Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek's disease virus in chickens.

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

Feral pigeons (Columba livia) as potential reservoirs of Salmonella sp. and Escherichia coli / Pombos de vida livre (Columba livia) e seu potencial como reservatórios de Salmonella sp. e Escherichia coli

This study aimed to review the scientific literature for information about free-living pigeons (Columba livia) as potential reservoirs of Salmonella sp. and Escherichia coli. Rock doves are currently adapted to the urban environment and distributed all over the world. These birds carry microorganisms that are pathogenic for man and other animals, such as bacteria, viruses, fungi and parasites. Among these microorganisms, Salmonella is a pathogenic genus that cause severe economic losses and it is zoonotic, causing foodborne infections in humans. In addition, Escherichia coli is an worrisome species involved in the poultry industry. However, this micro-organism is also a risk to the public health, considering pathotypes that are known to cause diseases in man have been isolated from feral pigeons. The infections caused by these bacteria depend on virulence factors that provide the necessary tools to develop the disease. These factors are encoded by genes that may be found in pathogenicity islands inside the bacterial genome. In addition, pigeons may harbor antimicrobial-resistant bacteria, which may pass this characteristic to other strains, and present a risk to the public health as well. In conclusion, pigeons are reservoirs of strains of Salmonella sp. and Escherichia coli that may present high levels of resistance to antibiotics.(AU)

O objetivo deste estudo foi revisar na literatura científica informações sobre os pombos de vida livre (Columba livia) e seu potencial como reservatórios de Salmonella sp. e Escherichia coli. Os pombos, atualmente adaptados ao meio urbano, encontram-se distribuídos por todo o mundo e carreiam micro-organismos patogênicos ao homem e a outros animais, podendo ser um dos responsáveis pela disseminação de bactérias, fungos, vírus e parasitas. Entre esses micro-organismos, a Salmonella é um patógeno que gera grande preocupação para a economia e para a saúde pública mundial, uma vez que cria transtornos para a indústria avícola quando ocorrem a contaminação dos plantéis e, consequentemente, o risco para a saúde humana por conta de surtos de toxi-infecções alimentares causados por essa bactéria. Outra bactéria preocupante para a indústria avícola e para a saúde pública é a Escherichia coli, uma vez que alguns patotipos patogênicos para o homem já foram isolados de pombos de vida livre. O desenvolvimento de infecções por essas bactérias depende de fatores que são codificados por genes de virulência que podem ser encontrados nas ilhas de patogenicidade de cepas patogênicas. Os pombos podem também albergar cepas com carga genética resistente a antibióticos, passíveis de serem transmitidas a outras bactérias e, portanto, de trazer riscos para a saúde pública. Dessa forma, com base nessas informações, conclui-se que os pombos são reservatórios de cepas de Salmonella sp. e Escherichia coli, que podem apresentar elevado potencial de virulência, com altos níveis de resistência antimicrobiana.(AU)

Phenotypic and molecular detection of Salmonella sp. on growing, rearing and production phases in a commercial group of laying hens / Detecção fenotípica e molecular de Salmonella sp. nas fases de cria, recria e produção em lote de poedeiras comerciais

This present study was developed with the objective of detect Salmonella sp. by conventional bacteriology and qPCR techniques in samples of flooring material from transport crates (meconium); raising environment (swab of cages and drinking fountains); cloacal swab; food and insects from growing, rearing and production phases in a commercial group of laying hens. A total of 864 samples were collected, among whom 248 originated from growing, 392 from rearing and 224 from production phase. Among the 864 samples, 2,8% where positives in bacteriologic technique and 15.3% in qPCR. Contamination was higher in growing and rearing phases and declined in production phase. Twenty four isolations of Salmonella where typified as Salmonella Agona (41.7%), Salmonella Livingstone (33.3%), Salmonella Cerro (16.7%), Salmonella Senftenberg (4.2%) and Salmonella Schwarzengrund (4.2%). During growing phase Salmonella Livingstone was identified. These findings suggest vertical contamination in the group. During rearing and production phases, isolated materials belong to serovars Agona, Cerro, Senftenberg and Schwarzengrund, pointing to horizontal contamination. It is possible to conclude that both vertical and horizontal contaminations are important during the cycle of commercial egg production and contamination in rearing phase is higher than in growing and production phases.(AU)

