Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

Neuropathological survey of fallen stock: Active surveillance reveals high prevalence of encephalitic listeriosis in small ruminants

This paper describes the prevalence of brain lesions in the Swiss fallen stock population of small ruminants. 3075 whole brains (75% sheep, 25% goats) were collected as part of a year-long active survey of transmissible spongiform encephalopathies (TSEs) in small ruminants conducted by the Swiss authorities between July 2004 and July 2005. All fallen stock brains were systematically examined by histopathology to obtain reliable data on histologically identifiable brain lesions. Lesions were found in an unexpectedly high number of animals (8.1% of all examined brains). A wide spectrum of diseases was detected showing that this approach provides an excellent opportunity to screen for the prevalence of neurological diseases. Encephalitic listeriosis was by far the most frequent cause of CNS lesions in both species and its prevalence was unexpectedly high when compared to notified confirmed cases. In conclusion, the prevalence of listeriosis as estimated by passive surveillance based on the notification of clinical suspects has been underestimated in the past.

Contamination routes and control of Listeria monocytogenes in Food Production

Listeria monocytogenes is the causative agent of the severe foodborne infection listeriosis. The number of listeriosis cases in recent years has increased in many European countries, including Finland. Contamination of the pathogen needs to be minimized and growth to high numbers in foods prevented in order to reduce the incidence of human cases. The aim of this study was to evaluate contamination routes of L. monocytogenes in the food chain and to investigate methods for control of the pathogen in food processing. L. monocytogenes was commonly found in wild birds, the pig production chain and in pork production plants. It was found most frequently in birds feeding at landfill site, organic farms, tonsil samples, and sites associated with brining. L. monococytogenes in birds, farms, food processing plant or foods did not form distinct genetic groups, but populations overlapped. The majority of genotypes recovered from birds were also detected in foods, food processing environments and other animal species and birds may disseminate L. monocytogenes into food chain. Similar genotypes were found in different pigs on the same farm, as well as in pigs on farms and later in the slaughterhouse. L. monocytogenes contamination spreads at farm level and may be a contamination source into slaughterhouses and further into meat. Incoming raw pork in the processing plant was frequently contaminated with L. monocytogenes and genotypes in raw meat were also found in processing environment and in RTE products. Thus, raw material seems to be a considerable source of contamination into processing facilities. In the pork processing plant, the prevalence of L. monocytogenes increased in the brining area, showing that the brining was an important contamination site. Recovery of the inoculated L. monocytogenes strains showed that there were strain-specific differences in the ability to survive in lettuce and dry sausage. The ability of some L. monocytogenes strains to survive well in food production raises a challenge for industry, because these strains can be especially difficult to remove from the products and raises a need to use an appropriate hurdle concept to control most resistant strains. Control of L. monocytogenes can be implemented throughout the food chain. Farm-specific factors affected the prevalence of L. monocytogenes and good farm-level practices can therefore be utilized to reduce the prevalence of this pathogen on the farm and possibly further in the food chain. Well separated areas in a pork production plant had low prevalences of L. monocytogenes, thus showing that compartmentalization controls the pathogen in the processing line. The food processing plant, especially the brining area, should be subjected to disassembling, extensive cleaning and disinfection to eliminate persistent contamination by L. monocytogenes, and replacing brining with dry-salting should be considered. All of the evaluated washing solutions decreased the populations of L. monocytogenes on precut lettuce, but did not eliminate the pathogen. Thus, the safety of fresh-cut produce cannot rely on washing with disinfectants, and high-quality raw material and good manufacturing practices remain important. L. monocytogenes was detected in higher levels in sausages without the protective culture than in sausages with this protective strain, although numbers of L. monocytogenes by the end of the ripening decreased to the level of < 100 MPN/g in all sausages. Protective starter cultures provide an appealing hurdle in dry sausage processing and assist in the control of L. monocytogenes.

Prevalence of Listeria monocytogenes in Idiazabal cheese

Este trabajo se encuentra bajo la licencia Creative Commons Attribution 3.0.

Exhausted CD8 T cells downregulate the IL-18 receptor and become unresponsive to inflammatory cytokines and bacterial co-infections.

