Étude de l’efficacité de la vaccination à Salmonella Enteritidis chez la poule pondeuse et de la protection contre l’infection

Les infections à Salmonella Enteritidis chez les humains sont associées à la consommation d’œufs ou d’ovoproduits contaminés. La vaccination est un outil utilisé pour diminuer les risques d’infection à SE chez la volaille, mais avec des résultats variables. Au Canada deux bactérines, MBL SE4C et Layermune, sont couramment utilisées pour lutter contre SE. Cependant, leur efficacité n’a pas été complètement déterminée chez les poules pondeuses plus âgées. Par ailleurs, la capacité de ces vaccins à prévenir la transmission verticale et horizontale n’a pas encore été étudiée. L’objectif principal de cette étude était d’évaluer l’effet des deux bactérines sur la réponse immunitaire chez les poules pondeuses, de vérifier la protection conférée par ces vaccins contre l’infection expérimentale à SE, et d’identifier des protéines immunogènes afin de développer un vaccin sous-unitaire. Les oiseaux ont été vaccinés avec deux protocoles d’immunisation en cours d’élevage (soit à 12 et 18, ou à 16 semaines d’âge). Le groupe contrôle a été injecté avec la solution saline. Les oiseaux ont été inoculés per os avec 2 x 109 CFU de la souche SE lysotype 4 à 55 ou à 65 semaines d’âge. Les anticorps (IgG et IgA) ont été mesurés à différents temps avec un ELISA maison en utilisant l’antigène entier de SE. La phagocytose, flambée oxydative, les populations des splénocytes B et T ont été analysées en utilisant la cytométrie en flux. Les signes cliniques, l’excrétion fécale, la contamination des jaunes d’œufs et l’invasion des salmonelles dans les organes ont été étudiés pour évaluer l’efficacité de protection. La transmission horizontale a aussi été étudiée en évaluant l’infection à SE chez les oiseaux mis en contact avec les oiseaux inoculés. Les protéines immunogènes ont été identifiées par SDS-PAGE et Western blot à l’aide d’antisérums prélevés suite à la vaccination et/ou à l’infection expérimentale/naturelle, puis caractérisées par la spectrométrie de masse. Le protocole de vaccination avec deux immunisations a généré un niveau élevé de séroconversion à partir de 3 jusqu’à 32-34 semaines post-vaccination par rapport à celui avec une seule immunisation (p < 0.02), mais il n’y avait plus de différence entre les groupes à 54 et 64 semaines d’âge. Il n’y a pas eu de corrélation entre les niveaux d’IgG et les taux d’isolement des salmonelles dans les organes et des jaunes d’œuf. La production des IgA n’a été observée que chez les oiseaux vaccinés avec 2 injections de MBL SE4C (p ≤ 0.04). Après l’infection expérimentale, la production des IgA a été significativement plus élevée aux jours 1 et 7 p.i dans l’oviducte des oiseaux vaccinés (sauf pour le groupe vacciné avec 2 injections de Layermune) par comparaison avec le groupe contrôle (p ≤ 0.03). Seule la bactérine MBL SE4C a eu un effet protecteur contre la contamination des jaunes d’œuf chez les oiseaux infectés. Ce vaccin réduit partiellement en utilisant deux immunisations, le taux d’excrétion fécale des salmonelles chez les oiseaux inoculés et les oiseaux horizontalement infectés (p ≤ 0.02). Cinq des protéines identifiées par la spectrométrie de masse sont considérées comme des protéines potentiellement candidates pour une étude plus approfondie de leur immonogénicité: Lipoamide dehydrogenase, Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase), Elongation factor Tu (EF-Tu), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et DNA protection during starvation protein. En général, les bactérines ont induit une immunité humorale (IgG et IgA) chez les poules pondeuses. Cette réponse immunitaire a protégé partiellement les oiseaux quant à l’élimination des salmonelles, la contamination des jaunes d’œuf, ainsi que la transmission horizontale. Dans cette étude, la bactérine MBL SE4C (avec deux immunisations) s’est montrée plus efficace pour protéger les oiseaux que la bactérine Layermune. Nos résultats apportent des informations objectives et complémentaires sur