O experimento associando probióticos no aleitamento e creche utilizou 144 leitões do nascimento até aos 62 dias de idade no Laboratório de Pesquisa em Suínos (FMVZ USP). No aleitamento, o delineamento experimental foi inteiramente casualizado com dois tratamentos e na creche em blocos casualizados formando o esquema fatorial 2x3 com seis tratamentos. A unidade experimental no aleitamento foi a baia maternidade constituída de grade de parição e na creche gaiola suspensa com 3 animais cada, constituindo, 8 repetições por tratamento. Aos 35 dias de idade os animais da creche foram inoculados com cepa de Salmonella Typhimurium via oral. Na maternidade os tratamentos foram: Placebo (PLA), 1mL de água destilada e Probiótico A (ProbA), 5g em 15mL de água destilada. O ProbA foi aplicado em duas ocasiões, ao nascimento e 12 horas antes do desafio e os animais que não receberam ProbA, receberam PLA. Os tratamentos na creche foram: Probiótico A Probiótico B (ProbA ProbB) 30g/tonelada de ProbB na ração; Controle Probiótico B (CTL ProbB): 30g/tonelada de ProbB na ração; Probiótico A Probiótico A(ProbA ProbA); Controle Controle (CTL CTL); Probiótico A Desafio (ProbA DES); Controle Desafio (CTL DES). As variáveis; peso, consumo, ganho de peso e conversão alimentar, foram analisadas em medidas repetidas no tempo com contrastes e o escore de fezes submetido à análise de variância pelo teste de Tukey. Foi utilizado o programa computacional Statistical Analysis System SAS. Na maternidade o peso médio do ProbA foi superior ao PLA, não observando-se diferenças no ganho de peso. Na creche o ProbA mostrou-se significativamente melhor em comparação aos demais tratamentos na conversão alimentar. No escore fezes, os animais desafiados apresentaram mais diarréia, e mais eliminação de S. Typhimurium, evidenciando o efeito positivo do desafio programado. A avaliação histológica aos 63 dias revelou aspecto similar nos diferentes grupos com e sem desafio. A eficiência econômica destacou o CTL ProbB.. O estudo indicou ação positiva dos probióticos quando aplicado logo ao nascimento, por influenciar diretamente na formação da microbiota intestinal, em fases mais adiantadas como na creche os efeitos são menos diretos. / The experiment involving probiotics in lactation and nursery were used one hundred and forty four piglets were used from birth to 62 days of age at Swine Research Laboratory (USP FMVZ). In the lactation, the experimental design was randomized with two treatments and in the nursery were randomized blocks forming a 2x3 factorial, consisting of six treatments. The experimental unit considered was the pen with 3 animals, with 8 repetitions per treatment. At 35 days of age the nursery animals were inoculated with a Salmonella Typhimurium strain orally. The treatments in the maternity were: Placebo (PLA): 1mL of distilled water and Probiotic A (ProbA): 5g in 15ml of distilled water. The ProbA was applied twice, at birth and 12 hours before the challenge and the animals that received no ProbA, received PLA. At the nursery twelve hours before the challenge, the same group of piglets that received ProbA and PLA in the maternity, received reinforcement for ProbA and PLA. In the nursery: Probiotic A Probiotic B (ProbA ProbB): 30g/ton of diet; Control Probiotic B (ProbB CTL): 30g/ton of diet; Probiotic A probiotic A (ProbA probA) Control Control (CTL CTL) Probiotic Challenge (DES ProbA) Control Challenge (CTL DES).The variables analyzed were weight, consumption, weight gain and feed conversion. The variables were analyzed with repeated measures contrasts and fecal score variable was subjected to analysis of variance by Tukey test. We used the computer program Statistical Analysis System SAS. In maternity, ProbA was superior to PLA treatment and did not observe differences in weight gain. At nursery the ProbA ProbA was significantly better compared to the other treatments in feed conversion. In the feces score, challenged animals showed high scores and greater elimination of S. Typhimurium, showing the effects of the programmed challenge. Histological evaluation at 63 days showed similar appearance within different groups (challenged or not). Economic evaluation shows CTL ProbB. The study indicated positive action of probiotics applied shortly after birth, by directly influencing the formation of the intestinal tract and in nursery as the effects are less direct.

