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Lawsonia intracellularis is an obligate intracellular bacterium and causative agent of proliferative enteropathy. The pathogenesis of L. intracellularis is not completely understood, including the endocytic mechanisms to access the host cell cytoplasm. In this study, we evaluated the mechanisms involved in endocytosis of L. intracellularis in vitro using intestinal porcine epithelial cells (IPEC-J2). Confocal microscopy was used to co-localize L. intracellularis and clathrin. Clathrin gene knockdown was then applied to verify whether L. intracellularis endocytosis is clathrin-dependent. Finally, internalization of viable and non-viable (bacteria were inactivated by heat) L. intracellularis organisms were assessed to study the role of the host cell during bacterial endocytosis. L. intracellularis organisms were observed co-localized with clathrin by confocal microscopy but the amount of L. intracellularis internalized in cells, with and without clathrin knockdown, did not differ statistically. The internalization of non-viable L. intracellularis showed a decrease in the internalization in cells with less clathrin synthesis (P<0.05). The present study is the first to elucidate the involvement of clathrin in the endocytosis of L. intracellularis. Clathrin-mediated endocytosis was shown to be an important, but not required, process for L. intracellularis internalization in porcine intestinal epithelial cells. Independence of bacterial viability for host cell internalization was also confirmed.

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The authors wish to make the following correction to this paper [...].

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Porcine circovirus type 3 (PCV3) has been recently described as a potential cause of abortions and systemic vasculitis in pigs. Although the virus has been detected by real-time PCR in several porcine tissues from countries worldwide, PCV3-associated diseases have not been satisfactorily clarified. The objective of this study was to investigate the association between the presence of PCV3 mRNA detected by in situ hybridization (ISH) within histological lesions and PCV3 DNA detected by real-time PCR in naturally infected pigs. A total of 25 PCV3 PCR-positive cases were analyzed. Formalin-fixed tissues from these cases were evaluated for histologic lesions and for ISH-RNA positive signals for PCV3. The most frequent tissue type with histopathologic lesions was heart, 76.2%, with lymphoplasmacytic myocarditis and epicarditis as the most frequent lesions observed. Lymphoplasmacytic interstitial pneumonia was also a frequent finding, 47.6%. There were also lesions in kidney, liver, spleen and lymph nodes. PCV3-ISH-RNA positive signals were mostly observed in association with lymphoplasmacytic inflammatory infiltrate in various tissues, including arteries. Based on our results, the minimum set of specimens to be submitted for histopathology and mRNA in situ hybridization to confirm or exclude a diagnosis of PCV3 are heart, lung and lymphoid tissues (i.e., spleen and lymph nodes), especially for differential diagnosis related with PCV2-associated diseases.

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Surtos recentes da Diarreia Epidêmica Suína (PED, em inglês) têm sido descritos em diversos países, sobretudo na China e Estados Unidos, e tem provocado significativo impacto econômico à suinocultura mundial. Como a doença se disseminou também pela América do Sul, afetando Peru, Colômbia e Equador, o conhecimento sobre a enfermidade é de extrema relevância para manutenção do status livre da doença no Brasil. A PED é causada por vírus da família Coronaviridae, caracterizando-se por diarreia líquida profusa e vômitos, de intensidade variável, sendo mais graves em leitões neonatos. A RT-PCR tem sido a técnica de diagnóstico mais empregada, mas a sorologia e a imuno-histoquímica também podem ser utilizadas. A vacina comercial disponível nos Estados Unidos é considerada de alto custo e efetividade intermediária e, por isso, protocolos rígidos de biossegurança são fundamentais para dificultar a entrada do agente no rebanho. Estudos recentes têm se intensificado no sentido de elucidar a epidemiologia da doença, uma vez que a via de disseminação viral entre rebanhos ainda não se encontra totalmente estabelecida e o impacto econômico causado pela alta mortalidade, sobretudo de animais lactentes, compromete sensivelmente a produção de carne suína nos países afetados.

