Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Alison P. Adams / Salmonella contamination of meat is a leading cause of foodborne illness around the world. Nontyphoidal Salmonella are responsible for an estimated 94 million infections and 155,000 deaths worldwide each year. Of these infections, 86% are estimated to be foodborne. Infection of dairy and beef cattle can lead to contamination of milk and milk products as well as processed beef. Once cattle are infected, Salmonella can be found in many organs of the animals. Peripheral lymph node infections are of particular interest, because these lymph nodes along with hides are the main culprits of meat contamination during processing. Vaccination of production food animals is one of several strategies of prevention and control of Salmonella infections and outbreaks. Vaccination is becoming even more important with the reduction of prophylactic antibiotic use that is driven by an increase in antibiotic resistant bacteria isolated from a variety of food production animals. There are limited commercially available vaccines for cattle that have shown effectiveness, but great strides are being made in this area of research. The vast number of Salmonella serovars with differences in vital virulence factors capable of infecting cattle makes developing vaccines that are cross protective very difficult. This report discusses the known virulence factors of Salmonella, the disease symptoms of bovine salmonellosis, prevention and control strategies, and the development of new vaccines.

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FoxO3a is a transcription factor that regulates various cellular functions such as cell cycle or cell death. However, its role in the innate immune response is not clear. I investigated the impact of FoxO3a signaling on the immune response during infection with Salmonella Typhimurium (ST). My results revealed that FoxO3a regulated the homeostasis of myeloid cells in the spleen and blood of mice during steady-state. Following infection of macrophages with ST, FoxO3a signaling promoted the expression of pro-inflammatory cytokines such as IL12 and TNFα, but inhibited the expression of the anti-inflammatory cytokine IL10. Phenotypic analysis revealed that FoxO3a signaling had no effect on classical macrophage polarization into M1 vs M2 phenotypes, although it appeared to regulate mitochondrial function during infection with ST. Inflammatory responses are critical during infection with virulent intracellular pathogens, and these results provide new insights into the role of FoxO3a signaling in inflammatory responses.

Tesis de Magíster en Bioquímica Área de Especialización, Bioquímica de Proteínas y Biotecnología; y Memoria para Optar al Título de Bioquímico / Los mecanismos moleculares que permiten a Salmonella Typhimurium causar enfermedades en mamíferos son numerosos y generan manifestaciones clínicas que abarcan desde una colitis hasta septicemia y la posterior muerte del hospedero. Estos mecanismos se relacionan con su capacidad de infectar en primera instancia células epiteliales del tracto digestivo y su posterior supervivencia y replicación en células fagocíticas. Esto es posible gracias a la existencia de factores de virulencia que se encuentran codificados en genes localizados mayoritariamente en islas de patogenicidad o en el plasmidio de virulencia de Salmonella. La gran mayoría de estos mecanismos han sido caracterizados en la infección del modelo murino o aviar. En este trabajo buscamos identificar los genes de S. Typhimirium que están implicados en la supervivencia de esta bacteria dentro de organismos eucariontes simples tales como amebas. Considerando que Salmonella podría interactuar con estos organismos en el medio ambiente, en este trabajo se usó como modelo de estudio la ameba social Dictyostelium discoideum, que de acuerdo a nuestras observaciones sería incapaz de degradar a Salmonella. Esto es interesante si se toma en cuenta que las amebas presentan una estrecha relación con los macrófagos, ya que ambas son células eucariontes que comparten muchos de los mecanismos implicados en el proceso de fagocitosis. Para estudiar la supervivencia de Salmonella al interior de D. discoideum implementamos un ensayo de competencia infectando esta ameba con S. Typhimurium 14028s y mutantes definidas. Nuestros resultados indican que las mutantes ΔinvA y ΔssaD (relacionadas directamente con la patogenicidad de