Devido à limitação do uso de antimicrobianos, alternativas que seguem a linha de controle biológico, como os bacteriófagos líticos e probióticos, vêm sendo propostos para o controle de Salmonella sp. em animais de produção. O objetivo deste estudo foi avaliar o efeito da administração oral de probiótico e bacteriófagos líticos sobre a ocorrência de infecção e excreção fecal de Salmonella sp. em suínos em fase de crescimento e terminação. O experimento foi realizado em dois períodos (blocos), com duração de 49 dias, em suínos com 43 dias de idade. Estes animais foram divididos em três tratamentos: T1 (controle), T2 (bacteriófagos) e T3 (probiótico), com 12 animais em cada tratamento e 10 animais controle. Após duas semanas de alojamento, os suínos foram inoculados com Salmonella Typhimurium (dia 0 P.I.). Os animais receberam uma suspensão de bacteriófagos líticos (CNPSA1, CNPSA3 e CNPSA4) por oito dias consecutivos, quatro dias antes e quatro dias após inoculação. O probiótico foi fornecido pela via oral nos dois primeiros dias de alojamento (108 Unidade Formadora de Colônia-UFC/ g), posteriormente o produto (107 UFC/g) foi fornecido diariamente na ração na concentração de 1%. Foram realizadas colheitas de sangue (-14, 0, 7, 14, 21, 28 e 35 PI) para pesquisa de IgG anti- Salmonella, e de fezes (-14, -7, 0, 3, 7, 14, 21 e 28) para pesquisa e quantificação de Salmonella, Enterococcus, Lactobacillus e coliformes totais. No dia 35PI os animais foram eutanasiados e fragmentos de órgãos foram coletados para pesquisa de Salmonella. Foram coletados segmentos do intestino delgado para análise de morfometria e pesquisa de IgA de mucosa. Os resultados indicam que o tratamento de suínos na fase de crescimento não foi capaz de impedir a infecção dos animais, e a soroconversão ocorreu a partir do dia 7PI. Não foi observada menor excreção de Salmonella nos grupos tratados. Não houve efeito sobre a população de Lactobacillus, Enterococcus e coliformes totais, nem diferença na morfometria de vilosidades entre grupos. O grupo tratado com probiótico apresentou uma tendência a maiores densidades óticas no teste de ELISA para pesquisa de IgA anti-Salmonella na mucosa intestinal. A partir disso, conclui-se que o tratamento com probiótico e fagos líticos testados de acordo com o protocolo proposto no presente estudo não influenciam a infecção e excreção de Salmonella sp. em suínos em fase de crescimento e terminação. / Due to the limited use of antimicrobial, alternatives that follow the line of biological control, as lytic bacteriophages and probiotics, have been proposed for the control of Salmonella sp. animal production. The aim of this study was to evaluate the effect of oral administration of probiotic and lytic bacteriophages on the occurrence of infection and fecal excretion of Salmonella sp. in growing pigs. The experiment was conducted in two periods (blocks), lasting 49 days, in 43 days old pigs. These animals were divided into three treatments: T1 (control), T2 (bacteriophages) and T3 (probiotic), with 12 animals in each treatment and 10 control animals. After two weeks of accommodation, the pigs were inoculated with Salmonella Typhimurium (day 0 PI). The animals received a suspension of lytic bacteriophages (CNPSA1, CNPSA3 and CNPSA4) for eight consecutive days, four days before and four days after inoculation. The probiotic was given orally in the first two days of lodging (108 of Colony Forming Units-CFU / g), then the product (107 CFU / g) was supplied daily in the diet at a concentration of 1%. Were collected blood (-14, 0, 7, 14, 21, 28 and 35 PI) for the detection of IgG anti-Salmonella, and feces (-14, -7, 0, 3, 7, 14, 21, 28) for research and quantification of Salmonella, Enterococcus, Lactobacillus and Coliforms. On 35PI the animals were euthanized and samples of organs were collected for Salmonella. We collected small intestinal segments for morphometric analysis and research of mucosal IgA. The results indicate that treatment of pigs in the growing phase was not able to prevent infection of animals, and seroconversion occurred on the day 7PI. There was no lower excretion of Salmonella in the treated groups. There was no effect on the population of Lactobacillus, Enterococcus and Coliforms, and no difference in the morphology of villi between groups. The group treated with probiotic showed a trend to higher optical densities in ELISA for the detection of IgA anti-Salmonella in the intestinal mucosa. From this, it is concluded that treatment with probiotic and lytic phages tested in accordance with the protocol proposed in this study did not influence the infection and excretion of Salmonella sp. in growing pigs.

