Immunopathogenesis of PRRS: comparative study in the lung and primary lymphoid organs of animals infected with different virulence PRRSV-1 strain

El PRRSV es el causante del síndrome reproductivo y respiratorio porcino; una enfermedad que, a pesar de los esfuerzos realizados por controlarla y disminuir su impacto económico, sigue siendo considerada como una de las enfermedades más importantes que afectan al sector porcino. Los dos genotipos del PRRSV, el genotipo Europeo o tipo 1 (PRRSV-1), representado por la cepa Lelystad (LV), y el genotipo Norteamericano o tipo 2 (PRRSV-2), cuyo prototipo es el aislado ATCC-VR-2332, presentan entre sí una elevada variabilidad genética y biológica. Inicialmente, se creía que todas las cepas del genotipo Europeo eran genéticamente homólogas. Sin embargo, el análisis de la secuencia de la ORF5 y OFR7 han justificado la división del genotipo Europeo en cuatro subtipos genéticos bien diferenciados: el subtipo 1 o Pan-europeo, y los subtipos 2, 3 y 4 de Europa del Este. La amplia variabilidad biológica, genética e incluso antigénica de los diferentes aislados del PRRSV-1, así como las elevadas pérdidas económicas que esta enfermedad ocasiona en el sector porcino, justifican la realización de estudios que tengan como objetivo aclarar los mecanismos patogénicos desencadenados por las diferentes cepas de este virus. En esta tesis doctoral hemos evaluado la expresión del antígeno viral, el cuadro lesional y la expresión de diferentes citoquinas proinflamatorias e inmunomoduladoras en el pulmón y en los órganos linfoides primarios (timo y médula ósea) tras la infección con una cepa...Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a disease of pigs, named porcine reproductive and respiratory syndrome (PRRS). Despite efforts in controlling it and reducing its economic impact, PRRS is still considered one of the most important diseases affecting the swine industry. PRRSV is divided into two distinct genotypes, European PRRSV or Type 1 (PRRSV-1) with the prototype Lelystad strain (LV), and North American or Type 2 (PRRSV-2), whose prototype is the ATCCVR- 2332 strain, with high genetic and biological diversity among both genotypes. Initially, PRRSV-1 was thought to be genetically homogenous. However, deep analysis of PRRSV-1 ORF5 and OFR7 sequences defined four distinct genetic subtypes: subtype 1 or Pan- European, and subtypes 2, 3 and 4 in Eastern Europe. The wide biological, genetic and antigenic diversity of the different PRRSV-1 strains, as well as the high economic losses to the pork industry associated with this disease justify studies that aim to clarify the pathogenic mechanisms triggered by different virus strains. In the present thesis we evaluated the expression of viral antigen, the morphologic lesion and the expression of several proinflammatory and immunomodulatory cytokines in lungs and primary lymphoid organs (thymus and bone marrow) after the infection with a prototype PRRSV-1 Lelystad (LV), two field strains (SU1-bel, of high virulence, and 215-06,..

Las células presentadoras de antígeno y su papel en el síndrome reproductivo y respiratorio porcino

Las células presentadoras de antígeno son aquellas células encargadas de capturar, procesar y presentar antígenos con la finalidad de lograr una respuesta inmune efectiva por parte del organismo. Su papel, como centinelas, es crucial durante el transcurso de diversas enfermedades infecciosas. El estudio de estas células tras la infección con el virus del Síndrome Reproductivo y Respiratorio Porcino nos da información para abordar nuevas estrategias de control frente a esta enfermedad.Antigen presenting cells are able to capture, process and present antigens in order to develop an effective immune response. The role of these cells during infectious diseases is crucial to control the disease. Thus, the study of these cells after the infection with Porcine Reproductive and Respiratory Syndrome Virus gives us useful information on how to control this disease

Multi-Etiological Nature of Tuberculosis-Like Lesions in Condemned Pigs at the Slaughterhouse

