Memoria para optar al Título Profesional de Médico Veterinario / Salmonella Enteritidis (S.E.) es una de las principales causas de toxiinfecciones alimentarias en el mundo, además de causar importantes pérdidas económicas a la industria avícola. Dentro de los alimentos implicados en los brotes de salmonelosis humana, el huevo cumple un rol fundamental en la transmisión de este enteropatógeno bacteriano. Debido al surgimiento de multiresistencia a los antimicrobianos, y a que ninguna de las medidas de control aplicadas por la industria avícola ha logrado eliminar esta bacteria del contenido de los huevos, es que nace la idea de utilizar bacteriófagos para el biocontrol de S.E. en huevos. El objetivo del presente estudio fue determinar la capacidad biocontroladora de una mezcla de tres bacteriófagos líticos, sobre S.E. contenida en huevos permeabilizados. Se analizaron las efectividades tanto de dos vías de administración de estos como de diferentes tiempos, sobre el biocontrol de S.E. en huevos experimentalmente contaminados. Para ésto, huevos SPF fueron contaminados, a través de la cámara de aire, con una suspensión de S.E. (1,28 x 101 UFC/mL); dos horas después se adicionaron los fagos (107 UFP/mL), mediante dos formas de administración: inmersión o aspersión de gota gruesa. Los huevos así tratados fueron mantenidos a temperatura ambiente hasta por cinco días, divididos según vía de administración en cinco grupos paralelos, se analizaron por bacteriologías cualitativa y cuantitativa (UFC/mL) cada 24 horas (D1 a D5). El diseño experimental incluyó, además, un grupo control de infección, inoculado sólo con S.E. y dos grupos controles de fagos que sólo recibieron la mezcla de fagos en estudio, uno por inmersión y el otro por aspersión. Los resultados de la bacteriología cualitativa mostraron que los grupos de huevos que recibieron terapia con fagos, tanto por inmersión como por aspersión, en ningún día de los análisis de las muestras (D1 a D5) tuvieron reducciones significativas (p > 0,05) en la incidencia de contaminación. Sin embargo, la bacteriología cuantitativa mostró que los fagos fueron capaces de lograr disminuciones significativas (p < 0,05) en los recuentos de S.E. de aproximadamente 3 log, independiente de las vías de administración y de los tiempos. Estos resultados sugieren que los bacteriófagos pueden ser una alternativa efectiva para el control de Salmonella Enteritidis en huevos, dosificados tanto por inmersión como por aspersión. Además, se comprobó que la actividad lítica de los bacteriófagos se mantuvo en el tiempo, por hasta 5 días

Memoria para optar al Título Profesional de Médico Veterinario / Actualmente la importancia en salud pública de las bacterias del género Salmonella es ocasionada por el aumento de resistencia antimicrobiana que ha tenido en los últimos años, debido al inadecuado uso de antimicrobianos. La infección se asocia al consumo de alimentos contaminados, sin embargo, cobra importancia el rol de las aves silvestres como reservorios y agentes diseminadores de genes de resistencia a antimicrobianos en seres humanos y animales. A pesar de que la mayor cantidad de casos clínicos en seres humanos ocurren durante verano, pocos estudios reportan si existe estacionalidad en el aislamiento de cepas de Salmonella desde aves silvestres. El objetivo de esta Memoria fue determinar la existencia de variación estacional en la detección de cepas de S. enterica aisladas desde heces de Larus dominicanus de la Región de Valparaíso y caracterizar fenotípica y genotípicamente aquellas cepas con resistencia antimicrobiana. Se analizó un total de 608 muestras obtenidas mediante torulado de heces frescas tomadas directamente desde el ambiente. Las cepas aisladas fueron confirmadas mediantes la Reacción de la Polimerasa en Cadena (PCR) para el gen invA. Para determinar la asociación entre el aislamiento de cepas de Salmonella y la estación del año se utilizó el programa Infostat®. La determinación de fenotipos de resistencia se realizó mediante el método de difusión en placa (Kirby Bauer). Los perfiles genéticos fueron evaluados mediante un PCR para los genes: tet(A), tet(B), tet(G), blaPSE-1, blaTEM, blaCMY, aadB, aacC e integrones clase 1. Se aislaron 7 cepas de Salmonella que no fueron asociadas estadísticamente a la estación del año en que fueron obtenidas. Se encontró un total de 5 perfiles fenotípicos, y 2 perfiles genotípicos de resistencia antimicrobiana, siendo blaTEM el único gen