le potentiel de deux bactérines pour lutter contre SE chez les poules pondeuses. Étant donné la protection partielle obtenue en utilisant ces vaccins, l’identification des antigènes immunogènes a permis de sélectionner des protéines spécifiques pour l’élaboration éventuelle d’un vaccin plus efficace contre SE chez les volailles.Contaminated eggs and egg products have been associated with outbreaks of human Salmonella Enteritidis (SE) infections. Killed bacteria (bacterins) have been used to control Salmonella infections in poultry but variation in the conferred protection has been observed. In Canada the bacterins MBL SE4C and Layermune are currently used to control SE. However, their efficacy in protecting older layers has not been fully determined. Furthermore, the capacity of these bacterins to prevent vertical and horizontal transmissions has not yet been investigated. The main objectives of this study were to evaluate the effect of two available commercial bacterins on the immune response of laying hens, to verify the protection conferred by these vaccines against SE challenge and to identify immunogenic proteins to develop an oral subunit vaccine. Laying hens were vaccinated with two immunization schedules prior to the lay cycle (either at 12 and 18, or 16 weeks of age). The control group was injected with a saline solution. Laying hens were later inoculated per os with 2 x 109 CFU of SE PT4 strain either at 55 or 65 weeks of age. Serum IgG and mucosal IgA antibodies were measured with an in-house SE whole cell antigen ELISA. The phagocytosis, oxidative burst, splenic T and B cells populations were analyzed using flow cytometry. Clinical signs, fecal shedding, egg yolks contamination and organ invasion by SE were assessed to evaluate vaccine protection. Potential horizontal transmission from inoculated laying hens to non-inoculated laying hens, housed in the same isolator unit, was also evaluated. Immunogenic proteins were identified by SDS-PAGE and Western blot with sampled antisera during vaccination and/or infection of poultry with SE and then subjected to mass spectrometry. The vaccination protocol with two immunizations showed a higher seroconversion level than the single vaccination at 3 until 32-34 weeks post vaccination (p < 0.02) but no difference before challenge (54 and 64 old weeks). There was no relationship between high IgG level and SE isolation rates in organs and egg yolks. Only the MBL SE4C vaccine elicited IgA antibody production at 3 weeks post vaccination in both immunization protocols (p ≤ 0.04). Significant higher mucosal IgA levels were observed at day 1 and 7 post challenge in oviduct of vaccinated birds (except for the twice vaccinated Layermune group) compared to the control group (p ≤ 0.03). Humoral efficacy to protect from SE contamination of egg yolk was only observed in MBL SE4C vaccinated group and only this bacterin administered twice reduced SE shedding rate in inoculated birds and their exposed cagemates (p ≤ 0.02). A set of 5 proteins were considered as putative protein candidates to further detailed study on their immunogenicity: Lipoamide dehydrogenase; Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase); Elongation factor Tu (EF-Tu); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and DNA protection during starvation protein. Overall, the commercial bacterins induced humoral immunity (IgG and IgA antibodies) in laying hens. This immune response partially protected for SE clearance, egg yolks contamination as well as horizontal transmission. In this study, MBL SE4C bacterin appeared to be more efficient in comparison to Layermune for protection of hens with a vaccination protocol comprising two immunizations. Our results provide additional and objective information on the potential of these vaccines for the control of SE in laying hens. Considering the partial protection achieved with the use of these bacterins, the identification of immunogenic antigens could help in the selection of specific proteins to elaborate a more efficient vaccine against SE in poultry