Tuberculosis-like lesions (TBL) in pigs have been associated with microorganisms other than mycobacteria. In this work a histopathological and microbiological evaluation of TBL in pigs is shown. A total of 352 samples belonging to 171 pigs totally condemned at slaughterhouse due to generalized TBL were sampled and selected for analysis. Pyogranulomatous (56.2%) and granulomatous lesions (20.2%) were observed in all analysed organs. Most of the granulomas observed in both lymph nodes and lungs belonged to more advanced stages of development (stages III and IV) whereas in the liver and the spleen most of lesions belonged to intermediate stages (stages II and III). Different microorganisms were simultaneously detected from TBL in the 42.7% of the animals. Mycobacterium tuberculosis complex (MTC) (38%), coryneform bacteria (40.3%) and streptococci (28.1%) were the main groups of microorganisms detected after bacteriological analysis, with Trueperella pyogenes and Streptococcus suis as the most frequently isolated species. Mycobacteria belonging to MTC were the most frequently detected pathogens in granulomatous and pyogranulomatous lesions in submandibular lymph nodes (32.7%) and coryneform bacteria were the microorganisms more frequently isolated from lungs (25.9%) and spleen samples (37.2%). These results may provide new insights into the pathogenesis and diagnosis of this pathology. The importance of coryneform bacteria and streptococci in such processes must be evaluated in future studie

Multi-Etiological Nature of Tuberculosis-Like Lesions in Condemned Pigs at the Slaughterhouse.

Tuberculosis-like lesions (TBL) in pigs have been associated with microorganisms other than mycobacteria. In this work a histopathological and microbiological evaluation of TBL in pigs is shown. A total of 352 samples belonging to 171 pigs totally condemned at slaughterhouse due to generalized TBL were sampled and selected for analysis. Pyogranulomatous (56.2%) and granulomatous lesions (20.2%) were observed in all analysed organs. Most of the granulomas observed in both lymph nodes and lungs belonged to more advanced stages of development (stages III and IV) whereas in the liver and the spleen most of lesions belonged to intermediate stages (stages II and III). Different microorganisms were simultaneously detected from TBL in the 42.7% of the animals. Mycobacterium tuberculosis complex (MTC) (38%), coryneform bacteria (40.3%) and streptococci (28.1%) were the main groups of microorganisms detected after bacteriological analysis, with Trueperella pyogenes and Streptococcus suis as the most frequently isolated species. Mycobacteria belonging to MTC were the most frequently detected pathogens in granulomatous and pyogranulomatous lesions in submandibular lymph nodes (32.7%) and coryneform bacteria were the microorganisms more frequently isolated from lungs (25.9%) and spleen samples (37.2%). These results may provide new insights into the pathogenesis and diagnosis of this pathology. The importance of coryneform bacteria and streptococci in such processes must be evaluated in future studies

Enzymatic and Molecular Identification of Meloidogyne Species in Tomato Orchards in Paraguay

Tomato is a major crop in Paraguay, where it provides a source of employment and income for households. Tomato production can be affected by root-knot nematodes, especially Meloidogyne spp. The unequivocal identification of Meloidogyne spp. in Paraguay has not been conducted yet. This study aims to identify Meloidogyne species in eight tomato production districts of this country by biochemical and molecular techniques. Females of Meloidogyne spp. were extracted from tomato roots and characterized using esterase isozyme phenotypes. In addition, DNA was extracted from nematode eggs, and species-specific SCARs (sequence-characterized amplified regions) were used to confirm the diagnosis. Nematodes were detected in 100% of studied samples (prevalence), of which M. incognita (Est: I2, Rm: 1.1;1.2) and M. javanica (Est: J3, Rm: 1.0, 1.20, 1.35) were present in 39.13% and 26.08% of samples, respectively. One population (8.69%) of Meloidogyne sp. presenting an atypical esterase profile (Rm: 1.0 and 1.3) was only detected in Julián Augusto Saldívar District. Mixed populations, mostly M. incognita and M. javanica, were observed in 26.08% of samples. The SCAR primers incK14F/incK14R amplified specific fragments for M. incognita (399 bp) and M. javanica (670 bp), confirming the enzymatic results. Here, we present the first study of root-knot nematode identification at the species level in Paraguay