encontrado en las cepas estudiadas. Aunque genotípicamente no coinciden los resultados, probablemente porque la resistencia encontrada a nivel fenotípico es codificada por otros genes no estudiados en esta Memoria, se evidencia el impacto que tienen las aves silvestres en la mantención y diseminación de bacterias resistentes a agentes antimicrobianos. / Nowadays, the importance in public health regarding the Salmonella genus bacteria is caused by the increase of the antimicrobial resistance in the last few years, given the injudicious use of antimicrobials. The infection is associated with the consumption of contaminated food, however, the role of wild birds takes an important place, for they behave as natural reservoirs and dissemination agents of resistance genes to antimicrobials in humans and animals. Despite that most of clinical cases in humans occur during the summer, little research has reported if there exists seasonality in the isolation of Salmonella strains from wild birds. The objective of this research was to determine the existence of seasonal variation in the detection of strains of S. enterica from faeces of Larus dominicanus in the Valparaíso Region, and, to characterise both phenotypically and genotypically those strains with antimicrobial resistance. A total of 608 fresh faecal samples were taken with swabs directly from the environment. The isolated strains were confirmed by the polymerase chain reaction (PCR) for the invA gene. To determine the association between the isolation of Salmonella strains and the season, the software Infostat® was used. The determination of resistance phenotypes was carried out by the agar diffusion test (Kirby Bauer). The genetic profiles were evaluated through PCR for the genes tet(A), tet(B), tet(G), blaPSE-1, blaTEM, blaCMY, aadB, aacC and class 1 integrons. Seven Salmonella strains were isolated, because they were not statistically associated with the season when they were taken. A total of 5 phenotypic profiles were found, as well as 2 genotypic profiles of antimicrobial resistance, being blaTEM the only gene found in the researched strains. Although the results do not match genotypically –probably perhaps the resistance found at a phenotypic level is codified by other genes which were not studied for this Project, the impact that wild birds have in the storage and spreading of bacteria resistant to antimicrobial agents is evident. / Financiamiento: Proyecto Fondecyt 11110398.

>Magister Scientiae - MSc / The aim of this study was to investigate the presence of Salmonella in the tea processing environment, to identify and detect potential virulence genes isolated from Salmonella in the tea, and to determine the antibiotic resistance levels of Salmonella isolated from fermented Rooibos. / National Research Foundation (NRF)

Analiza el efecto de la lactoferrina en la cinética de crecimiento de Salmonella typhimurium cepa SL 1344 ΔhilA, evalúa el efecto citotóxico del tratamiento con gentamicina a células HEp-2, determina el efecto in vitro de la lactoferrina sobre la adhesión de Salmonella typhimurium SL 1344 ΔhilA y especifica el tratamiento y concentración de lactoferrina que permita mayor disminución de la adherencia e invasión a las células HEp-2. / Tesis

Despite their beneficial effects, concerns have been raised about the role of antimicrobial growth promoters (AGP) in the emergence of antibiotic resistant bacteria. This study evaluated the effects of approved AGP on the emergence of antibiotic resistance in commensal E. coli and foodborne pathogen Salmonella. A survey of antibiotic resistance levels in commercial broiler chicken farms in the Fraser Valley (B.C.) and an experimental feeding trial were conducted from May 2004 to February 2005 and May to November 2005, respectively. The latter examined the effects of ten AGP formulations (bambermycin, penicillin, salinomycin, bacitracin, combination of salinomycin and bacitracin, chlortetracycline, virginiamycin 11ppm, virginiamycin 22ppm, monensin and narasin) on bird performance as well. Multiple antibiotic resistant commensal E. coli and Salmonella carrying virulence genes were found at commercial broiler chicken farms and therefore may serve as reservoirs for these genes. There was no significant difference between feed formulations on the phenotypic or genotypic characteristics of the isolates, except for tetracycline resistance gene tet(B). In the experimental feeding trial, broiler chickens were fed a diet including or excluding AGP. Birds were sampled prior to and weekly during feeding of the control and the AGPP containing diets. Although not detected on day 0, E. coli increased after day 7 to more than 9.9 log10 CFU/g in ceca. Multi-drug resistant E. coli were isolated from birds fed the ten AGP containing diets as well as the control diet. Except for penicillin, none of the AGP containing diets significantly improved bird performance compared to the control diet (P>0.05). Good management practices can significantly improve broiler chickens performance and decrease the mortality rate. / Land and Food Systems, Faculty of / Graduate

