Evaluation of the role of clathrin and bacterial viability in the endocytosis of Lawsonia intracellularis.

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.

Correction: De Conti et al. Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs. Animals 2021, 11, 1520.

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

Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs.

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.

Diarreia Epidêmica Suína (PED), enfermidade exótica no Brasil mas emergente nas Américas / Porcine Epidemic Diarrhea (PED), exotic disease in Brazil but emerging in the Americas / Diarrea Epidemica Suina (PED), enfermedad exotica en Brasil, pero emergente el las Américas

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)

Diarreia Epidêmica Suína (PED), enfermidade exótica no Brasil mas emergente nas Américas / Porcine Epidemic Diarrhea (PED), exotic disease in Brazil but emerging in the Americas / Diarrea Epidemica Suina (PED), enfermedad exotica en Brasil, pero emergente el las Américas

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.

Survival of Lawsonia intracellularis in porcine peripheral blood monocyte-derived macrophages.

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.

Intestinal absorption and histomorphometry of Syrian hamsters (Mesocricetus auratus) experimentally infected with Lawsonia intracellularis.

The objective of this study was to evaluate the intestinal absorption and histomorphometry of hamsters experimentally infected with Lawsonia intracellularis and correlate these parameters with severity of infection based on immunohistochemistry. Sixty hamsters were equally divided into control and inoculated groups which were orally infected with intestinal mucosa homogenate from pigs naturally infected with L. intracellularis. The intestinal absorption of glucose, sodium, potassium and chloride was evaluated in live animals (25 inoculated and 25 control) on day 26 after inoculation. In this procedure, a standard solution was infused into the cranial jejunum and collected at the terminal ileum. The experimental infection was confirmed by gross and histopathological examination and L. intracellularis antigen labeling by immunohistochemistry. Histomorphometry analysis demonstrated positive correlation between intestinal crypt depth and severity of infection based on immunohistochemistry. Infected animals had significantly lower intestinal absorption of glucose, potassium and chloride. These results indicate a lower intestinal absorption as an important mechanism of diarrhea in hamsters experimentally infected with L. intracellularis. Therefore, malabsorption should be considered as the main mechanism involved in the physiopathology of the diarrhea in L. intracellularis infected animals.

Adenocarcinoma pulmonar em um bovino / Pulmonary adenocarcinoma in a bovine

Um bovino Guzerá, fêmea, adulto, com histórico de insuficiência cardíaca congestiva direita de duração de duas semanas, morreu durante o transporte ao hospital veterinário. À necropsia, o lobo pulmonar cranial esquerdo estava moderadamente aumentado de tamanho e firme. O parênquima do lobo afetado era branco e continha múltiplas áreas de 0,3 a 1,5cm de diâmetro, amareladas e caseosas. Alterações semelhantes foram observadas nos linfonodos mediastínicos e brônquicos, no pericárdio parietal, no epicárdio e na adventícia da artéria pulmonar. Histologicamente, a massa tecidual do lobo pulmonar era constituída por células epiteliais neoplásicas de padrão acinar, com duas ou mais camadas celulares, algumas com projeções papilares intraluminais. A anisocariose era acentuada, e o índice mitótico, moderado (dois a três por campo de maior aumento). Envolvendo as neoformações, observava-se abundante tecido conjuntivo fibroso. Focos de necrose e mineralização eram multifocais moderados. Alterações histológicas semelhantes foram observadas nos linfonodos brônquicos, nos mediastínicos, nos pericárdios visceral e parietal e na adventícia da artéria pulmonar. Com exceção do fígado com congestão generalizada crônica, não foram observadas alterações macro e microscópicas em outros órgãos. Os achados histológicos foram compatíveis com adenocarcinoma pulmonar, com metástases regionais. O quadro de insuficiência cardíaca congestiva direita provavelmente foi decorrente do impedimento da drenagem linfática pelas metástases.

An adult Guzera cow, dysplaying for two weeks signs of right-sided congestive heart failure died during the transport to the veterinary hospital. At necropsy, the left cranial lung lobe was moderately increased in volume and firm. The parenchyma of the affected lung lobe was white and contained multiple 0.3 to 1.5cm in diameter, yellow, dry, friable nodules. Similar changes were observed in bronchial and mediastinal lymph nodes, parietal pericardium, epicardium, and adventitia of the pulmonary artery. Microscopically, the pulmonary tissue mass was composed of neoplastic epithelial cells arranged in acini lined by two or more layers, some with intraluminal papillary projections. Anisokaryosis was marked, and mitotic index was moderate (2-3 mitosis in high field). Abundant fibrous connective tissue surrounded the neoplastic cell aggregates. Foci of necrosis and mineralization were moderate. Similar microscopic changes were observed in bronchial and mediastinal lymph nodes, visceral and parietal pericardium and adventitia of the pulmonary artery. With liver chronic generalized congestion exception, no other macro or microscopic lesions were observed. Microscopic findings were consistent with pulmonary adenocarcinoma with regional metastases. The right-sided congestive heart failure was probably due to obstruction of lymphatic drainage by metastases.

Fisiopatologia das diarréias em suínos / Pathophysiology of swine diarrhea

A diarréia é a principal manifestação clínica das infecções entéricas. O desequilíbrio entre os processos de absorção e secreção intestinal decorrentes de alterações no transporte de água e eletrólitos é determinante na patogênese dos processos diarréicos. Os mecanismos fisiopatológicos estão associados às características particulares de cada patógeno. Dessa forma, a interação específica entre os patógenos entéricos e o epitélio intestinal resulta em diferentes tipos de diarréias. Na produção de suínos, o conhecimento da fisiopatologia das infecções entéricas é fundamental no desenvolvimento de métodos de diagnóstico e, em um futuro próximo, na elaboração de estratégias de prevenção e controle dessas afecções. Com base nos processos fisiológicos característicos do trato gastrointestinal, esta revisão tem como objetivo descrever aspectos relevantes e atuais dos mecanismos fisiopatológicos das diarréias em suínos. Serão enfatizadas as alterações patológicas no transporte intestinal de íons e nutrientes decorrentes da ação de patógenos entéricos importantes na produção suinícola.

Diarrhea is the main clinical manifestation of enteric infections. Imbalance between absorption and intestinal secretion process due to changes in the water and electrolytes transport is critical in the diarrheal diseases pathogenesis. The pathophysiological mechanisms are associated to specific characteristics of each pathogen. Therefore, specific interaction between enteric pathogens and intestinal epithelium result in different types of diarrhea. In swine production, the knowledge about the enteric infections pathophysiology is important for the development of diagnostic methods and, in a near future improvement of prevention and control strategies. The aim of this review is to describe different relevant aspects of the swine diarrhea pathophysiology based on the physiological processes typical of gastrointestinal tract. Pathological changes in the intestinal transport of ions and nutrients due to infectious relevant for swine production will be emphasized.