No período de 1999 a 2002, uma linhagem de Salmonella Enteritidis esteve envolvida em mais de 90% das salmoneloses ocorridas no RS. Este trabalho teve por objetivo avaliar a cinética de crescimento, a resistência ácida e a resistência térmica dessa linhagem e compará-la com S. Typhimurium e S. Bredeney não envolvidas em surtos alimentares, porém isoladas na mesma região. Em uma primeira etapa, a cinética de crescimento foi avaliada semeando-se cada sorovar em caldo nutriente (CN) e em salada de batata com maionese caseira (SMC), os quais foram mantidos a 30°C e 9,5°C. Em CN, a cinética de crescimento a 30°C foi semelhante para todos os sorovares, porém, em SMC a S. Enteritidis apresentou maior quantidade de células nas primeiras 6 horas de crescimento, sendo que somente depois de 12 horas todos os sorovares atingiram quantidades semelhantes de células. Em CN e em SMC, na temperatura de 9,5°C, não foi detectado crescimento de nenhum dos sorovares de Salmonella durante as primeiras 24 horas, sugerindo que essa temperatura foi suficiente para controlar a multiplicação desses microrganismos. Em uma segunda etapa, avaliou-se a resistência ácida e térmica dos diferentes sorovares de Salmonella. Para tanto, os três sorovares foram inoculados separadamente em CN e CN enriquecido com 1% de glicose (CNG), este último utilizado para produção de culturas ácido-adaptadas. Em seguida, os microrganismos foram submetidos a diferentes pH (3,5; 4,0 e 4,5) e temperaturas (52, 56 e 60ºC). Os resultados demonstraram que a S. Bredeney apresentou maior resistência para os pH 3,5 e 4,0, porém a S. Enteritidis demonstrou maior capacidade de adaptação ácida do que S. Typhimurium e S. Bredeney. Em pH 4,5 todos os sorovares, tanto não adaptados quanto ácido-adaptados, mantiveram a mesma quantidade de células viáveis durante 300 minutos. Quando expostas a 52ºC, S. Bredeney apresentou maior resistência, entretanto somente a S. Enteritidis foi protegida com a adaptação ácida. Para 56 e 60ºC, a S. Enteritidis, não adaptada e ácido-adaptada, apresentou maior resistência. A análise por SDS-PAGE demonstrou diferenças no perfil protéico de células não adaptadas e ácidoadaptadas para todos os sorovares testados. Com base nestes resultados, a capacidade de multiplicação mais rápida nas primeiras horas de cultivo em SMC, a maior capacidade de adaptação ácida e a maior resistência térmica demonstradas pela S. Enteritidis podem estar relacionadas ao freqüente envolvimento desse sorovar nas salmoneloses do RS. / During the period of 1999 to 2002, a strain of Salmonella Enteritidis was involved in more than 90 % of foodborne salmonellosis occurred in Rio Grande do Sul (RS) State. This work aimed to evaluate the growth kinetics, and the acid and the thermal resistance of this strain, comparing with S. Typhimurium and S. Bredeney, which were not involved in foodborne outbreaks, but were isolated in the same region. In the first stage of this study, the growth kinetics was assessed. Each serovar was inoculated separately in nutrient broth (CN) and in potato salad prepared with homemade mayonnaise (SMC), and then incubated at 30 ºC and 9.5 ºC. In CN, at 30 ºC, similar growing characteristics were found for all serovars, however in SMC S. Enteritidis demonstrated higher counts at the first 6 hours. Only after 12 hours of incubation, all serovars reached similar counts. In CN and in SMC, at 9.5 ºC, during the first 24 hours, there was no detectable growth of any Salmonella serovar, suggesting that such temperature was adequate to control the multiplication of tested Salmonella serovars. In the second stage of the study, the acid and the thermal resistances of Salmonella serovars were evaluated. The three serovars were cultivated separately in Nutrient Broth and Nutrient Broth supplemented with 1 % glucose (NBG). The latter medium was used to induce acid-adapted cells. Then, the three serovars were exposed to different pH (3.5, 4.0, and 4.5) and temperatures (52, 56, and 60 ºC). Results indicated that S. Bredeney presented higher resistance to pH 3.5 and 4.0, but S. Enteritidis presented a better capacity of acid adaptation than S. Typhimurium and S. Bredeney. At pH 4.5, all serovars demonstrated a similar behavior, remaining at same levels of viable cells until 300 minutes. At 52 ºC, S. Bredeney presented greater survivor rates, however acid adaptation protected only S. Enteritidis. At 56 ºC and 60 ºC, non-adapted and acidadapted S. Enteritidis were more thermally resistant than other serovars tested. SDS-PAGE analysis demonstrated differences in protein profile of non-adapted and acid-adapted cells of all serovars. The capacity of rapid multiplication in the first hours of cultivation, the greater acid adaptation and thermal resistance presented by S. Enteritidis, may be related to the frequent involvement of this strain in salmonellosis cases in the RS.