Salmonella en otros modelos de infección) y ΔaroA (mutante metabólica), presentan problemas de supervivencia en D. discoideum en comparación con la cepa silvestre parental. Posteriormente, para identificar el conjunto de genes involucrados en la supervivencia intracelular de S. Typhimurium en este organismo modelo realizamos infecciones de D. discoideum utilizando una genoteca de ~3690 mutantes de S. Typhimurium 14028s por deleción de genes individuales, que contienen un cassette de resistencia a kanamicina. Desarrollamos un protocolo que permite amplificar y secuenciar las regiones adyacentes a la inserción de este cassette e identificar a nivel genómico cada una de las mutantes presentes en la población recuperada desde D. discoideum. Mediante este análisis a gran escala fue posible identificar un total de 81 mutantes bajo selección negativa. Entre las mutantes identificadas podemos mencionar a ΔorgB, gen que codifica un componente esencial para el funcionamiento del sistema de secreción tipo III (T3SS) codificado en la isla de patogenicidad SPI-1 y 3 mutantes (ΔssrA, ΔssaR y ΔpipB2) cuya función se asocia al T3SS codificado en la isla de patogenicidad SPI-2. El análisis a escala genómica también nos permitió tener una idea general del ambiente en el que se encuentra S. Typhimurium al interior de D. discoideum. Esta idea surge de la selección negativa que presentan mutantes en genes como rpoE y creB, que codifican proteínas que participan de la regulación transcripcional de genes asociados con la respuesta al estrés extracitoplasmático y al crecimiento en medio mínimo, respectivamente. En conjunto, los resultados de esta tesis constituyen un primer paso para comprender la interacción entre Salmonella y la ameba D. discoideum a nivel molecular / The molecular mechanisms that allow Salmonella Typhimurium to cause disease in mammals are numerous, and are responsible for clinical traits ranging from a self-limited colitis to septicemia and eventually the death of the host. These mechanisms are related to the ability of the pathogen to infect epithelial cells in the small intestine and its intracellular survival and growth in phagocytic cells of the host. This ability is the result of genes coding for virulence factors generally located in pathogenicity islands or in the Salmonella virulence plasmid. The vast majority of these mechanisms have been characterized using murine and avian infection models. In this work, we aimed to identify S. Typhimurium genes involved in the survival of this pathogen in simple eukaryotic organisms like amoebas. Considering that Salmonella can interact with these organisms in the environment, in this work we used the social amoeba Discoideum discoideum as a model. According to our data, this organism is unable to degrade Salmonella after phagocytosis. This is remarkable considering that amoebas show a close relationship with macrophages, both being eukaryotic cells that share many phagocytosis mechanisms. To study the intracellular survival of Salmonella in D. discoideum we developed a competition assay where the amoeba was infected with S. Typhimurium 14028s and selected derivative mutants. Our data show that mutants ΔinvA and ΔssaD (directly related to pathogenicity in other infection models) and ΔaroA (metabolic mutant) have an impaired intracellular survival in D. discoideum as compared to the wild-type strain. Later, in order to identify the complement of genes involved in the intracellular survival of S. Typhimurium in this organism, we infected D. discoideum using a single-gene deletion mutant library of S. Typhimurium 14028s (~3690 mutants), containing a kanamycin resistance cassette. We developed a protocol to amplify and sequence the genomic region adjacent to the resistance cassette. This protocol allowed us to identify at the genomic level the mutants present in the population of bacteria recovered from D. discoideum. Using this genomic analysis, we identified 81 mutants under negative selection. Relevant mutants in this group include ΔorgB, a gene that encodes an essential component required for the function of the T3SS encoded in SPI-1, and 3 mutants (ΔssrA, ΔssaR and ΔpipB2) in genes associated to the T3SS encoded in SPI-2. Furthermore, our genomic analysis allowed us to have a general idea of the environment faced by Salmonella within D. discoideum. This notion comes from the negative selection observed for mutants in genes such as rpoE and creB, coding proteins involved in the transcriptional regulation of genes associated to extracitoplasmatic stress and growth in minimal media, respectively. Taken together, the results in this work are the first step in the understanding of the molecular mechanisms involved in the interaction between Salmonella and D. discoideum / Conicyt; Fondecyt