Maternal tolerance during pregnancy increases the risk of infection with certain intracellular pathogens such as Salmonella enterica serovar Typhimurium (S.Tm). Systemic S.Tm infection during pregnancy in normally resistant 129X1/SvJ mice, with a functional natural resistance-associated macrophage protein-1 (Nramp1), leads to severe placental infection followed by fetal and maternal death. We hypothesized infection-induced inflammatory trophoblast cell death contributes to adverse pregnancy outcomes. We therefore investigated the kinetics of systemic and oral S.Tm infection in wild-type and gene deficient mice with defects in specific inflammatory pathways. Systemic infection with S.Tm resulted in preferential placental replication compared to other tissues in Nramp1+/+ mice. At 24 hours, <25% of individual placentas per mouse were infected, progressively increasing to >75% by 72 hours which correlated with a steady increase in resorption rates. Moreover, placental infection was associated with increased neutrophils, macrophages and natural killer cells whereas neutrophil numbers in the spleen remained unchanged, suggesting dichotomous modulation of inflammation in the systemic compartment compared to the feto-maternal interface. Oral infection resulted in systemic dissemination of the bacteria, substantial placental colonization and fetal loss five days post-infection in C57BL/6J mice. Systemic infection in pregnant cell death deficient Rip3-/-Nramp1+/+ mice (with defective necroptosis) resulted in decreased fetal demise relative to Nramp1+/+ and Caspase-1,11-/-Nramp1+/+ mice (with defective pyroptosis) suggesting a role for necroptotic inflammation. This study provides insight into the kinetics and mechanism of inflammation and cell death during placental S.Tm infection. Such studies may assist in the rational management of foodborne pathogens contracted during pregnancy.

Salmonella typhimurium (ST) infection not only causes salmonellosis in humans, but also can result in great economic losses in the typically narrow-margin, high-volume broiler business due to reduced growth rates and mortalities. Over the last decade, the use of antibiotics and attenuated vaccines to restrain or prevent bacterial infections in domestic animals has been criticised because of the possible development of antibiotic resistance and the potential dangers of residual antibiotics and vaccines in animal-derived food products for human consumption. For these reasons, many countries have begun phasing out growth promoting antibiotics in broiler diets. It is therefore essential for the poultry production industry to develop feed additives and processing techniques as alternatives for sub-therapeutic dietary supplementation of antibiotics. However, innovative research is needed to evaluate the efficacy of new and existing alternative products. The general aim of this trial was to determine the effects of Salmonella typhimurium colonisation of the gastrointestinal tract of broiler chicks on gastrointestinal health and production performance. The effect of Zinc-Bacitracin (Zn-BC), a commonly used antibiotic growth promoter in the poultry industry, on Salmonella colonisation was also measured. A pilot trial was first conducted to determine the level of Salmonella typhimurium required to infect broiler chicks, and the necessity of administering an immunosuppressive agent in order to obtain infection. The main trial followed to determine the effects of Salmonella typhimurium on gastrointestinal health and function. The ultimate aim of the study was to obtain baseline values of various parameters that could be used in future trials for the evaluation of antibiotic alternative products. The results obtained from the pilot trial showed that it was not necessary to administer cyclophosphamide