During many chronic infections virus-specific CD8 T cells succumb to exhaustion as they lose their ability to respond to antigenic activation. Combinations of IL-12, IL-18, and IL-21 have been shown to induce the antigen-independent production of interferon (IFN)-γ by effector and memory CD8 T cells. In this study we investigated whether exhausted CD8 T cells are sensitive to activation by these cytokines. We show that effector and memory, but not exhausted, CD8 T cells produce IFN-γ and upregulate CD25 following exposure to certain combinations of IL-12, IL-18, and IL-21. The unresponsiveness of exhausted CD8 T cells is associated with downregulation of the IL-18-receptor-α (IL-18Rα). Although IL-18Rα expression is connected with the ability of memory CD8 T cells to self-renew and efflux rhodamine 123, the IL-18Rα(lo) exhausted cells remained capable of secreting this dye. To further evaluate the consequences of IL-18Rα downregulation, we tracked the fate of IL-18Rα-deficient CD8 T cells in chronically infected mixed bone marrow chimeras and discovered that IL-18Rα affects the initial but not later phases of the response. The antigen-independent responsiveness of exhausted CD8 T cells was also investigated following co-infection with Listeria monocytogenes, which induces the expression of IL-12 and IL-18. Although IL-18Rα(hi) memory cells upregulated CD25 and produced IFN-γ, the IL-18Rα(lo) exhausted cells failed to respond. Collectively, these findings indicate that as exhausted T cells adjust to the chronically infected environment, they lose their susceptibility to antigen-independent activation by cytokines, which compromises their ability to detect bacterial co-infections.

Synergism between high-pressure processing and active packaging against Listeria monocytogenes in ready-to-eat chicken breast

The effects of high-pressure processing (HPP) in conjunction with an essential oil-based active packaging on the surface of ready-to-eat (RTE) chicken breast were investigated as post-processing listericidal treatment. Three different treatments were used, and all samples were vacuum packed: (i) HPP at 500. MPa for 1. min (control), (ii) active packaging based on coriander essential oil, and (iii) active packaging and HPP. When applied individually, active packaging and pressurisation delayed the growth of Listeria monocytogenes. The combination of HPP and active packaging resulted in a synergistic effect reducing the counts of the pathogen below the detection limit throughout 60. days storage at 4. °C. However, when these samples were stored at 8. °C, growth did occur, but again a delay in growth was observed. The effects on colour and lipid oxidation were also studied during storage and were not significantly affected by the treatments. Active packaging followed by in-package pressure treatment could be a useful approach to reduce the risk of L. monocytogenes in cooked chicken without impairing its quality. Industrial relevance: Ready-to-eat products are of great economic importance to the industry. However, they have been implicated in several outbreaks of listeriosis. Therefore, effective ways to reduce the risk from this pathogenic microorganism can be very attractive for manufacturers. This study showed that the use of active packaging followed by HPP can enhance the listericidal efficiency of the treatment while using lower pressure levels, and thus having limited effects on colour and lipid oxidation of RTE chicken breast.

Surto de listeria monocytogenes na Região de Lisboa e Vale do Tejo : 2009-2011

RESUMO - O género Listeria contém oito espécies (L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, e a L. rocoutiae), das quais duas são patogénicas. L. monocytogenes é patogénica para humanos e animais; L. ivanovii primeiramente infecta animais e raramente causa doenças em humanos. A Listeria monocytogenes é uma bactéria patogénica Gram-positiva facultativa intracelular, ubíqua na natureza. Nos últimos anos o número de casos de listeriose tem vindo a aumentar. Pode causar uma doença rara e grave chamada listeriose, especialmente nas mulheres grávidas, nos idosos ou em indivíduos com o sistema imunitário debilitado, de maneira esporádica ou em forma surtos. Realizou-se um estudo observacional, descritivo com o objectivo de se fazer a descrição e a caracterização do surto de listeriose ocorrido na região de Lisboa e Vale do Tejo entre 2009-2011. O período de maior número de casos diagnosticados de listeriose ocorreu entre o mês de Abril e Agosto de 2010. Mas a janela temporal em que ocorreu o surto estendeu-se de Março de 2009 a Janeiro de 2012. Ocorreram 51 casos de internamento com diagnóstico de listeriose, dos quais 25 casos foram confirmados, pela técnica de PFGE, pertencer à mesma estirpe I, sorotipo 4b e pulsotipo 070 e 0101. Na maioria dos casos eram do sexo feminino, com uma média de 57,14 anos de idade e com residência na região de Lisboa e Vale do Tejo. Em 96,08% dos doentes internados com listeriose apresentavam factores de predisposição, comorbilidade e /ou imunossupressão. A bacteriemia foi a manifestação clínica mais frequente, seguida da meningite. A letalidade da listeriose foi de 15,69%.