O aumento na poluição ambiental é na atualidade uma das grandes preocupações em nível mundial. Especificamente, a poluição atmosférica por material particulado tem demostrado ser um fator determinante no desenvolvimento de doenças cardiopulmonares e câncer de pulmão nas populações expostas. O material particulado é constituído por uma mistura complexa de compostos orgânicos e inorgânicos, muitos dos quais possuem potencial mutagênico e genotóxico. As características destes compostos variam em função da suas propriedades físicas, químicas e em razão das condições meteorológicas prevalecentes. A maior parte dos estudos tem se focado em avaliar o potencial genotóxico da fração orgânica do material particulado e poucos estudos têm explorado a fração solúvel em água e a contribuição diferencial das diversas espécies químicas presentes nesta fração para o dano genotóxico. O objetivo deste trabalho foi avaliar os efeitos mutagênicos e genotóxicos in vitro da fração orgânica e da fração solúvel em água de material particulado (MP10) coletado em três locais diferentes do estado de São Paulo e estabelecer a relação entre a composição química e o efeito biológico observado. Para isto, realizou­-se a extração orgânica e solúvel em água de 12 amostras de MP10. A mutagenicidade e genotoxicidade foram avaliadas usando o ensaio de Salmonella/microssoma e o teste de micronúcleos (MN) em células A549 e 16 hidrocarbonetos Policíclicos Aromáticos (HPAs) e 15 metais hidrossolúveis presentes nas amostras foram determinados quimicamente. Adicionalmente, foi determinada a metodologia de extração da fracção solúvel em água e se avaliou a estabilidade química e biológica desta fração. Os resultados indicam que a extração assistida por micro-ondas é um método eficiente para a extração da fração solúvel em agua do MP e que um tempo superior a 60 dias de armazenamento e congelamento deste tipo de extrato tem um efeito significativo sobre os resultados analíticos e a resposta biológica. Foi demonstrado ainda que as duas frações de MP estudadas são responsáveis pela indução do dano ao DNA e que não existe uma relação direta entre a concentração de MP e o efeito genotóxico observado, confirmando a importância do uso de bioensaios na avaliação da genotoxicidade de misturas complexas como o MP. Os HPAs prevalecentes nas amostras de PM10 foram fluorantene e benzo(ghi)perileno. Nos extratos solúveis em água, as maiores concentrações de metais foram determinadas para zinco, ferro e cobre. Confirmou-se que a indução de MN e o ensaio de Salmonella/microssoma representam uma poderosa ferramenta na avaliação da polução atmosférica e que as análises químicas por si só não são suficientes para a proteção e predição dos efeitos biológicos em populações expostas. / The increase of environmental pollution is today one a major concern worldwide. Specifically, air pollution by particulate matter has been shown to be a determining factor in the development of cardiopulmonary disease and lung cancer in exposed populations. The particulate material consists of a complex mixture of organic and inorganic compounds, many of which have mutagenic and genotoxic activity. The characteristics of these compounds vary according to their physical and chemical properties and also to the prevailing weather conditions. Most studies have focused on evaluating the genotoxic potential of the organic fraction of particulate material, but few studies have explored the water­-soluble fraction, and the differential contributions of different chemical species present in this fraction to genotoxic damage. The aim of this study was to evaluate the in vitro mutagenic and genotoxic effects of organic and water-soluble fractions of 12 samples of particulate matter (PM10) collected at three different sites in the state of São Paulo and establish the relationship between the chemical composition and the biological effect observed. The mutagenicity and genotoxicity were evaluated using the Salmonella/microssome test and the micronucleus assay (MN) in A549 cells and 16 Polycyclic Aromatic Hydrocarbons (PAHs) and 15 water-soluble metals present in the samples were chemically determined. Additionally, the extraction method of water- soluble fraction was determined and the chemical and biological stability of this fraction evaluated. The results indicate that the microwave­-assisted extraction is an efficient method for the extraction of the water-soluble compounds of PM and that the freezing and storage of the extract over 60 days has a significant effect on the mutagenic and analytical results of PM samples. It was demonstrated that the two PM fractions studied are responsible for