as the Salmonella typhimurium proved to be highly virulent. The cloacal swabs taken in the second trial showed that the use of Zn-BC as an antibiotic did not inhibit Salmonella colonisation in the challenged birds. The inclusion of Zn-BC in this trial inhibited the growth of the gut microflora allowing the Salmonella to proliferate in the body of the chicken, which lead to the conclusion that the routine inclusion of Zn-BC at sub-clinical levels as a growth promoter may be detrimental when the bird gets exposed to Gram(-) bacteria, such as Salmonella. In both of the trials, Salmonella challenge resulted in enlargement of the organs with a consequent increase in the organ weights. In the pilot trial there was a significant difference (P<0.0033) of the control weights for the duodenum, ileum, caeca and liver and those of the Salmonella infected birds. Control birds that did not receive CY had duodenum weights of 1.00 (±0.236) while the birds infected with 1 x 108 CFU/mL had weights of 1.99 (±0.310), while the control birds that did receive CY had duodenum weights of 0.98 (±0.244) with the Salmonella infected birds having weights of between 1.79 (±0.299) and 2.13 (±0.006). Significant results (P<0.016) in the main trial were found to occur predominantly at 7 days of age for the duodenum, jejenum, ileum and caeca weights. Control birds in the group that did receive antibiotics had 7 day duodenum weights of 1.80 (±0.301) compared to the Salmonella infected bird which had weights of between 2.33 (±0.376) and 2.51 (±0.424). In general Salmonella did not affect the growth and performance of the challenged birds. Birds challenged with Salmonella showed a tendency to have enlarged livers, possibly due to hepatic damage. In the main trial there was a significant difference (P<0.016) in liver weights at 28 days of age between the control and Salmonella infected groups regardless of whether the birds received antibiotics or not. The control birds that received antibiotics had liver weights of 3.24 (±0.234) while the birds infected with the higher level of Salmonella had weights of 3.86 (±0.542). This finding, together with the noticeable, although mainly insignificant, trend of decreased serum albumin levels and increased serum globulin and total serum protein levels noted in infected birds can be used in conjunction to measure the effect of ST on liver damage. Salmonella colonisation resulted in an increase in the severity of lesions seen in the gastrointestinal tract (P<0.0016). Histopathology results proved to be inconsistent and did not provide any conclusive evidence on the effect of Salmonella on the organs. Villi measurements taken in the second trial showed that Salmonella significantly (P<0.016) shortened the length of the villi in the duodenum and jejenum of challenged birds when measured at 28 days of age. Control birds had duodenum villi length of 662.5 (±56.79) while those birds infected with Salmonella had lengths of between 558.9 (±77.74) and 537.0 (±51.66). There was a significant difference in the duodenum villi length regardless of antibiotic inclusion into the diet. In the birds that did receive antibiotics, there was a significant difference (P<0.016) in the jejenum villi length with the control birds having the longest villi 725.7 (±90.92) while the birds infected with the higher level of Salmonella having the shortest villi 557.2 (±124.5). It would appear that using all of the information and results obtained for liver weights, broiler performance, serum biochemical level, lesion scoring, histopathology and villous morphological measurements should be used in conjunction with one another to measure the effect of Salmonella on the broiler chicken. The results obtained in this trial clearly show just how significant a problem Salmonella infection can be in the poultry industry due to seemingly healthy adult birds displaying little or no systemic disease being non-symptomatic carriers. Many of the Para-typhoid salmonellae do not always produce clinical signs in chicks, and their presence in the poultry industry may go unrecognised for this reason. / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Animal and Wildlife Sciences / unrestricted