Detección de Listeria monocytogenes y Listeria spp en puntos críticos establecidos en una planta procesadora de productos congelados para rápido consumo

Tesis ( Maestro en Ciencias con Especialidad en Microbiología) U.A.N.L.

Étude prospective des causes de mortalité chez l'espèce caprine avec emphase sur la lymphadénite caséeuse

En Amérique du Nord, et particulièrement au Canada, il y a très peu de données sur l’incidence des causes de mortalité chez l’espèce caprine. Le premier objectif de cette étude était de déterminer les principales causes de mortalité chez les chèvres au Québec. Depuis 2006, avec l’arrêt de la vaccination contre la lymphadénite caséeuse, les éleveurs de caprins laitiers et de boucherie du Québec ont rapporté une recrudescence des abcès chez leur bétail. Le second but de cette étude était de déterminer l’importance de la lymphadénite caséeuse dans le dépérissement et la mortalité des chèvres du Québec. Cent-cinquante-deux chèvres provenant de 13 élevages différents ont été soumises pour nécropsie et la cause de mortalité, de même que la présence d’abcès (s’il y a lieu), leur localisation et leur cause furent compilés. Les mortalités proportionnelles étaient, par ordre décroissant : l’entérotoxémie de type D (n= 26; 17,1%), la pneumonie (n= 21; 13,8%), la paratuberculose (n= 16; 10,5%), listériose encéphalitique (n= 10; 6,6%), la toxémie de gestation (n= 8; 5,3%), l’arthrite-encéphalite caprine (n= 7; 4,6%) et la lymphadénite caséeuse (n= 6; 3,9%). La lymphadénite caséeuse a été diagnostiquée chez 24,3% des chèvres soumises, mais sans être une cause majeure de dépérissement ou de mortalité. Les abcès étaient internes dans 54,1% des cas. Au total, la paratuberculose a été diagnostiquée chez 29 chèvres (16 en étant décédées) et fut considérée comme une cause majeure de dépérissement, d’émaciation et de mortalité. Le développement et l’implantation de mesures préventives contre cette maladie seraient donc à envisager dans le futur.

Functional γ-aminobutyrate shunt in Listeria monocytogenes: role in acid tolerance and succinate biosynthesis

Listeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whether L. monocytogenes has this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis in L. monocytogenes. Using crude protein extracts from L. monocytogenes EGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities, argD (GABA-AT) and lmo0913 (SSDH). Crude protein extracts prepared from a mutant lacking a functional argD gene significantly reduced GABA-AT activity, while an lmo0913 mutant lost all detectable SSDH activity. The deletion of lmo0913 increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genus Listeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle of Listeria.

Listeria monocytogenes in ready-to-eat, sliced, cooked ham and salami products, marketed in the city of Sao Paulo, Brazil Occurrence, quantification, and serotyping

The preference for ready-to-eat sliced foods may pose an increased risk for food-borne diseases and a major concern is the presence of Listeria monocytogenes L monocytogenes was assessed in two types of products cooked ham and salami One hundred and thirty samples of each product were acquired in retail shops in the city of Sao Paulo and submitted to laboratory analysis The rate of positives was significantly higher in salami samples than in ham samples (62% and 0 8% respectively) L. monocytogenes counts in salami samples varied between <10 and 1900 colony-forming units per gram (CFU/g) The serotypes found in both products were as follows according to incidence 4b (37 5%) 1/2b (25%) 3b (25%) and 1/2c (12 5%) Based on the results of the present study the authors suggest that the risk of listeriosis resulting from the consumption of salami is higher than that associated with the consumption of cooked ham (C) 2010 Elsevier Ltd All rights reserved

Padronização da técnica de imunoistoquímica para identificação de Listéria monocytogenes em placentas humanas e tecido nervoso central de ruminantes.

