isolated from broilers' GI tract.

The antibacterial activity of the isolates was determined against common intestinal pathogenic organisms as shown in Table 2. All the isolates demonstrated a certain inhibitory effect, and the inhibition zone was found to be in the range of 9–23 mm. Lactobacillus reuteri XC1, L. reuteri XC3, and L. reuteri XC4 showed a better antibacterial effect against Bacillus cereus than others according to the diameter of the inhibition zone. The antibacterial activity of the cell-free extracts treated with organic acids (acetic acid and lactic acid) was significantly higher than that in the untreated group (P < 0.05). Moreover, the antibacterial activity of the cell-free extracts treated with acetic acid was better than those treated with lactic acid. But, the antibacterial activity of all isolates disappeared when culture supernatants were treated with proteinase K and catalase.

As shown in Table 3, all the isolates were tested for antibiotic susceptibilities to nine antibiotics that were commonly used in the laboratory for constructing vector and screening recombinant strains. The results showed that all isolates were susceptible to neomycin, ampicillin, and chloromycetin, moderately susceptible to tetracycline, not resistant to any antibiotic.

The growth and survival curves of all the isolates tested at different pH values for 24 h at 37°C are shown in Fig. 3A. At pH 4.0–6.0, the growth and survival of all the isolates were not affected; but when the pH was <4, the growth of all isolates was significantly decreased. At pH values 2.0 and 3.0, the growth and survival of L. reuteri XC3 and L. reuteri XC1 were significantly better than those of other isolates (P < 0.05). At pH 2.0, the growth and survival of L. reuteri XC2, L. reuteri XC4, L. reuteri XC5, and L. reuteri XC6 were almost zero. When lactobacilli smoothly passed through the stomach into the small intestine, bile salt would become another barrier to lactobacilli growth and survival. The growth curves of the isolates in the presence of 0.3 and 0.5% bile salt are shown in Fig. 3B,C, respectively. The results showed that the growth of all isolates was only delayed, but not ended in 0.3 and 0.5% bile salt, which suggested that they were bile resistant and tolerant to some extent. The tolerance to bile salt of L. reuteri XC1 was better than that of other isolates (P < 0.05).

Different GI tract regions of chickens play different roles in feed digestion, nutrient absorption, and intestinal health, all of which depend on the ecological balance of the different microorganisms in different GI tract regions [30]. Lactobacilli are commonly agreed to dominate the anterior small intestine, crop, duodenal and jejunal epithelial cells, and digesta of chickens. As an important part of the intestinal microbiota of chickens, the account and diffusion of Lactobacilli in animals' GI tract play an important role in maintaining the ecological balance of the different microorganisms [31]. In the present study, it was found that Lactobacilli had the highest diversity in the crop and the lowest one in the cecum. Compared with the lower GI tract, the upper one colonized more Lactobacilli. Similar observations have been reported with molecular analysis of 16S rRNA genes [30]. Some reports have also investigated the composition of the ileal and cecal microflora of broilers by analysis of 16S rRNA gene sequences or bacteriological culture and culture-independent methods. Lactobacilli are numerous in the ileum, whereas the cecal microflora is dominated by obligately anaerobic bacteria and uncultivated bacteria [32–34]. However, Bjerrum et al. [35] investigated the bacterial counts in the contents of different intestinal segments in conventional and organic broiler chickens by conventional culture techniques. Their results showed that the lactobacilli counts of the crop were almost the same as ileum, cecum, and rectum in conventional broiler chickens, and the lactobacilli numbers of the crop seemed higher than the ileum, cecum and rectum in organic birds. Amit-Romach et al. [10] examined the intestinal microflora of Cobb chickens by 16S ribosomal DNA (rDNA) targeted probes, and the results indicated that in young chicks the major species present in the small intestines and ceca was Lactobacilli, with a Bifidobacteria population becoming more dominant in the ceca at older age. It suggested that the different origins and locations of the chickens or the composition and structure of the feed or the housing conditions as well as the analyzing methods influence the composition of the intestinal microbial community of chickens [31,35].