Recent outbreaks of Porcine Epidemic Diarrhea (PED) has been described in several countries, mainly in China and the United States, and it has causing significative economic impact to the swine industry worldwide. As the virus spread also in South America, affecting Peru, Colombia and Ecuador, knowledge about the disease is extremely important to maintain the free status of the disease in Brazil. PED is caused by a virus from Coronaviridae family, and is characterized by profuse liquid diarrhea and vomit, varying in severity, being more severe in newborn piglets. The RT-PCR has been the most used diagnostic technique, but serology and immunohistochemistry can also be used. The commercial available vaccine is considered costly and with intermediate effectiveness, consequently complementary biosecurity rules are important to hinder the entry of the agent into the herd. Many studies have been conducted in order to elucidate the epidemiology of the disease, since the route of viral spread among herds is not yet fully established and the economic impact due to high mortality, especially in young animals, significantly impairs the production of pork in affected countries.

Brotes recientes de la Diarrea Epidémica Suína (PED, en inglés) han sido descritos en diversos países, sobre todo en China y Estados Unidos, y ha provocado un significativo impacto económico a la suinocultura mundial. Como la enfermedad también se diseminó en América del Sur, afectando a Perú, Colombia y Ecuador, el conocimiento sobre la enfermedad es de extrema relevancia para el mantenimiento del estatus libre de la enfermedad en el Brasil. La PED es causada por el virus de la familia Coronaviridae, caracterizándose por diarrea liquida profusa y vómito, de intensidad variable, siendo más grave en lechones neonatos. La RT-PCR ha sido la técnica de diagnóstico más utilizada, pero la serología e inmunohistoquímica también pueden ser utilizadas. La vacuna comercial disponible en los Estados Unidos es considerada de alto costo y efectividad intermedia, por eso, protocolos rígidos de bioseguridad son fundamentales para dificultar la entrada del agente en el rebaño. Estudios recientes se han intensificado en el sentido de dilucidar la epidemiologia de la enfermedad, una vez que la vía de diseminación viral entre rebaños aún no se encuentra completamente establecida y el impacto económico causado por la alta mortalidad, sobre todo de animales lactantes, compromete sensiblemente la producción de carne suína en los países afectados.

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Surtos recentes da Diarreia Epidêmica Suína (PED, em inglês) têm sido descritos em diversos países, sobretudo na China e Estados Unidos, e tem provocado significativo impacto econômico à suinocultura mundial. Como a doença se disseminou também pela América do Sul, afetando Peru, Colômbia e Equador, o conhecimento sobre a enfermidade é de extrema relevância para manutenção do status livre da doença no Brasil. A PED é causada por vírus da família Coronaviridae, caracterizando-se por diarreia líquida profusa e vômitos, de intensidade variável, sendo mais graves em leitões neonatos. A RT-PCR tem sido a técnica de diagnóstico mais empregada, mas a sorologia e a imuno-histoquímica também podem ser utilizadas. A vacina comercial disponível nos Estados Unidos é considerada de alto custo e efetividade intermediária e, por isso, protocolos rígidos de biossegurança são fundamentais para dificultar a entrada do agente no rebanho. Estudos recentes têm se intensificado no sentido de elucidar a epidemiologia da doença, uma vez que a via de disseminação viral entre rebanhos ainda não se encontra totalmente estabelecida e o impacto econômico causado pela alta mortalidade, sobretudo de animais lactentes, compromete sensivelmente a produção de carne suína nos países afetados.(AU)

Recent outbreaks of Porcine Epidemic Diarrhea (PED) has been described in several countries, mainly in China and the United States, and it has causing significative economic impact to the swine industry worldwide. As the virus spread also in South America, affecting Peru, Colombia and Ecuador, knowledge about the disease is extremely important to maintain the free status of the disease in Brazil. PED is caused by a virus from Coronaviridae family, and is characterized by profuse liquid diarrhea and vomit, varying in severity, being more severe in newborn piglets. The RT-PCR has been the most used diagnostic technique, but serology and immunohistochemistry can also be used. The commercial available vaccine is considered costly and with intermediate effectiveness, consequently complementary biosecurity rules are important to hinder the entry of the agent into the herd. Many studies have been conducted in order to elucidate the epidemiology of the disease, since the route of viral spread among herds is not yet fully established and the economic impact due to high mortality, especially in young animals, significantly impairs the production of pork in affected countries.(AU)