Este projeto foi desenvolvido no Laboratório de Patologia Experimental do Hospital de Clínicas de Porto Alegre (HCPA), com a aprovação da Comitê de Ética em Pesquisa do HCPA e com apoio financeiro parcial do Fundo de Incentivo à Pesquisa e Eventos do HCPA (FIPE). O experimento 1, chamado de projeto piloto, teve como objetivo implementar a técnica de IHQ para identificar a Listeria monocytogenes (L.m.), utilizando anticorpo policlonal antilisteria monocytogenes (Biodesig ). Vários testes foram realizados para acertar a diluição (1:1000) que foi diferente da preconizada pelo fabricante. Os blocos de parafina, de dez placentas provenientes de parto prematuro ou aborto foram utilizados para os cortes histológicos e a preparação das lâminas para a coloração Hematoxilina e Eosina (HE) e imunoistoquímica (IHQ). As lâminas foram identificadas por números para resguardar a identidade das pacientes. O resultado do HE mostrou alterações inflamatórias em oito placentas e L. m. foi identificada pelo IHQ em cinco dessas placentas. O objetivo do 2º experimento foi identificar a L. m. em tecido nervoso cerebral de ruminantes, utilizando a técnica implementada no projeto piloto. O material utilizado neste trabalho foi cedido pelo Setor de Patologia Veterinária do Departamento de Patologia da Universidade Federal de Santa Maria. Os casos estudados (2 ovinos, 1 caprino e 2 bovinos) tinham suspeitas clínicas diversas e as necropsias dos animais evidenciaram aspectos sugestivos da doença. Os cinco casos foram confirmados pelo IHQ, comprovando a importância da utilização desta técnica para o diagnóstico da listeriose no SNC de ruminantes. O 3º experimento objetivou identificar a L. m. em placentas encaminhadas ao Serviço de Patologia do HCPA no ano 2000. Da mesma forma que no experimento 1, as lâminas foram identificadas por números. Após o levantamento realizado nos registros dos exames anatomopatológicos (AP) deste setor, observou-se que 714 AP eram de placentas provenientes de aborto, parto prematuro e nascimento a termo examinados naquele período. Foram sorteados 254 AP para análise através de HE, revelando que 148 desses AP apresentavam alterações inflamatórias (corioamnionite, vilite e deciduite). Os blocos destas placentas foram utilizados para fazer as lâminas e realizar IHQ. A consulta aos prontuários dos casos com alterações inflamatórias permitiu observar que um deles tinha a confirmação bacteriológica de L. m. na placenta, tornando-se este o controle positivo. O controle negativo foi selecionado entre aqueles sorteados que não apresentavam alterações inflamatórias. A presença de L. m. foi identificada em 33,78% das placentas analisadas pela técnica IHQ. Corioamnionite e vilite foram as alterações inflamatórias que mostraram diferença estatística significativa nas placentas positivas. L. m. estava presente nas placentas de 1º, 2º e 3º trimestres gestacionais. A idade das gestantes, casos de aborto e/ou parto prematuro não mostraram diferença estatística significativa com a presença ou ausência de L. m. nas placentas. Abortos habituais ocorreram em pacientes com ou sem L. m. no tecido placentário. Conclusão: a técnica de imunohistoquímica pode ser utilizada para confirmar o diagnóstico histopatológico de listeriose em placentas e tecido nervoso central de ruminantes.

The effects of Chlorella vulgaris in the protection of mice infected with Listeria monocytogenes. Role of natural killer cells

In this work we have demonstrated the effects of oral administration of Chlorella vulgaris (CV) on Natural Killer cells (NK) activity of mice infected with a sublethal dose of viable Listeria monocytogenes. The treatment with C. vulgaris produced a significant increase on NK cells activity in normal (non-infected) animals compared to the animals that received only vehicle (water) (p < 0.0001). Similarly, the infection alone produced a significant increase on NK cells activity, which was observed at 48 and 72 hours after the inoculation of L. monocytogenes. Moreover, when CV was administered in infected animals, there was an additional increase in NK cells activity which was significantly higher than that found in the infected groups (p < 0.0001) CV treatment (50 and 500mg/Kg) of mice infected with a dose of 3x105 bacteria/animal, which was lethal for all the non- treated controls, produced a dose-response protection which led to a 20% and 55% survival, respectively (p < 0.0001).

Produção de biofilme por Listeria monocytogenes isoladas de diferentes alimentos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)