Evaluation of rapid methods for diagnosis of tuberculosis in slaughtered free-range pigs

Free-range pigs can be infected by Mycobacterium tuberculosis complex (MTC) and may contribute to the spread of bovine tuberculosis (bTB). In the present study, the diagnostic values of bacteriological culture, a duplex real-time quantitative PCR and an antibody ELISA were evaluated in an abattoir study of submandibular lymph nodes and serum samples from 73 pigs with and without lesions consistent with bTB. The duplex qPCR was an accurate method for diagnosis of TB in pigs (specificity 100%; sensitivity 80%). Combining qPCR with histopathology improved sensitivity and had very good concordance (κ = 0.94) with the reference method. Serological results suggest that the antibody ELISA can be used for monitoring herds but not individuals

Distribution of granulomas per examined organ and stage of development.

<p>^aSLN: submandibular lymph nodes.</p><p>Distribution of granulomas per examined organ and stage of development.</p

Microorganisms isolated from lesions.

<p>^<b>a</b><i>Rhodococcus boritolerans</i>, <i>Dietzia timorensis</i>, <i>Pseudoclavibacter</i> spp, <i>Brevibacterium</i> spp and <i>Actinomyces masicol</i> (1 isolate/each)</p><p>^b<i>Streptococcus</i> spp. (3 isolates), <i>S</i>. <i>mitis</i>, <i>S</i>. <i>rattus</i> and <i>S</i>. <i>bovis</i> (1 isolate/each)</p><p>^c<i>E</i>. <i>faecium</i> (8 isolates) <i>E</i>. <i>durans</i> (3 isolates), <i>E</i>. <i>faecalis</i> (6 isolates), <i>E</i>. <i>gallinarum</i> and <i>E</i>. <i>avium</i> (1 isolate/each)</p><p>^d<i>C</i>. <i>maltaromaticum</i> (16 isolates) and <i>C</i>. <i>divergens</i> (1 isolate)</p><p>^e<i>A</i>. <i>urinae</i> (7 isolates), <i>A</i>. <i>viridans</i> (4 isolates) and <i>A nurinaequi</i> (2 isolates)</p><p>^f<i>S</i>. <i>sciuri</i>, <i>S</i>. <i>xylosus</i> (2 isolates/each), <i>Staphylococcus</i> spp., <i>S</i>. <i>aureus</i> and <i>S</i>. <i>haemolyticus</i> (1 isolate/each)</p><p>^<b>g</b><i>Leuconostoc</i> spp. (4 isolates), <i>Escherichia coli</i> (2 isolates) <i>Mezorhizobium</i> spp., <i>Halospirulina</i> spp., <i>Glanulicatella</i> spp. and <i>Lactococcus lactis</i> (1 isolate/each)</p><p>Microorganisms isolated from lesions.</p

A-G.

<p><b>A)</b> TBL in the submandibular lymph node of an affected pig. Bar, 1cm. B) TBL in the spleen of an affected pig. Bar, 1cm. C) Microscopic image of a TBL lesions in the lymph node of an affected animal showing a profuse infiltrate of degenerated neutrophils. HE. Bar, 200μm. D) Clustered epithelioid macrophages surrounded by lymphocytes and erythrocytes in a stage I granuloma in the liver. HE. Bar, 50μm. E) Coalescent stage II granulomas in the lymph node of a pig showing epithelioid macrophages completely enclosed by a thin capsule, with peripheral infiltration of scattered lymphocytes. HE. Bar, 100μm. F) Stage III granuloma with a central necrotic core, partially mineralized, surrounded by a dense connective tissue capsule infiltrated by lymphocytes and scattered neutrophils. HE. Bar, 100μm. G) Thickly encapsulated, large, irregular, multicentric granulomas with prominent caseous necrosis and multifocal islands of mineralization (stage IV granulomas). HE. Bar, 500μm.</p