Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.Fil: Hiriart, Yanina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Serradell, Maria de Los Angeles. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martinez, Araci. Universidad de la República; UruguayFil: Sampaolesi, Sofia. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez Maciel, Maria Dolores. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chabalgoity, Jose Alejandro. Universidad de la República; UruguayFil: Yim, Lucia. Universidad de la República; UruguayFil: Algorta, Gabriela. Universidad de la República; UruguayFil: Rumbo, Martín. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Extended-spectrum β-lactamases, transferable quinolone resistance, and virulotyping in extra-intestinal E. coli in Uruguay

Introduction: To characterize extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli isolates obtained from extra-intestinal samples in three Uruguayan hospitals. Methodology: Fifty-five ESBL-producing E. coli isolates were studied. Virulence genes, ESBLs, and PMQR genes were detected by polymerase chain reaction. ESBL-producing isolates were compared by pulsed-field gel electrophoresis. Multi-locus sequence typing was also performed on 13 selected isolates. Results: Thirty-seven isolates harbored blaCTX-M-15 (67.3%), eight blaCTX-M-2 (14.6%), five blaCTX-M-14 (9.1%), three carried both blaCTX-M-2 and blaCTX-M-14, one blaCTX-M-9, and one blaCTX-M-8. Among the CTX-M-15 producers, 92% belonged to sequence types ST131 and ST405, and carried aac(6’)Ib-cr as well. Isolates harboring blaCTX-M-2, blaCTX-M-14, blaCTX-M-9, or blaCTX-M-8 were found to be genetically unrelated. Conclusions: The successful dissemination of CTX-M-15-producing E.coli isolates seems to be linked to the spreading of high-risk clones and horizontal gene transfer. A trade-off between carrying more antibiotic resistance and less virulence-related genes could partially account for the evolutionary advantages featured by successful clones.Fil: Vignoli, Rafael. Universidad de la República; UruguayFil: García Fulgueiras, Virginia. Universidad de la República; UruguayFil: Cordeiro, Nicolás F.. Universidad de la República; UruguayFil: Bado, Inés. Universidad de la República; UruguayFil: Seija, Verónica. Universidad de la República; Uruguay. Hospital Pasteur de Montevideo; UruguayFil: Aguerrebere, Paula. Universidad de la República; UruguayFil: Laguna, Gabriel. Universidad de la República; UruguayFil: Araújo, Lucía. Universidad de la República; UruguayFil: Bazet, Cristina. Universidad de la República; UruguayFil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Chabalgoity Rodríguez, José Alejandro. Universidad de la República; Urugua

Early Peritoneal Immune Response during Echinococcus granulosus Establishment Displays a Biphasic Behavior

Cystic echinococcosis is a zoonotic disease caused by the larval stage of the cestode Echinococcus granulosus and shows a cosmopolitan distribution with a worldwide prevalence of roughly 6 million infected people. Human cystic echinococcosis can develop in two types of infection. Primary infection occurs by ingestion of oncospheres, while secondary infection is caused by dissemination of protoscoleces after accidental rupture of fertile cysts. Murine experimental secondary infection in Balb/c mice is the current model to study E. granulosus-host interaction. Secondary infection can be divided into two stages: an early stage in which protoscoleces develop into hydatid cysts (infection establishment) and a later stage in which already differentiated cysts grow and eventually become fertile cysts (chronic infection). During infection establishment parasites are more susceptible to immune attack, thus our study focused on the immunological phenomena triggered early in the peritoneal cavity of experimentally infected mice. Our results suggest that early and local Th2-type responses are permissive for infection establishment

TLR7 agonist in combination with Salmonella as an effective antimelanoma immunotherapy