O presente estudo foi desenvolvido com objetivo de detectar Salmonella sp. pelas técnicas de bacteriologia convencional e PCR em tempo real em amostras de forros de caixas de transporte (mecônio), de ambientes de criação (suabes de gaiola e de bebedouro), suabes de cloaca, ração e insetos oriundos das fases de cria, recria e produção de um lote de poedeiras comerciais. Foram coletadas 864 amostras das quais 248 foram originadas da cria, 392 da recria e 224 da produção. Das 864 amostras 2,8% foram positivas na técnica bacteriológica e 15,3% no PCR em tempo real. A contaminação aumentou da fase de cria para a recria e declinou na fase de produção. Vinte e quatro isolados de Salmonella foram tipificados como Salmonella Agona (41,7%), Salmonella Livingstone (33,3%), Salmonella Cerro (16,7%), Salmonella Senftenberg (4,2%) e Salmonella Schwarzengrund (4,2%). Na fase de cria identificou-se Salmonella Livingstone. Esses achados sugerem a contaminação vertical do lote. Nas fases de recria e produção os isolados pertenceram aos sorovares Agona, Cerro, Senftenberg e Schwarzengrund apontando para a contaminação horizontal. Pode-se concluir com este estudo que tanto a contaminação vertical como a horizontal são importantes no ciclo de produção de ovos comerciais e que a contaminação na fase de recria é maior que na fase de cria e de produção.(AU)

SummonChimera infers integrated viral genomes with nucleotide precision from NGS data.

BACKGROUND: Viral integration into a host genome is defined by two chimeric junctions that join viral and host DNA. Recently, computational tools have been developed that utilize NGS data to detect chimeric junctions. These methods identify individual viral-host junctions but do not associate chimeric pairs as an integration event. Without knowing the chimeric boundaries of an integration, its genetic content cannot be determined. RESULTS: Summonchimera is a Perl program that associates chimera pairs to infer the complete viral genomic integration event to the nucleotide level within single or paired-end NGS data. SummonChimera integration prediction was verified on a set of single-end IonTorrent reads from a purified Salmonella bacterium with an integrated bacteriophage. Furthermore, SummonChimera predicted integrations from experimentally verified Hepatitis B Virus chimeras within a paired-end Whole Genome Sequencing hepatocellular carcinoma tumor database. CONCLUSIONS: SummonChimera identified all experimentally verified chimeras detected by current computational methods. Further, SummonChimera integration inference precisely predicted bacteriophage integration. The application of SummonChimera to cancer NGS accurately identifies deletion of host and viral sequence during integration. The precise nucleotide determination of an integration allows prediction of viral and cellular gene transcription patterns.

Genes de virulência e diversidade genética em Salmonella spp. isoladas de amostras de origem suína / Virulence genes and genetic diversity in Salmonella spp. isolated from samples of swine origin

A diversificação da produção industrial de alimentos de origem suína e o intercâmbio comercial de animais e seus derivados destinados ao consumo humano podem ser importantes disseminadores de sorovares de Salmonella spp. na cadeia alimentar. Objetivou-se avaliar em 86 cepas de Salmonella spp., isoladas em granja de terminação e no abate de suínos, a ocorrência de três genes de virulência (invA, agfA e lpfA), bem como a similaridade genética entre elas. A ocorrência do gene invA foi verificada em 100% das amostras. O gene lpfA foi detectado em 80,23% (69/86) das cepas, não foi detectado em S. Panama e estava presente em todas as cepas de S. Infantis. O gene agfA foi detectado em 63,95% (55/86) das amostras. S. Agona apresentou positividade para todos os genes de virulência estudados. A análise de homologia entre as cepas agrupou os diferentes sorovares em clusters. A similaridade foi independente do local de isolamento, o que demonstra a presença de clones ao longo da cadeia de produção e a existência de multiplicidade de fontes para a infecção dos animais, como a ração, e a contaminação cruzada das carcaças. A pesquisa de genes de virulência e a avaliação da proximidade gênica permitem a caracterização e um maior entendimento sobre cepas de Salmonella circulantes na cadeia produtiva de suínos e, assim, podem subsidiar medidas de controle durante o processo produtivo com o objetivo de garantir a saúde do consumidor...