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias / La enfermedad infecciosa causada por Salmonella spp., se denomina salmonelosis, que es una de las enfermedades transmitidas por los alimentos más comunes. El aislamiento e identificación de Salmonella se basa el esquema de Kaufmann-White, y está compuesto por reacciones serológicas que usan anticuerpos contra las aglutininas de LPS, que se demora varios días para obtener los resultados. En este trabajo se ha implementado el método de genotipificación por PCR múltiple de 46 aislados de S. enterica aisladas de cerdos desde 5 planteles comerciales. Este método consiste en dos reacciones de cinco pares de partidores y una reacción de dos pares, basada en seis loci genéticos de S. enterica serovar Typhimurium y cuatro loci de S. enterica serovar Typhi. Como resultado, se determinaron 20 genotipos distintos que se relacionaron al origen de las cepas. Además se determinó la frecuencia relativa de los genes de virulencia spvC (54%); pagK (96%); sirA (96%); gipA(63%); SEN1417(37%); prot6e(0%) y pefA(80%) formando 12 perfiles genéticos y la frecuencia de genes asociados a resistencia antimicrobiana tetA(85%); tetB(7%); tetG(0%); blaPSE-1(0%); blaTEM(72%); blaCMY(0%); aadB(0%) y aacC(0%), constituyendo en total de 6 perfiles. Se construyó la matriz de datos para determinar la diversidad de estas cepas en base a perfiles genéticos asociados a virulencia y resistencia antimicrobiana, en donde se determinó 22 combinaciones o virulotipos distintos, los cuales se agruparon en 2 clústeres relacionados principalmente al origen de las muestras, confirmando así la hipótesis de circulación de múltiples cepas al interior de estos planteles, lo que representa una amenaza para el estatus sanitario de los animales y para la salud pública por su efecto en la inocuidad alimentaria / The infectious disease caused by Salmonella spp., is called salmonellosis, which is a disease transmitted by the most common foods. The isolation and identification of Salmonella is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies against LPS agglutinins, which lasts several days to obtain the results. In this work, the PCR-based genotyping method has been implemented to analyze 46 S. enterica isolates from pigs belonging to 5 commercial establishments. This method consists of two reactions of five pairs of primers and a two-pair reaction, based on six genetic loci of S. enterica serovar Typhimurium and four loci of S. enterica serovar Typhi. As a result, 20 different genotypes were determined that maintained great closeness between them and their origin. In addition, the relative frequency of virulence genes was determined spvC (54%); pagK (96%); sirA (96%); gipA (63%); SEN1417 (37%); prot6e (0%) and pefA (80%) forming 12 genetic profiles and the frequency of genes associated with antimicrobial resistance tetA (85%); tetB (7%); tetG (0%); blaPSE-1 (0%); blaTEM (72%); blaCMY (0%); aadB (0%) and aacC (0%), constituting a total of 6 profiles. The data matrix was constructed to determine the diversity of these strains based on genetic profiles associated with virulence and antimicrobial resistance, where 22 different combinations or virulotypes were determined, which were grouped into 2 clusters related mainly to the origin of the samples. These results confirm the working hypothesis of circulation of multiple strains within these farms, which represents a threat to the sanitary status of animals and public health due to its effect on food safety