the induction of DNA damage and that there is no direct relationship between the MP concentration and the genotoxic effect observed, confirming the importance of using bioassays in the genotoxicity evaluation of complex mixtures as PM. The PAHs prevailing in our samples were fluoranthene and benzo(ghi)perylene. In the water-soluble extracts, highest concentrations of the elements studied were found for zinc, iron, and copper in the three places of sampling. We confirmed that MN induction and Salmonella/microsome assay represents a powerful tool to evaluate the atmospheric air pollution and that the total concentration of PM and the chemical analyses alone would not be sufficient for the prognosis of biological effects in exposed populations

No presente estudo avaliou-se o potencial de aplicação da bacteriocina 2a produzida pelo Lactobacillus sake 2a em combinação com outras substâncias antimicrobianas na inibição de cepas de Salmonella isoladas de linguiça: S. Derby SD2, S. Enteritidis SE3, S. Hadar SH4, S. Panama SP5 e S. Typhimurium ST6. A partir de cultura de L. sake 2a em caldo MRS, obteve-se por extração ácida e concentração em liofilizador, um extrato protéico de bacteriocina de cerca de 500 UA/ml. Verificou-se que esse extrato protéico de bacteriocina 2a tem atividade contra Listeria monocytogenes Scott A Cmr Emr nos meios de cultura BHI, MRS e TSB com 0,1% de glicose, independente de temperatura e atmosfera de incubação. O extrato protéico de bacteriocina 2a foi utilizado só e em combinação com EDTA, ácido cítrico, ácido lático ou lisozima sobre "pool" de cepas de Salmonella. Todos os antimicrobianos testados apresentaram efeito inibidor contra Salmonella, no entanto, quando combinados à bacteriocina 2a esse efeito foi mais acentuado. Entre os tratamentos realizados, o que apresentou efeito mais potente na eliminação ou inibição de Salmonella foi a combinação bacteriocina 2a mais ácido lático 0,1%. Bacteriocina 2a também se mostrou mais eficiente que a nisina (usada como padrão) em combinações com lisozima e EDTA. O estudo permitiu verificar que existe um bom potencial de uso da bacteriocina 2a ou do L. sake 2a bac+ em alimentos em associação com os antimicrobianos ácido lático, ácido cítrico, EDTA ou lisozima. Entretanto, penas a combinação de antimicrobianos não é suficiente para eliminar ou inibir a multiplicação de Salmonella. As condições de temperatura, concentração e forma de aplicação dos tratamentos combinados podem variar e são pontos importantes quando se visa à ação direta sobre as células do patógeno ou quando este está associado a um substrato. / The objective of this study was to analyse the potential of application of bacteriocin 2a produced by Lactobacillus sake 2a in combination with other antimicrobials substances on inhibition of strains of Salmonella isolated from raw Brazilian sausages (lingüiça): S. Derby SD2, S. Enteritidis SE3, S. Hadar SH4, S. Panama SP5 and S. Typhimurium ST6. Culture of L. sake 2a grown at 30ºC in MRS broth was used to obtain a cell-free supernatant after centrifugation. This cell-free supernatant was submitted to acid extraction method for bacteriocin and water reduction by liofilization process. After this, a final fraction was obtained and denominated proteic extract of bacteriocin 2a and had a final concentration of approximately 500 A.U/mL. Proteic extract of bacteriocin 2a obtained from cultures of L. sake 2a grown in broths: BHI, MRS, and TSB with 0.1% of glucose showed bactericidal effect against Listeria monocytogenes Scott A Cmr Emr, in any condition of temperature and atmosphere. The proteic extract of bacteriocin 2a was used alone and in combination with EDTA, citric acid, lactic acid or lyzozyme on pool of strains of Salmonella. All antimicrobials tested showed inhibitory effect against Salmonella, but in combination to bacteriocin 2a this effect was more efficient on inhibition or elimination of Salmonella. Bacteriocin 2a showed be more efficient than nisin when in association with lyzozyme and EDTA. This study was able to verify that exist a great potential of application of bacteriocin 2a and/or L. sake 2a bac+, in foods in combination with the antimicrobials: lactic acid, citric acid, EDTA or lyzozyme. However, the simple combination of these antimicrobials is not sufficient to eliminate or inhibit the growth of Salmonella. Temperature conditions, concentration of antimicrobials and form of application of treatments can change and they are very important points; mainly when the aim of the study is to evaluate the direct action of antimicrobials on cells of pathogen or action of these substances on pathogen into a specific substrate.