Aim: We evaluated a novel approach combining the use of attenuated Salmonella immunotherapy with a Toll-like receptor agonist, imiquimod, in B16F1 melanoma-bearing mice. Materials & methods: B16F1 melanoma-bearing mice were daily treated with topical imiquimod in combination with one intratumoral injection of attenuated Salmonella enterica serovar Typhimurium LVR01. Results: The combined therapy resulted in retarded tumor growth and prolonged survival. Combination treatment led to an enhancement in the expression of pro-inflammatory cytokines and chemokines in the tumor microenvironment, with a Th1-skewed profile, resulting in a broad antitumor response. The induced immunity was effective in controlling the occurrence of metastasis. Conclusion: Salmonella LVR01 immunotherapy in combination with imiquimod is a novel approach that could be considered as an effective antimelanoma therapy

Prion Replication in the Hematopoietic Compartment Is Not Required for Neuroinvasion in Scrapie Mouse Model

Fatal neurodegenerative prion diseases are caused by the transmissible PrPSc prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion

Comparative genomics of Salmonella enterica serovar Enteritidis ST-11 isolated in Uruguay reveals lineages associated with particular epidemiological traits

Salmonella enterica serovar Enteritidis is a major cause of foodborne disease in Uruguay since 1995. We used a genomic approach to study a set of isolates from different sources and years. Whole genome phylogeny showed that most of the strains are distributed in two major lineages (E1 and E2), both belonging to MLST sequence type 11 the major ST among serovar Enteritidis. Strikingly, E2 isolates are over-represented in periods of outbreak abundance in Uruguay, while E1 span all epidemic periods. Both lineages circulate in neighbor countries at the same timescale as in Uruguay, and are present in minor numbers in distant countries. We identified allelic variants associated with each lineage. Three genes, ycdX, pduD and hsdM, have distinctive variants in E1 that may result in defective products. Another four genes (ybiO, yiaN, aas, aceA) present variants specific for the E2 lineage. Overall this work shows that S. enterica serovar Enteritidis strains circulating in Uruguay have the same phylogenetic profile than strains circulating in the region, as well as in more distant countries. Based on these results we hypothesize that the E2 lineage, which is more prevalent during epidemics, exhibits a combination of allelic variants that could be associated with its epidemic ability

Genotypic and phenotypic analysis of Salmonella enterica serovar Derby, looking for clues explaining the impairment of egg isolates to cause human disease

Salmonella enterica serovar Derby causes foodborne disease (FBD) outbreaks worldwide, mainly from contaminated pork but also from chickens. During a major epidemic of FBD in Uruguay due to S. enteritidis from poultry, we conducted a large survey of commercially available eggs, where we isolated many S. enteritidis strains but surprisingly also a much larger number (ratio 5:1) of S. Derby strains. No single case of S. Derby infection was detected in that period, suggesting that the S. Derby egg strains were impaired for human infection. We sequenced fourteen of these egg isolates, as well as fifteen isolates from pork or human infection that were isolated in Uruguay before and after that period, and all sequenced strains had the same sequence type (ST40). Phylogenomic analysis was conducted using more than 3,500 genomes from the same sequence type (ST), revealing that Uruguayan isolates clustered into four distantly related lineages. Population structure analysis (BAPS) suggested the division of the analyzed genomes into nine different BAPS1 groups, with Uruguayan strains clustering within four of them. All egg isolates clustered together as a monophyletic group and showed differences in gene content with the strains in the other clusters. Differences included variations in the composition of mobile genetic elements, such as plasmids, insertion sequences, transposons, and phages, between egg isolates and human/pork isolates. Egg isolates showed an acid susceptibility phenotype, reduced ability to reach the intestine after oral inoculation of mice, and reduced induction of SPI-2 ssaG gene, compared to human isolates from other monophyletic groups. Mice challenge experiments showed that mice infected intraperitoneally with human/pork isolates died between 1–7 days p.i., while all animals infected with the egg strain survived the challenge. Altogether, our results suggest that loss of genes functions, the insertion of phages and the absence of plasmids in egg isolates may explain why these S. Derby were not capable of producing human infection despite being at that time, the main serovar recovered from eggs countrywide