The diversification of industrial food production of swine origin and trade of animals and their derivatives for human consumption may be important disseminators of serovars of Salmonella spp. in the food chain. This study aimed to evaluate 86 strains of Salmonella spp. isolated form in the finishing and slaughter of pigs, the occurrence of three virulence genes (invA, agfa and lpfA), as well as the genetic similarity between them. The occurrence of gene invA was observed in 100% of the samples. The gene lpfA was detected in 80.23% (69/86) strains and is not detected in S. Panama, but present in all strains of S. Infantis. The gene agfA was detected in 63.95% (55/86). S. Agona was positive for all virulence genes studied. The analysis of homology between the different serovars grouped the isolates in clusters. The similarity was regardless of the location of isolation, demonstrating the presence of clones along the production chain and that there are multiple sources for the infection of animals, such as feed, and cross-contamination of carcasses. A survey of virulence genes and evaluation of gene proximity allow characterization and better understanding of Salmonella strains circulating in the pig production chain, thus being able to support control measures during the production process in order to ensure consumer health...

Aspectos físico-químicos e microbiológicos do queijo tipo coalho comercializado em estados do nordeste do Brasil / Physical-chemical and microbiological aspects of the rennet cheese sold in the Northeast States of Brazil

O objetivo deste trabalho foi avaliar o perfil das características físico-químicas e microbiológicas dos queijos de coalho comercializados em seis estados do nordeste brasileiro. Foram realizadas análises físico-química e microbiológica de 104 amostras de queijo de coalho comercializadas nos estados de Pernambuco, Piauí, Ceará, Rio Grande do Norte, Sergipe e Paraíba, sendo 50 amostras inspecionadas e 54 sem inspeção. Não foi observada diferença significativa (p > 0,05) para os parâmetros físico-químicos entre as amostras. De acordo com os parâmetros legislativos, nas 104 amostras analisadas de queijos tipo coalho, verificaram-se que 100 (96,15%) estavam fora dos limites para Staphylococcus coagulase positiva; 32 amostras (31%) também não seguiam a padronização exigida para coliformes termotolerantes; e, em apenas uma amostra do total, constatou-se a presença de Salmonella sp. Não foi observada diferença significativa (p > 0,05) entre as amostras com e sem inspeção para as análises microbiológicas. A semelhança encontrada na avaliação dos parâmetros físico-químicos do queijo de coalho entre os estados avaliados demonstra uniformidade no processamento desse produto. Altos níveis de contaminação por agentes patogênicos, como o Staphylococcus coagulase positiva e coliformes termotolerantes, além da presença de Salmonella sp., demonstram que o consumo de queijo de coalho pode representar risco à saúde dos consumidores.(AU)

The objective of this study was to evaluate the physical-chemical and microbiological profile of the rennet cheese sold in six states in northeastern Brazil. Physical-chemical and microbiological analyses of 104 samples of rennet cheese sold in the states of Pernambuco, Piauí, Ceará, Rio Grande do Norte, Paraíba and Sergipe were analyzed, being 50 samples inspected and 54 without inspection. There was no significant difference (p > 0.05) for physical-chemical parameters between samples. In accordance with the parameters of the legislation, in the 104 examined samples of rennet cheese, it was observed that 100 (96.15%) were outside the limits for Staphylococcus coagulase positive; 32 samples (31%) did not follow the standardization required for coliforms; and only one sample showed the presence of Salmonella sp. There was no significant difference (p > 0.05) between samples with and without inspection for microbiological analyses. The similarity found in the assessment of physical-chemical parameters of rennet cheese between the evaluated states demonstrates uniformity in the processing of this product. High levels of contamination by pathogens such as Staphylococcus coagulase positive and thermo-tolerant coliforms, as well as the presence of Salmonella sp., demonstrate that the consumption of rennet cheese may present a health risk for consumers.(AU)

Salmonella spp. en manipuladores de alimentos en los comedores de una universidad venezolana / Salmonella spp. among food handlers in the dining room at a venezuelan university