In the present study, we also determined the counts and diffusion of different Lactobacillus species in different GI tract regions, and found that L. reuteri, L. johnsonii, L. acidophilus, L. crispatus, L. salivarius, and L. aviarius were the predominant Lactobacillus species in the GI tract of chickens, and L. reuteri was the most abundant Lactobacillus species. Moreover, the diffusion of some Lactobacilli in the remainder of the gut was almost similar to the diffusion of Lactobacilli in the crop, which were present throughout the chicken digestive tract, consistent with previous studies [31,36,37] which proposed that the crop microflora acts as a bacterial inoculum for the remainder of the gut, which is critical to understanding the contribution of the microflora members to the well-being of the avian host and in the selection of species for probiotics chicken intestine [38]. Van Coillie et al. [39] found that all the isolates in the cloaca and vagina of 22–44 weeks old laying hens, belongs to the L. acidophilus, L. reuteri or L. salivarius phylogenetic groups, with the L. reuteri group being the most predominant group. Bjerrum et al. [35] reported that in the organic 41-day-old (Scan Labelle 657) chickens, 61% of the ileal lactobacilli clones were closely related to L. crispatus and 20% to L. salivarius, in conventional 40-day-old (Ross 208) chickens, and the dominating phylotype (44% of the total) was related to L. salivarius, followed by L. johnsonii (30%). Gong et al. [30] found that both L. aviarius and L. salivarius were the predominant species among Lactobacilli in the GI tract of 5-week-old (Ross × Ross) broiler chickens. The results of Hilmi et al. [31] showed that L. reuteri, L. crispatus, and L. salivarius were the most abundant species in the crops of chickens originating from four different farms using two different commercial feeds. Therefore, we proposed that L. reuteri, L. acidophilus, L. crispatus, and L. salivarius were four typical and predominant Lactobacillus species and present throughout the chicken digestive tract.

Lactobacillus reuteri has been used to express heterologous genes as the delivery vector or host in previous studies [19,20], and its genome-wide sequence has also been reported by Morita et al. [29]. Moreover, our results indicated that L. reuteri was the most abundant Lactobacillus species in the GI tract of chickens. Therefore, we are interested in this species for analyzing parts of probiotic properties to select a potential target for genetic engineering.

It was very hazardous for probiotic strains to carry potentially transmissible plasmid-encoded antibiotic resistance genes, which could be horizontally transferred into recipient bacteria of the gut to generate new antibiotic-resistant pathogens [40]. Rushdy and Gomaa [41] found that L. brevis (B23) strain was susceptible to most of the antibiotics tested and resistance was observed in the case of cefixime, nalidixic acid, vancomycin, and oxacillin. Zhou et al. [42] and Mourad et al. [43] found that most strains were susceptible to the antibiotics tested, which were used in human clinical therapy. In our study, all isolates were susceptible to neomycin, ampicillin, and chloromycetin, mildly susceptible to tetracycline, and not resistant to any antibiotics tested, which provided convenience to construct vector and screen recombinant strains in the laboratory.

Probiotic strains with the antibacterial activity depend on acid, hydrogen peroxide, or bacteriocins produced by themselves [44]. In this study, the antibacterial activity of the cell-free extracts treated with acetic acid was better than those treated with lactic acid. But, the antibacterial activity of all isolates disappeared when culture supernatants were treated with proteinase K and catalase. This indicated that the ingredients with the inhibitory effect may be the organic acids or other substances depended on organic acids, and acetic acid may play a major role. Ehrmann et al. [12] and Van Coillie et al. [39] have reported that the ingredients with the inhibitory effect on different Salmonella and Escherichia coli serotypes are related to organic acids produced by Lactobacilli strains isolated from chicken intestine.

It is very crucial for probiotics to survive and grow in the stomach with low pH and the upper GI tract containing high bile salts, which is a prerequisite for colonization and metabolic activity of probiotic bacteria in the small intestine of the host [45]. Jin et al. [46] reported that the pH of the gastric juice in chickens and ducks can be as low as 0.5–2.0, and the survival of L. fermentum isolated from chicken intestine was <30% at pH 2.0 and 3.0 for 3 h [46]. In this study, at pH 2.0 and 3.0, the growth and survival of L. reuteri XC3 and L. reuteri XC1 were significantly better than those of other isolates. At pH 2.0, the growth and survival of L. reuteri XC2, L. reuteri XC4, L. reuteri XC5, and L. reuteri XC6 were almost zero. It is suggested that L. reuteri XC1 and L. reuteri XC3 have good acid tolerance. But the results of acid tolerance from the previous report showed that ∼35 or 50% of all the L. reuteri strains tested survived after 180 minute incubation at pH 2.0 and 3.0, respectively [47]. It indicated that the acid tolerance ability of the Lactobacilli was different from different species and sources. When Lactobacilli smoothly passed through the stomach into the small intestine, bile salt would become another barrier to Lactobacilli growth and survival. Gilliland et al. [48] reported that 0.3% ox-bile is considered to be a crucial concentration to evaluate bile-tolerant probiotic LAB. In the present study, the growth of all L. reuteri was only delayed, but not ended in 0.3 and 0.5% bile, which was similar to the results described by Raghavendra and Halami [49]. But, Ehrmann et al. [12] reported that some Lactobacilli isolated from ducks' GI tract could survive in 2% oxgall. The survival of Lactobacilli is thought to be relevant to the ability to de-conjugate bile acids and the cell membranes consisting of lipids and fatty acids that are very susceptible to destruction by bile salts.

This work was supported by the grants from the National Natural Science Foundation of China (No. 31072060), China Agriculture Research System (No. CARS-40-13), the National Special Research Fund for Non-profit Sector (Agriculture) (No. 20090300606), and the National Special Research Fund for Non-profit Sector (Agriculture) (No. 20130305905).