A Simple Procedure for Extraction of Surface Protein of Salmonella Serotypes and Escherichia coli Strains Isolated from Poultry and Pigs

Salmonella and E.coli possess different surface protein structures that can induce protective immune responses. Identification of these proteins capacitates development of diverse applications in prevention and diagnosis that contribute to effectively control disease-causing enterobacteria pathogens such as Salmonella and E.coli. A simple procedure for obtaining protein complexes of Salmonella serotypes and E.coli is performed in this study. A sonication process with heat treatment of whole bacteria induced the release of protein complexes. Concentration of the protein extract was quantified using protein quantification Kits-Rapid, and protein complex profile was obtained by SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and silver staining. The concentrations of protein ranged from 29.45 to 45.35 µg/mL in the  Salmonella protein extracts, and from 25.35 to 36.72 µg/mL in the E.coli protein extracts. Six major groups of proteins from E. coli (YfiO, NipB, OmpF, YfgL, Talc, YaeT) and four major groups of proteins from Salmonella (Flagellin, OmpA, Porin, SEF21) were preliminarily determined by a simple procedure of extraction based on the molecular weight

Antibacterial and Antioxidant Activity of Rhodomyrtus Tomentosa and Cinnamomum Zeylanicum Crude Extracts

The aim of this study was to investigate the extraction method for R. tomentosa and C. zeylanicum leaves and the evaluation of antibacterial and antioxidant activities of crude extracts. The results of the study showed that the active ingredients of crude extracts were clearly separated by Thin-layer chromatography and the presence of rhodomyrtone in R. tomentosa crude extract and cinnamaldehyde in C. zeylanicum crude extract. R. tomentosa crude extract was antibacterial activity against Staphylococcus aureus with 13.1 mm of inhibition zone, but is not effective against Salmonella Typhimurium. C. zeylanicum leaf extract did not show antibacterial activity on both S. aureus and S. Typhimurium. At a dilution of 1/2 of the R. tomentosa crude extract can completely inhibit S. aureus growth. This study also indicated the presence of antioxidant compounds such as flavonoids, tannins, phenols and terpenoids in C. zeylanicum and R. tomentosa crude extracts. The results showed that R. tomentosa and C. zeylanicum crude extracts should be used as a biotherapy alternative to antibiotic therapy. However, further study would be needed to investigate the antibacterial activity of crude extracts in vivo

Using learning games for fourth-grade students to teach Vietnamese lessons and teachers' perspectives on learning games

Students can learn &nbsp;&nbsp;in&nbsp;&nbsp; a&nbsp;&nbsp; meaningful way through the use of educational games. When students apply their skills and information in order to progress through a game, a huge degree of engagement is generated among them in the classroom as a result both their capabilities and their learning &nbsp;&nbsp;increase. 153 children from primary schools in Vietnam participated in this research. The children were split into two groups: an experimental and a control group. Both groups participated in two sessions of word and sentence practice as well as spelling instruction. According to the findings of the study, the educational outcomes of the students are greatly improved when games are used in their classrooms. In addition, research has shown that some teachers are against the use of games in the classroom since it makes it more difficult to prepare educational materials. Some educators believe that playing educational games is a more effective method of instruction as it allows for greater flexibility during the learning process. This makes a contribution to the theoretical framework that is necessary to guide educators and instructors in the process of improving learning game programs for children in primary schools and other learners

Development of a diagnostic scar marker for Vibrio shilonii caused acute hepatopancreatic necrosis disease in whiteleg shrimp

Background: In a previous report, we showed that Vibrio shilonii was found on whiteleg shrimp (Litopenaeus vannamei) with acute hepatopancreatic necrosis disease in Thua Thien Hue province, Vietnam. This study was performed to develop a diagnostic molecular marker generated by random amplified polymorphic DNA (RAPD) for V. shilonii rapid detection.Methods: Pathogen Vibrio spp. were isolated from shrimps and fishes, and were identified by 16S rRNA sequencing. Genetic diversity of Vibrio strains was analysis by RAPD technique. Specific PCR product for V. shilonii was cloned and sequenced. SCAR marker was developed from specific PCR product.Result: Twenty random primers were evaluated for RAPD to identify DNA polymorphisms between Vibrio species. The random primer OPN-06 generated a 468-bp DNA fragment specific for V. shilonii. This was then converted into a sequence-characterized amplified region (SCAR) marker designated N6-441.Conclusion: Specific primers (Vshi-441F/Vshi-441R) amplified a unique DNA fragment in all V. shilonii isolates but not in the other Vibrio spp. This PCR assay showed significantly sensitive to the target DNA and reliably for the amplification the V. shilonii genome.Keywords: AHPND; RAPD; SCAR; Vibrio shilonii; Vietna

Evaluation of Xpert MTB/RIF and MODS assay for the diagnosis of pediatric tuberculosis

BACKGROUND: Tuberculosis (TB) in children is rarely confirmed due to the lack of effective diagnostic tools; only 10 to 15% of pediatric TB is smear positive due to paucibacillary samples and the difficulty of obtaining high-quality specimens from children. We evaluate here the accuracy of Xpert MTB/RIF in comparison with the Micoroscopic observation drug susceptibility (MODS) assay for diagnosis of TB in children using samples stored during a previously reported evaluation of the MODS assay. METHODS: Ninety-six eligible children presenting with suspected TB were recruited consecutively at Pham Ngoc Thach Hospital in Ho Chi Minh City Viet Nam between May to December 2008 and tested by Ziehl-Neelsen smear, MODS and Mycobacterial growth Indicator (MGIT, Becton Dickinson) culture. All samples sent by the treating clinician for testing were included in the analysis. An aliquot of processed sample deposit was stored at −20°C and tested in the present study by Xpert MTB/RIF test. 183 samples from 73 children were available for analysis by Xpert. Accuracy measures of MODS and Xpert were summarized. RESULTS: The sensitivity (%) in detecting children with a clinical diagnosis of TB for smear, MODS and Xpert were 37.9 [95% CI 25.5; 51.6], 51.7 [38.2; 65.0] and 50.0 [36.6; 63.4], respectively (per patient analysis). Xpert was significantly more sensitive than smear (P=0.046). Testing of additional samples did not increase case detection for MODS while testing of a second sputum sample by Xpert detected only two additional cases. The positive and negative predictive values (%) of Xpert were 100.0 [88.0; 100.0] and 34.1 [20.5; 49.9], respectively, while those of MODS were 96.8 [83.3; 99.9] and 33.3 [19.6; 49.5]. CONCLUSION: MODS culture and Xpert MTB/RIF test have similar sensitivities for the detection of pediatric TB. Xpert MTB RIF is able to detect tuberculosis and rifampicin resistance within two hours. MODS allows isolation of cultures for further drug susceptibility testing but requires approximately one week to become positive. Testing of multiple samples by xpert detected only two additional cases and the benefits must be considered against costs in each setting. Further research is required to evaluate the optimal integration of Xpert into pediatric testing algorithms