Records of bovine samples submitted for salmonella cultures at four regional diagnostic laboratories in the state of Virginia were used to investigate the association of weather conditions and the diagnosis of salmonellosis in cattle. Spearman's correlation coefficients were calculated for the correlations between the monthly number of samples positive for salmonella culture and weather parameters: temperature, precipitation, and relative humidity. Significant correlation coefficients between the monthly average temperature and the monthly number of positive samples were found to be negative in one laboratory (rs= -0.38, p=0.03) and positive in one laboratory (rs=0.30, p=0.02). The latter correlation coefficient was found between the monthly average temperature and the monthly number of positive samples the following month. The same laboratories that had significant correlation of the monthly number of positive samples and the monthly average temperature also had significant correlation with the monthly average relative humidity (rs= -0.39, p=0.03 and rs=0.37, p=0.004). The monthly average relative humidity was more highly correlated to the number of positive samples reported in the same month for both laboratories that had significant correlation coefficients. None of the correlations between the monthly precipitation and the monthly number of positive samples were significant (p>0.05). The inconsistent directions of correlation coefficients need to be investigated further to find a reason for the discrepancy between regions of the state. / Master of Science

The foodborne, facultative intracellular pathogen Salmonella enterica serovar Typhimurium has the capability to remodel the host endosomal system and establish a network consisting of tubular membranous structures connected with the Salmonella-containing vacuole (SCV). However, despite its ability to form these Salmonella-modified membranes (SMM), the cellular origin and composition of SMM are mostly unknown. Thus we developed a novel approach facilitating the isolation of SMM. Therefore, we combined differential fractionation and affinity-based enrichment using an epitope-tagged SPI2-T3SS effector SseF with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This enabled us to probe the composition of SMM during infection of the epithelial cell line HeLa and mouse macrophage RAW264.7 cells. The identified SMM proteome consists of 247 host proteins in HeLa cells and 262 proteins in macrophages, respectively. The protein compositions revealed the importance of cytoskeletal and host trafficking proteins for SMM and indicated a redirection of host trafficking towards SMM. In addition our results suggested a contribution of the traffic between ER and Golgi apparatus, e.g. COPI and COPII vesicles, that has not been assumed as origin for SMM before. A selection of proteins involved in trafficking and cytoskeleton formation were validated by confocal light microscopy. Furthermore we analysed the effect of IFNgamma activation on the SMM proteome in RAW264.7 macrophages and revealed a strong impact on the protein composition. The identification of IFNgamma-inducible proteins and a higher percentage of ER proteins implied that Salmonella is able to adjust its SMM according to the cell status. Comparison of the SMM proteome with host compartments of other intracellular pathogens indicated communalities that might be important for the biogenesis of pathogen-containing vacuoles despite of their individual replication niches. Furthermore, the methods were adopted for a non-membrane integral Salmonella effector SseJ. Proteome analyses were conducted using the sensitive Velos Orbitrap system. This data set revealed not only the impact of the bait protein during the SMM probing but also differences between MS platforms. Nevertheless, interesting host and Salmonella proteins were identified using this approach, which are starting points for further research. Altogether, this work provides new insights into the origin of Salmonella-modified membranes and serves as a rich source for new theories that will help to understand the biogenesis and function of the Salmonella-containing vacuole and its connected tubular membrane network.

Se evaluó el efecto protectivo del uso combinado de exclusión competitiva (EC) y vacunación en gallinas de postura desafiadas experimentalmente con una cepa de campo de S.gallinarum. En dos ensayos se emplearon 200 pollitas de postura de la línea Hyline Brown de 1 día de edad provenientes de aves reproductoras libres de Salmonella sp. Para cada ensayo las aves fueron distribuídas en cinco grupos de 20 aves cada uno. En el primer ensayo, 100 aves fueron desafiadas a las 18 semanas de edad con 1 x 109 UFC/ ml de S. gallinarum y en el segundo ensayo con 1 x 10 8 UFC/ ml de la misma cepa a las 28 semanas de edad Los grupos A y C recibieron tres aplicaciones de una vacuna conteniendo la cepa 9R de S. gallinarum (Vacuna 9R) a la 4ta, 8va y 10ma semana de edad, mientras que los grupos B y D recibieron una aplicación de una vacuna inactivada