The disposal of sewage sludge is becoming an ever-increasing problem and a range of re-use options are being developed, with traditional composting and advanced alkaline stabilisation emerging as priority re-use alternatives in NSW. However, concerns have been raised regarding the dissemination of sludge related pathogens in the environment. Salmonella spp pose the greatest risk since they have the ability to proliferate in the absence of human and animal hosts. Composting processes eliminate salmonellae from sludge, but the opportunity for post-processing recontamination is considerable. This project examined the significance of post-processing recontamination of Salmonella broughton, introduced into composted sludge and N-Virosoil. In compost, inactivation rates of S. broughton showed an inverse relationship with simulated processing temperatures, with competitive exclusion by autocthonous compost flora thought to be the major mechanism of inhibition. S. broughton numbers were reduced to below the limits of detection after several weeks. S. broughton inactivation was also assessed in processed N-Virosoil and was found to be more immediate. It was concluded that both compost and N.Virosoil products have a low potential to support the regrowth of Salmonella spp. / Master of Science (Hons)(Environmental Science)

Broiler flocks become frequently asymptomatically contaminated by the enteric bacteria Salmonella sp. and Campylobacter sp. which are human pathogens. Among the strategies developed at farm level to reduce the incidence of these pathogens, some lactic acid bacteria have been shown to be interesting because of their antimicrobial activity and their stimulatory properties on the immune system of poultry. The aim of this thesis was to select bacteria with antagonistic activity against Salmonella or Campylobacter, and to improve their inhibitory effect by the combination with enzymes of polysaccharidase type. The first step of the thesis was an epidemiological study carried out in the Walloon region in order to determine the contamination way of broilers by Campylobacter in free range production. Results showed that the major way of contamination is the open-air range to which the animals have access during the rearing period. A preventive treatment of the open-air range and the straw litter with an antagonistic strain in combination with an enzyme seems thus to be suitable in this case. The second step of the work aimed at the selection of a xylanase for using as a dietary additive in combination with an antagonistic bacterial strain against Salmonella. Four xylanases were studied in vivo for their effect on growth performances of broiler chickens. Diet supplementation with enzyme led to an increased final body weight and daily weight gain (P < 0.05), without difference according to the bacterial or fungal origin of the xylanase. The Belfeed B1100MP xylanase, which is commercialized in he Walloon region, was selected in order to develop a probiotic-xylanase feed additive. The purpose of the third part was to select a bacterial strain with antagonistic activity against Campylobacter for applying on open-air range and broiler litter. An in vitro screening of 12 lactic acid bacteria was realised using a co-culture assay with a growth medium based on straw and dehydrated poultry excreta, supplemented with different cellulase concentrations. Lactobacillus pentosus and Enterococcus faecium showed inhibitory effect against Campylobacter without enzyme which was intensified by cellulose from 200 ppm. Finally, the effect of dietary supplementation with a L. plantarum strain combined with the Belfeed B1100MP (PE treatment) on growth performance, microflora, and faecal Salmonella Typhimurium concentrations, was studied with experimentally infected broiler chickens. The PE diet allowed to partially overcome the negative effects associated with the infection on growth performance and microflora, and to significantly reduce faecal Salmonella concentration.

The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.