Orientadores: Marcelo Brocchi, Gustavo Bueno Gregoracci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T09:27:26Z (GMT). No. of bitstreams: 1 SartiSprogis_AdrianeCristina_M.pdf: 1852459 bytes, checksum: 397af9ee41d085346189ca76eee83f96 (MD5) Previous issue date: 2014 / Resumo: A salmonelose é uma zoonose que representa um sério problema de saúde pública mundial, devido: à sua alta prevalência, à dificuldade de seu controle, ao seu caráter endêmico, à morbidade e à mortalidade. O conhecimento da ocorrência das diferentes sorovariedades de S. enterica em diferentes regiões e países pode ajudar no rastreamento e reconhecimento de patógenos emergentes, e assim implementar políticas de tratamento e prevenção. A grande maioria das sorovariedades expressa dois tipos diferentes de antígenos flagelares codificados pelos genes fliC (fase 1) e fljB (fase 2), sendo assim denominadas bifásicas. Contudo, algumas sorovariedades expressam apenas uma das fases, e são denominadas monofásicas. É possível que a variação de fase flagelar em S. enterica esteja associada a uma função de escape do sistema imunológico, por aumentar o repertório de antígenos expressos pela célula bacteriana, evitando temporariamente a resposta imune celular. Assim sendo, S. enterica bifásicas possuiriam uma vantagem seletiva sobre as monofásicas, porém isso não é totalmente verificado nos estudos epidemiológicos, pois no Estado de São Paulo, S. enterica I,4,[5],12:i:-, (monofásica) é uma das mais comumente associadas aos casos de diarreia e/ou infecções sistêmicas em pacientes humanos. De fato, a partir da década de 1990 houve um aumento significativo da S. enterica I,4,[5],12:i:- em muitos países. Sequências de profagos são muito comuns em S. enterica, sendo de conhecimento que esses fagos codificam vários fatores que contribuem para patogenicidade, diversidade genética e/ou características que aumentam o fitness. Coculturas experimentais de linhagens de S. enterica podem induzir espontaneamente profagos, que matam bactérias sensíveis, e assim a indução espontânea de fagos em uma população lisogênica acentua a competitividade entre populações. Neste estudo foram analisadas culturas puras de S. enterica Enteritidis (bifásica) adicionadas de fagos líticos induzidos de S. enterica I,4,[5],12:i:-, bem como coculturas entre as duas sorovariedades citadas, nas quais foram observadas induções espontâneas de fagos associados à alta densidade populacional e alterações das taxas de crescimento em ambos os estudos, corroborando a hipótese de que S. enterica monofásica pode alterar a dinâmica populacional a seu favor, pela liberação de fagos líticos à outra sorovariedade, interferindo no crescimento populacional de S. enterica Enteritidis, e que o sucesso evolutivo de S. enterica I,4,[5],12.:i:- pode estar associado a fagos líticos atuando como um regulador na ecologia bacteriana. Esses dados podem mudar nosso conhecimento sobre a interação bactéria-fago de uma simples relação parasita-hospedeiro para uma coevolução de duas vias entre seus genomas / Abstract: Salmonellosis is a zoonosis that is a serious public health problem worldwide, due to its high prevalence, difficulty controlling, their endemicity, morbidity and mortality. The knowledge of the occurrence of different serovars of S. enterica in different regions and countries can help in tracking and recognition of emerging pathogens and thus implement policies for treatment and prevention. The majority of serovars express two different types of flagellar antigens encoded by genes: fliC (phase 1) and fljB (phase 2), so called biphasic. However, some serovars express only one of the phases and are termed monophasic. It is possible that flagellar phase variation of S. enterica is associated with an escape function of the immune system to increase the repertoire of antigens expressed by the bacterial cell temporarily preventing cellular immune response. Thus, S. enterica biphasic would have a selective advantage over monophasic, but this is not fully verified in epidemiologic studies, because in the State of São Paulo, S. enterica I,4,[5],12:i:-, (monophase) is the one most commonly associated with cases of diarrhea and/or systemic infections in human patients, in fact, from the 1990s there was a significant increase of S. enterica I,4,[5],12:i:- in many countries. Prophages sequences are very common in S. enterica, with the knowledge that these phages encode several factors that contribute to pathogenicity, genetic diversity and/or characteristics that increase fitness. Cocultures experimental strains of S. enterica prophages can induce spontaneous, killing susceptible bacteria, and thus the spontaneous induction in a population of lysogenic phage