Brotes recientes de la Diarrea Epidémica Suína (PED, en inglés) han sido descritos en diversos países, sobre todo en China y Estados Unidos, y ha provocado un significativo impacto económico a la suinocultura mundial. Como la enfermedad también se diseminó en América del Sur, afectando a Perú, Colombia y Ecuador, el conocimiento sobre la enfermedad es de extrema relevancia para el mantenimiento del estatus libre de la enfermedad en el Brasil. La PED es causada por el virus de la familia Coronaviridae, caracterizándose por diarrea liquida profusa y vómito, de intensidad variable, siendo más grave en lechones neonatos. La RT-PCR ha sido la técnica de diagnóstico más utilizada, pero la serología e inmunohistoquímica también pueden ser utilizadas. La vacuna comercial disponible en los Estados Unidos es considerada de alto costo y efectividad intermedia, por eso, protocolos rígidos de bioseguridad son fundamentales para dificultar la entrada del agente en el rebaño. Estudios recientes se han intensificado en el sentido de dilucidar la epidemiologia de la enfermedad, una vez que la vía de diseminación viral entre rebaños aún no se encuentra completamente establecida y el impacto económico causado por la alta mortalidad, sobre todo de animales lactantes, compromete sensiblemente la producción de carne suína en los países afectados.(AU)

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Lawsonia intracellularis, an obligately intracellular enteric bacterium, infects intestinal epithelial cells, but may also be found within macrophages in the intestinal lamina propria of affected pigs. Macrophages play an important role in host defense against infectious agents, but the role of this cell in L. intracellularis infection is not well understood. The aim of this study was to evaluate the permissibility of macrophages to L. intracellularis infection in vitro. Pure culture of L. intracellularis was added to swine peripheral blood monocyte-derived macrophages. Viability of intracytoplasmic L. intracellularis was evaluated at different time points by transmission electron microscopy (TEM). Potential replication of L. intracellularis in macrophages was also evaluated by qPCR. By TEM, phagocytosis L. intracellularis within of phagolysosomes were observed 1-hour post-infection (hpi) and bacterial structures in binary fission at 48 hpi. The number of intracellular bacteria was determined at 1, 4, 24, 48, and 72 hpi by qPCR in infected macrophages and compared to the number of intracellular bacteria from culture in McCoy cells. In both cell lines, the amount of L. intracellularis was decreased at 4 hpiand increased at 24 hpi. The number of intracellular bacteria continued to increase in McCoy cells over time. This is the first study showing interaction, survival and propagation of L. intracellularis in macrophages. These findings are critical to establish an experimental model for future studies of the pathogenesis of porcine proliferative enteropathy and the potential persistence of L. intracellularis in macrophages during chronic infections.

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BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

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BACKGROUND: Lawsonia intracellularis is the etiologic agent of proliferative enteropathy, which causes diarrhea in several animal species, including swine. Serology can be used both to determine the prevalence of antibodies against a specific pathogen in a herd and to obtain the serological profile, which is used to determine the dynamics of infection in the herd. The objective of this study was to determine the serological profile and seroprevalence of anti-L. intracellularis antibodies in swine herds from intensive production regions of Minas Gerais, Brazil, and to identify the risk factors related to the herd-level seropositivity. RESULTS: A total of 2999 serum samples were collected for this cross-sectional study in the four major regions of intensive swine production in Minas Gerais, Brazil. To obtain better estimates and increase the external validity of the seroprevalence, the sample data were weighted based on the pig population of each herd, the stratum in which the herd was classified and the swine population of the region where each herd was located. A questionnaire was used to identify potential risk factors related to this herd-level seropositivity. The overall weighted prevalence in Minas Gerais was 34.7% (95% confidence interval: 32.12 - 37.20%), and there was no significant difference among the sampled regions, with the seroprevalence rates ranging between 32.06 and 37.66%. Finishing pigs were the most prevalent among the sampled categories. Among the evaluated risk factors, "cleaning before disinfecting" had a negative impact in the seroprevalence (p < 0.05) and was considered a protective factor. CONCLUSIONS: The anti-L. intracellularis antibodies were detected in all of the investigated herds in Minas Gerais, which indicated a wide distribution of the agent in the state. The predominant serological profile was consistent with the dynamics of infection previously observed in pig herds in other countries with similar antimicrobial usage, in which the nursery pigs usually show the lowest seroprevalence and the finishing pigs exhibit the highest. Herds that adopt the practice of "cleaning before disinfection" can decrease their L. intracellularis antibody seropositivity.

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