La relación existente entre una inadecuada manipulación de los alimentos y la producción de infecciones gastrointestinales a través de éstos, ha sido ampliamente demostrada y una gran variedad de microorganismos entre ellos Salmonella spp. está asociada a esta transmisión. El propósito de esta investigación fue detectar la prevalencia de Salmonella a partir de muestras de heces en manipuladores de alimentos que laboran en dos comedores universitarios del estado Zulia. Entre los meses abril y julio del año 2009, se cultivaron 40 muestras de heces de individuos asintomáticos de ambos sexos y diferentes edades. El aislamiento e identificación bioquímica y serológica se realizó siguiendo la metodología convencional. Las pruebas de susceptibilidad a los agentes antimicrobianos se efectuaron según el método de difusión del disco siguiendo los criterios establecidos por el CLSI. Del total de muestras procesadas 4 de ellas (10%) resultaron positivas para el género Salmonella, 3 (75%) correspondientes al serogrupo B y 1 (25%) resultó ser al serogrupo E1. El 50% de las cepas mostró resistencia a ampicilina. La presencia de Salmonella en las heces de los manipuladores constituye un grave problema de salud pública, que no debe pasar desapercibido debido a su elevada infectividad y a su asociación a brotes importantes

The relationship between inadequate food handling and the production of gastrointestinal infections has been amply demonstrated, and a variety of organisms, including Salmonella, are associated with this transmission. The purpose of this research was to detect the prevalence of Salmonella in stool samples from food handlers who work in two dining rooms at the Zulia state university. Between April and July of 2009, 40 samples were cultured from stools of asymptomatic individuals of both sexes and different ages. Isolation, biochemical and serological identification were performed using conventional methodology. Tests for susceptibility to antimicrobial agents were done using the disk diffusion method, following criteria established by the CLSI. Of the total samples processed, four of them (10%) were positive for Salmonella 3, (75%) for serogroup B and 1 (25%) for serogroup E1. 50% of the strains were ampicillin resistant. The presence of Salmonella in the stools of food handlers is a major public health problem that should not go unnoticed due to its high infectivity and association with major outbreaks

Detección de serotipos de salmonella entérica de origen animal en habitantes de cuatro regiones de colombia

Salmonella enterica es un patógeno involucrado en enfermedades transmitidas por alimentos y animales. El tracto gastrointestinal de los animales sacrificados para consumo, como vacas, cerdos, pollos, entre otros, es fuente de contaminación de los productos derivados, constituyendo una de las principales fuentes de infección por Salmonella en humanos (1). Los humanos pueden desarrollar cuadros clínicos que varíandesde gastroenteritis hasta bacteremia y sepsis, y después del cuadro clínico gastrointestinal pueden permanecer como portadores asintomáticos.

Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy.

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

Salmonella y Shigella a partir de muestras fecales en la población Santa Rosa Maracaibo-Venezuela / Salmonella and Shigella in faecal specimen in the Santa Rosa population Maracaibo-Venezuela

Las infecciones gastrointestinales, representan a nivel mundial unas de las mayores causas de morbi-mortalidad, principalmente en los países subdesarrollados, siendo comunes en aquellas poblaciones con escasas condiciones socio-sanitarias como la población indígena de Santa Rosa. El propósito de esta investigación fue detectar la presencia de Salmonella y Shigella a partir de muestras fecales en la población de Santa Rosa. Se procesaron 245 muestras de heces de individuos entre edades comprendidas entre 2 meses a 83 años con o sin diarrea, en los cuales se realizó la técnica del coprocultivo (8) y las pruebas de susceptibilidad a los agentes antimicrobianos siguiendo la metodología descrita por el CLSI (5). Del total de muestras procesadas, 7 de ellas (2,9%) resultaron positivas para los géneros Salmonella y Shigella. Salmonella se detectó en 85,7%, identificándose serogrupos Salmonella enterica grupo B en 66,7% y Salmonella enterica grupo C1 en 33,3%; mientras que Shigella se aisló en un 14,3%, siendo S. flexneri la única especie encontrada. En las pruebas de resistencia antimicrobiana para Salmonella resultó ser resistente a Ampicilina, Tetraciclina y Amoxicilina/ Acido Clavulánico con un 16,7% para cada uno. Shigella mostró un patrón de resistencia a Ampicilina, Tetraciclina, Cloranfenicol, Amoxicilina /Acido Clavulánico y Trimetoprim Sulfametoxazol. A pesar de la precarias condiciones del sector de Santa Rosa, la incidencia de patógenos bacterianos es baja, en comparación a otros sectores con características similares de vida.

Impact of in vitro assembly defects on in vivo function of the phage P22 portal.