Temporal Trends in Tobacco Smoking Prevalence During the Period 2010–2020 in Vietnam: A Repeated Cross-Sectional Study

Objectives: This study used repeated cross-sectional data from three national surveys in Vietnam to determine tobacco smoking prevalence from 2010 to 2020 and disparities among demographic and socioeconomic groups.Methods: Tobacco smoking temporal trends were estimated for individuals aged 15 and over and stratified by demographic and socioeconomic status (SES). Prevalence estimates used survey weights and 95% confidence intervals. Logistic regression models adjusted for survey sample characteristics across time were used to examine trends.Results: Tobacco smoking prevalence dropped from 23.8% in 2010 to 22.5% in 2015 and 20.8% in 2020. The adjusted OR for 2015 compared to 2010 was 0.87, and for 2020 compared to 2010 was 0.69. Smoking decreased less for employed individuals than unemployed individuals in 2020 compared to 2010. Smoking was higher in the lower SES group in all 3 years. Higher-SES households have seen a decade-long drop in tobacco use.Conclusion: This prevalence remained constant in lower SES households. This highlights the need for targeted interventions to address the specific challenges faced by lower-SES smokers and emphasizes the importance of further research to inform effective policies

Changing epidemiology and antimicrobial susceptibility of bloodstream infections at a Vietnamese infectious diseases hospital (2010–2020)

Bloodstream infection (BSI) poses a global health problem, with diverse organisms and rising antimicrobial resistance (AMR). Here, we characterized trends in BSI prevalence, AMR, and antibiotic use at a Vietnamese infectious diseases hospital from 2010 to 2020. Among 108,303 cultured blood samples, 8.8% were positive, yielding 7995 pathogens. Of 7553 BSI cases, 86.4% were community-acquired. BSI prevalence varied from 17 to 35 cases/1000 admissions/year, highest in HIV/hepatitis wards and patients >60. The in-hospital mortality or hospice discharge outcome was 21.3%. The top three pathogens, E. coli (24%), K. pneumoniae (8.7%) and S. aureus (8.5%) exhibited increasing prevalence and multidrug resistance. Pathogens like Cryptococcus neoformans (8.4%), Talaromyces marneffei (6.7%), and Salmonella enterica (6.5%) declined. E. coli and K. pneumoniae were prevalent in older adults with community-acquired BSIs. Antibiotic use reached 842.6 DOT/1000 PD and significantly reduced after an antibiotic control policy. Enhanced surveillance and antimicrobial stewardship are crucial for managing BSIs in Vietnam

The transfer and decay of maternal antibody against Shigella sonnei in a longitudinal cohort of Vietnamese infants.

BACKGROUND: Shigella sonnei is an emergent and major diarrheal pathogen for which there is currently no vaccine. We aimed to quantify duration of maternal antibody against S. sonnei and investigate transplacental IgG transfer in a birth cohort in southern Vietnam. METHODS AND RESULTS: Over 500-paired maternal/infant plasma samples were evaluated for presence of anti-S. sonnei-O IgG and IgM. Longitudinal plasma samples allowed for the estimation of the median half-life of maternal anti-S. sonnei-O IgG, which was 43 days (95% confidence interval: 41-45 days). Additionally, half of infants lacked a detectable titer by 19 weeks of age. Lower cord titers were associated with greater increases in S. sonnei IgG over the first year of life, and the incidence of S. sonnei seroconversion was estimated to be 4/100 infant years. Maternal IgG titer, the ratio of antibody transfer, the season of birth and gestational age were significantly associated with cord titer. CONCLUSIONS: Maternal anti-S. sonnei-O IgG is efficiently transferred across the placenta and anti-S. sonnei-O maternal IgG declines rapidly after birth and is undetectable after 5 months in the majority of children. Preterm neonates and children born to mothers with low IgG titers have lower cord titers and therefore may be at greater risk of seroconversion in infancy