© The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Subcellular localisation of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and their ability to form SNARE complexes are critical for determining the specificity of vesicle fusion. NPSN11, a Novel Plant SNARE (NPSN) gene, has been reported to be involved in the delivery of cell wall precursors to the newly formed cell plate during cytokinesis. However, functions of NPSN genes in plant–pathogen interactions are largely unknown. In this study, we cloned and characterized three NPSN genes (TaNPSN11, TaNPSN12, and TaNPSN13) and three plant defence-related SNARE homologues (TaSYP132, TaSNAP34, and TaMEMB12). TaSYP132 showed a highly specific interaction with TaNPSN11 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. We hypothesize that this interaction may indicate a partnership in vesicle trafficking. Expressions of the three TaNPSNs and TaSYP132 were differentially induced in wheat leaves when challenged by Puccinia striiformis f. sp. tritici (Pst). In virus-induced gene silencing (VIGS) assays, resistance of wheat (Triticum aestivum) cultivar Xingzi9104 to the Pst avirulent race CYR23 was reduced by knocking down TaNPSN11, TaNPSN13 and TaSYP132, but not TaNPSN12, implying diversified functions of these wheat SNARE homologues in prevention of Pst infection and hyphal elongation. Immuno-localization results showed that TaNPSN11 or its structural homologues were mainly distributed in vesicle structures near cell membrane toward Pst hypha. Taken together, our data suggests a role of TaNPSN11 in vesicle-mediated resistance to stripe rust.

So far, the physiological roles of NPSN genes in plant–pathogen interactions have not been well characterised. In this study, three NPSNs (TaNPSN11, TaNPSN12, and TaNPSN13) and three plant-defence related SNARE homologues (TaSYP132, TaSNAP34, and TaMEMB12) were cloned from common wheat (Triticum aestivum) cultivar Xingzi9104 and their possible interactions were investigated by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The transcriptional regulation of the three TaNPSNs and TaSYP132 in wheat response to Puccinia striiformis f. sp. tritici (Pst) was characterized using qRT-PCR and their functions were further tested by virus-induced gene silencing (VIGS) assay. Localization of TaNPSN11 on vesicle structures near cell membrane toward Pst hypha was clarified by immuno-cytochemical methods.

The Novel Plant SNARE (NPSN) genes are a family of SNARE genes that have no homologues in mammalian or yeast genomes (Sanderfoot et al., 2000). Three NPSN genes, NPSN11, NPSN12, and NPSN13, were identified in both Arabidopsis and rice (Oryza sativa) genomes and reported to locate on the plasma membrane (Zheng et al., 2002; Uemura et al., 2004; Bao et al., 2008a). Further research demonstrated that AtNPSN11 in Arabidopsis was immuno-fluorescently localized on the cell plate where it interacted with a t-SNARE protein KNOLLE during cytokinesis (Zheng et al., 2002). Interestingly, a recent study illustrated that AtNPSN11 might be involved in one of the two KNOLLE-containing tetrameric SNARE complexes, which jointly mediate membrane fusion during cytokinesis (El Kasmi et al., 2013).

Several SNARE genes in plants have been shown to be involved in plant resistance against various pathogens (Inada and Ueda, 2014). For instance, HvSNAP34 in barley (Hordeum vulgare) was reported to participate in callose deposition during non-host resistance to powdery mildew (Collins et al., 2003). Further research on AtSNAP33, the homologue of HvSNAP34 in Arabidopsis thaliana, has revealed the functional SNARE complex PEN1-SNAP33-VAMP721/722 (Wick et al., 2003; Lipka et al., 2008). Recent study shows that SEC11 from Arabidopsis modulates PEN1-dependent vesicle traffic by dynamically competing for PEN1 binding with VAMP721 and SNAP33 (Karnik et al., 2013). Tobacco (Nicotiana tabacum) NbSYP132 has been implicated in plant resistance against bacterial pathogen by mediating the secretion of pathogenesis-related protein 1 (Kalde et al., 2007). Another Golgi SNARE AtMEMB12 was targeted by miR393b* and involved in the accumulation of PR1 (Zhang et al., 2011). By interacting with potyviral 6K2 integral membrane protein, the Arabidopsis SNARE protein Syp71 is an essential host factor for successful Turnip mosaic potyvirus infection (Wei et al., 2013).

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells (Heese et al., 2001; Wick et al., 2003). Four different SNAREs form a SNARE complex to determine the specificity of intracellular fusion (Fukuda et al., 2000). In detail, one helix of the SNARE bundle is formed by one vesicle membrane-anchored SNARE (v-SNARE) and three target membrane-anchored SNAREs (t-SNAREs) through their R-, Qa-, Qb- and Qc-SNARE domains (Antonin et al., 2000; Fukuda et al., 2000).