The podovirus P22, which infects O-antigen strains of Salmonella, incorporates a dsDNA translocating channel (portal dodecamer) at a unique vertex of the icosahedral capsid. The portal subunit (gp1, 82.7 kDa) exhibits multiple S-Hcdots, three dots, centeredX hydrogen bonding states for cysteines 153, 173, 283 and 516 and these interactions are strongly perturbed by portal ring formation. Here, we analyze in vivo activities of wild type (wt) and Cys-->Ser mutant portals, demonstrate that in vivo activity is correlated with in vitro assembly kinetics, and suggest mechanistic bases for the observed assembly defects. The C283S portal protein, which assembles into rings at about half the rate of wt, exhibits significantly diminished infectivity ( approximately 50% of wt) and manifests its defect prior to DNA packaging, most likely at the stage of procapsid assembly. Conversely, the C516S mutant, which assembles at twice the rate of wt, is more severely deficient in vivo ( approximately 20% of wt) and manifests its defect subsequent to capsid maturation and DNA packaging. Both C153S and C173S portals function at levels close to wt. The results suggest that C283S and C516S mutations may be exploited for improved characterization of the folding and assembly pathway of P22 portal protein.

Factors affecting iron sulfide-enhanced bacteriophage plaque assays in Salmonella.

Reaction of ferric ions with hydrogen sulfide (H(2)S) enhances contrast of phage plaques in H(2)S+ Salmonella, but contrast diminishes in weak H(2)S+ strains. H(2)S was affected by concentrations of peptones, glucose, ferric ammonium citrate (FAC) and sodium thiosulfate (ST), and by FAC:ST ratio, temperature, pH, air, and host strain. Increasing peptone levels was most important for improving contrast in weak H(2)S+ strains.

Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage.

The tailspike protein of Salmonella phage P22 is a viral adhesion protein with both receptor binding and destroying activities. It recognises the O-antigenic repeating units of cell surface lipopolysaccharide of serogroup A, B and D1 as receptor, but also inactivates its receptor by endoglycosidase (endorhamnosidase) activity. In the final step of bacteriophage P22 assembly six homotrimeric tailspike molecules are non-covalently attached to the DNA injection apparatus, mediated by their N-terminal, head-binding domains. We report the crystal structure of the head-binding domain of P22 tailspike protein at 2.3 A resolution, solved with a recombinant telluromethionine derivative and non-crystallographic symmetry averaging. The trimeric dome-like structure is formed by two perpendicular beta-sheets of five and three strands, respectively in each subunit and caps a three-helix bundle observed in the structure of the C-terminal receptor binding and cleaving fragment, reported here after full refinement at 1.56 A resolution. In the central part of the receptor binding fragment, three parallel beta-helices of 13 complete turns are associated side-by-side, while the three polypeptide strands merge into a single domain towards their C termini, with close interdigitation at the junction to the beta-helix part. Complex structures with receptor fragments from S. typhimurium, S. enteritidis and S. typhi253Ty determined at 1.8 A resolution are described in detail. Insertions into the beta-helix form the O-antigen binding groove, which also harbours the active site residues Asp392, Asp395 and Glu359. In the intact structure of the tailspike protein, head-binding and receptor-binding parts are probably linked by a flexible hinge whose function may be either to deal with shearing forces on the exposed, 150 A long tailspikes or to allow them to bend during the infection process.

Ocorrência de salmonella e coliformes de origem fecal na canela em pó (Cinnamomum cassia Blume e Cinnamomum zeylanicum Nees) comercializada em Florianópolis, Santa Catarina, Brasil / Salmonella and fecal coliforms in Cinnamon (Cinnamomum cassia Blume and Cinnamomum zeylanicum Nees) sold in the city of Florianopolis, Santa Catarina, Brazil

Cem amostras de canela em pó de dez marcas diferentes comercializadas na cidade de Florianópolis, SC, foram submetidas à análise microbiológica, pesquisando-se salmonella e coliformes de origem fecal. Em nenhuma amostra foi detectada salmonella. Coliformes de origem fecal foram encontrados entre os valores <3 a 2.400NMP/g. Das amostras analisadas, 63 por cento apresentaram valores de <3 NMP/g; 34 por cento apresentaram valores entre 3 e 100 NMP/g, limite este permitido pela legislaçäo brasileira em vigor; e 3 por cento apresentaram valores superiores a 100 NMP/g. A presença de coliformes de origem fecal foi detectada em 37 por cento das amostras de canela em pó.