enhances the competitiveness between populations. This study analyzed pure cultures of S. enterica Enteritidis ( biphasic ) added lytic phage induced from S. enterica I,4,[5],12:i:-, as well as cocultures between the two serovars cited where inductions were observed spontaneous phage associated with high population density and changes in growth rates in both studies, supporting the hypothesis that S. enterica monophase can alter the population dynamics to their advantage by releasing lytic phage to another serovar, interfering with the population growth of S. enterica Enteritidis and the evolutionary success of S. enterica I,4,[5],12:i:- may be associated with lytic phages acting as a regulator in bacterial ecology. These data may change our understanding of bacteria- phage from a simple parasite-host coevolution for a two-way between their genomes / Mestrado / Clinica Medica / Mestra em Clínica Médica

In the United States, illnesses associated with fresh produce are increasing in frequency.  While contamination risks are present at every aspect of the farm to fork continuum, post-harvest practices holds the potential for cross-contamination of large amounts of product.  Post-harvest contamination risks for hydroponically grown lettuce packaged with intact roots and sold as "living lettuce"" are poorly understood.  In this study, transmission of Salmonella enterica serotype Enteritidis to the roots and leaves of butterhead lettuce was studied when contamination was introduced during typical handling practices.  The effectiveness of random sampling strategies for selection of Salmonella contaminated leaves was assessed by co-inoculating the Salmonella solution with Glo Germ™ and comparing recovery from blacklight selected leaves.  The recovery of Salmonella was improved by only 0.5 log CFU/g when blacklight was used to select Glo Germ™ contaminated leaves (P=0.05). This suggests random leaf selection as described by current FDA protocols is adequate. In addition, this study showed rapid transfer of Salmonella from liquid to the roots and sub-sequentially to the leaves of living lettuce.  Salmonella persisted but did not grow on leaves when stored at 4˚C for 18-days. Storage at 12˚C was associated with 2 log CFU/g increases in Salmonella on roots after 18-days storage (P=0.0002), while 4˚C storage was associated with a decrease of 0.4 log CFU/g Salmonella on roots (P=0.0001). Growth occurred only under temperature abuse conditions.  This reinforces the need for maintaining temperature control and highlights the importance of identifying risks associated with post-harvest handling during hydroponic production and distribution. / Master of Science in Life Sciences

Salmonella is a facultative intracellular bacteria which can grow and replicate within the infected host cells as well as extracellulary. The use of intracellular bacteria that have access to the host cell cytosol may allow a more specific targeting of DNA vaccine vectors to professional Antigen presenting cells (APC). The strategy of using live attenuated Salmonella to deliver plasmid-encoded antigens under the transcriptional control of eukaryotic promoters has been used successfully in vaccination. Another strategy, heterologous antigens can be expressed in Salmonella as fusions with recombinant or native proteins. This approach has been used mainly to direct the expression of the desired antigen to a particular location of the bacterial cell and increase the immunogenicity of foreign antigens by fusing them to proteins that could exert a carrier effect. Salmonella type III secretion system (TTSS)-mediated translocation can be used for efficient delivery of heterologous antigens to the cytosol of antigen-presenting cells leading to prominent both CD4 and CD8 T-cells. In this work we tried SPI2 membranal translocated effectors antigen fusions such as SseJ, SifA, SseL and SteC. Our In-vitro and in-vivo experiments prove that SseJ effector fusion is the best candidate for vaccination. In previous work it was shown that SifB promoter was the most efficient in-vivo inducible promoter. Here we show that SseJ antigen fusion protein under control of SifB is the most efficient in comparison to other effector fusions under control of other invivo inducible promoters. htrA/purD douple mutant S.typhimurium was used as attenuated carrier for vaccination, in this study we find that delta SifA mutant can stimulate in-vitro T-cell proliferation to same level.