de S. enteritidis en emulsión oleosa (Bacterina) a la 13va semana de edad. Adicionalmente los grupos A y B recibieron un producto de EC al día de edad en planta de incubación. En cada ensayo un grupo de aves que no recibió ninguna protección fue usado como control. El experimento se inició con la crianza de las aves en una granja comercial al norte de Lima bajo condiciones normales de manejo y bioseguridad. Las aves fueron trasladadas a la unidad experimental del Laboratorio de Patología Aviar de la FMV-UNMSM para el desafío y evaluación clínica por un periodo de observación de 22 días post reto. Fueron evaluados signos clínicos, mortalidad, lesiones, respuesta serológica y recuperación de la cepa de desafío. Para el análisis estadístico se usó la curva de supervivencia de Kaplan Meier y las diferencias estadísticas fueron evaluadas por Regresión de Cox. En ambos ensayos la mejor protección fue obtenida en los grupos vacunados con vacuna 9R sola o combinada con Exclusión competitiva (p<0.05). Diferencias estadísticas fueron observadas entre el grupo control y grupo A (vacuna 9R más EC) y grupo C (vacuna 9R). En ambos ensayos los grupos vacunados con bacterina sola o combinada con EC no fueron protegidos, no existiendo diferencias estadísticas con el grupo control (p>0.05). En el primer y segundo ensayo las aves del grupo A tuvieron una media de supervivencia significativamente diferente comparada con el grupo control y los grupos con bacterina y EC más bacterina (p<0.05). / The protective effect of the combined use of vaccination and competitive exclusion (CE) in layer hens experimentally challenged with a S. gallinarum field strain was evaluated. Two hundred Hyline Brown pullets since one day old and obtained from salmonella- free breeders were used. We divided this study in two trials in order to do two challenges in two different ages. Half of the birds were challenged at 18 weeks old (Trial 1) with 1 x 109 UFC/ ml de S. gallinarum and half remained were challenged at 25 weeks old (Trial 2) with 1 x 108 UFC/ ml . The birds were divided in five groups of 20 birds each. The groups A and C received three applications of a live Salmonella gallinarum 9R strain vaccine (9R vaccine) at 4th , 8th and 10th weeks old subcutaneously. Groups B and D received a Salmonella enteritidis oil emulsion commercial bacterine (bacterine SE) at 13 weeks old, subcutaneously. Group E unvaccinated and without competitive exclusion was the control group. The study started with the birds breed in a commercial poultry farm at the north of Lima under normal management and bio security conditions .The birds were moved to the Avian Pathology Laboratory of the College of Veterinary Medicine, San Marcos University to challenge and clinic evaluation about 22 days post-challenge. Clinical signs, mortality rates, lesions, serologic response and field strain recovery were evaluated. Kaplan Meier design and Cox regression were used to determine statistical differences. In both trials the best protection was performed in 9R vaccine groups single or combined with CE. (p<0.05). Statistical differences were observed between the control group and group A (9R vaccine plus CE) and group C (9R vaccine) .In both trials the bacterine SE vaccinated groups were not protected and there were no differences with the control group (p>0.05). In trial 1 and 2 the group A had a higher survival mean than the control group and bacterine SE group and bacterine plus CE (p<0.05). / Tesis

Antimicrobial efficacy of recently approved lytic bacteriophage preparation Salmofresh™ against Salmonella was evaluated on chicken breast fillets as dip and surface application, which reduced Salmonella by 0.7-0.9 log CFU/g and 0.8-1 log CFU/g, respectively. Surface application of Salmofresh™ on Salmonella inoculated chicken breast followed by storage under modified atmosphere packaging (95% CO2/ 5% O2) reduced Salmonella by 1.2 log CFU/g. The combined application of Salmofresh™ with cetylpyridinium chloride (CPC) and lauric arginate (LAE) reduced Salmonella on chicken breast fillets by 1.2-1.4 log CFU/g and 0.9-1 log CFU/g, respectively. The sequential application of chemical antimicrobial (CPC, LAE, chlorine and peracetic acid) and Salmofresh™ in reducing Salmonella was tested in a chicken skin model. Dip treatment in peracetic acid (400ppm) followed by surface application of phage revealed the highest reduction of Salmonella up to 2.5 log CFU/cm2 on chicken skin.