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Tuberculosis in animals is caused by members of the Mycobacterium tuberculosis complex (MTC), with the tuberculous granuloma being the main characteristic lesion. The macrophage is the main cell type involved in the development of the granuloma and presents a wide plasticity ranging from polarization to classically activated or pro-inflammatory macrophages (M1) or to alternatively activated or anti-inflammatory macrophages (M2). Thus, this study aimed to analyze macrophage polarization in granulomas from cattle and pig lymph nodes naturally infected with MTC. Tuberculous granulomas were microscopically categorized into four stages and a panel of myeloid cells (CD172a/calprotectin), M1 macrophage polarization (iNOS/CD68/CD107a), and M2 macrophage polarization (Arg1/CD163) markers were analyzed by immunohistochemistry. CD172a and calprotectin followed the same kinetics, having greater expression in late-stage granulomas in pigs. iNOS and CD68 had higher expression in cattle compared with pigs, and the expression was higher in early-stage granulomas. CD107a immunolabeling was only observed in porcine granulomas, with a higher expression in stage I granulomas. Arg1+ cells were significantly higher in pigs than in cattle, particularly in late-stage granulomas. Quantitative analysis of CD163+ cells showed similar kinetics in both species with a consistent frequency of immunolabeled cells throughout the different stages of the granuloma. Our results indicate that M1 macrophage polarization prevails in cattle during early-stage granulomas (stages I and II), whereas M2 phenotype is observed in later stages. Contrary, and mainly due to the expression of Arg1, M2 macrophage polarization is predominant in pigs in all granuloma stages.
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BACKGROUND: Vitamin D may improve innate antimicrobial response and the integrity of the intestinal mucosal barrier representing an alternative to antibiotics for improving pig health. Therefore, benefits of dietary supplementation with a product based on vitamin D3 metabolite-rich plant extracts were assessed in 252 purebred Iberian piglets for a period of 60 days. The study group received 1,25 dihydroxyvitamin D (1,25(OH)2D) (100 ppm) in the conventional feed, which already included vitamin D (2000 IU in the starter and 1000 IU in the adaptation diets, respectively). Average daily gain (ADG), feed conversion ratio (FCR) and coefficient of variation of body weight (CV-BW) were assessed along the study. Blood samples, from 18 animals of the study group and 14 animals of the control group, were collected at selected time points to determine white blood cell count, concentration of vitamin D3 and its metabolites, and IgA and IgG in serum. Histopathology, morphometry, and immunohistochemistry (IgA and FoxP3) from small intestine samples were performed on days 30 and 60 of the study from 3 animals per group and time point. RESULTS: The ADG (493 vs 444 g/day) and FCR (2.3 vs 3.02) showed an improved performance in the supplemented animals. Moreover, the lower CV-BW indicated a greater homogeneity in the treated batches (13.17 vs 26.23%). Furthermore, a mild increase of IgA and in the number of regulatory T cells in the small intestine were observed in treated pigs. CONCLUSIONS: These results highlight the benefits of this supplementation and encourage to develop further studies along other production stages.
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The diagnosis of bovine tuberculosis (bTB) is based on the single intradermal tuberculin test (SIT), interferon gamma, and compulsory slaughter of reactor animals. Culture and PCR from fresh tissue are regarded as gold standard techniques for post-mortem confirmation, with the former being time-consuming and presenting moderate to low sensitivity and the latter presenting promising results. Histopathology has the advantage to identify and categorize lesions in both reactor and non-reactor animals. Therefore, this study aims to highlight the role of histopathology in the systematic diagnosis of bTB to shorten the time to disclose positive animals. Blood (212) and lymph node (681) samples were collected for serological, bacteriological, and histopathological analyses from a total of 230 cattle subjected to the Spanish bTB eradication program. Seventy-one lymph nodes and 59 cattle yielded a positive result to bacteriology, with 59 lymph nodes and 48 cattle presenting a positive result in real-time PCR from fresh tissue. Roughly 19% (40/212) of sera samples gave a positive result to ELISA. Tuberculosis-like lesions (TBLs) were observed in 11.9% (81/681) of the lymph nodes and 30.9% (71/230) of cattle. Noteworthy, TBLs were evidenced in 18 out of 83 SIT- and real-time PCR and bacteriology negative animals, with 11/18 disclosing a positive result to Ziehl-Neelsen technique and two of them to ddPCR from paraffin blocks targeting IS6110. Six out of these 11 ZN+ corresponded with mesenteric LN and were confirmed positive to paratuberculosis. Histopathology yielded a sensitivity of 91.3% (CI95 83.2-99.4%) and a specificity of 84.4% (CI95 78.6-89.3%) with good agreement (κ = 0.626) when compared with real-time PCR. Our results confirm that histopathology allows a rapid confirmation of real-time PCR and bacteriology, emphasizing its contribution to bTB control and monitoring.
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Virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains, such as the Lena strain, have demonstrated a higher thymus tropism than low virulent strains. Virulent PRRSV strains lead to severe thymus atrophy, which could be related to marked immune dysregulation. Impairment of T-cell functions through immune checkpoints has been postulated as a strategy executed by PRRSV to subvert the immune response, however, its role in the thymus, a primary lymphoid organ, has not been studied yet. Therefore, the goal of this study was to evaluate the expression of selected immune checkpoints (PD1/PDL1, CTLA4, TIM3, LAG3, CD200R1 and IDO1) in the thymus of piglets infected with two different PRRSV-1 strains. Thymus samples from piglets infected with the low virulent 3249 strain, the virulent Lena strain and mock-infected were collected at 1, 3, 6, 8 and 13 days post-infection (dpi) to analyze PRRSV viral load, relative quantification and immunohistochemical staining of immune checkpoints. PD1/PDL1, CTLA4, TIM3, LAG3 and IDO1 immune checkpoints were significantly up-regulated in the thymus of PRRSV infected piglets, especially in those infected with the virulent Lena strain from 6 dpi onwards. This up-regulation was associated with disease progression, high viral load and cell death. Co-expression of these molecules can affect T-cell development, maturation and selection, negatively regulating the host immune response against PRRSV.
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Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Timo/inmunología , Timo/virología , Animales , Síndrome Respiratorio y de la Reproducción Porcina/virología , Porcinos , Regulación hacia Arriba , VirulenciaRESUMEN
Melanosis coli is a well-described condition in humans, characterized by the accumulation of lipofuscin-laden macrophages in the lamina propria of the colon, giving it a dark tone. An increased apoptosis rate of colonic epithelial cells appears to be the underlying pathogenesis. In pigs, oxidative damage has been proposed as one of the causes for melanosis coli. In this article, we report a series of cases of melanosis coli in pigs affecting several finishing units in the south of Spain. Large intestines had dark green to brown pigmentation of the mucosa. Histological, histochemical, and ultrastructural studies confirmed a high number of lipofuscin-laden macrophages in the lamina propria of the rectum and colon, which additionally stained positive for the apoptosis marker cleaved caspase-3. Of note, all affected finishing units utilized water supply with a high content of sulfates, which may be one of the causes for melanosis coli development in pigs.
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Enfermedades del Colon , Agua Potable , Melanosis , Enfermedades de los Porcinos , Animales , Enfermedades del Colon/veterinaria , Melanosis/veterinaria , Sulfatos , PorcinosRESUMEN
Tuberculosis like lesions (TBL) in free-range pigs are characterised by presenting a marked heterogeneity in pathology and microbiology features, with a notorious role of Mycobacterium tuberculosis complex (MTC), Trueperella pyogenes and different Streptococcus species. However, the capacity of these microorganism to spread to different organic cavities leading to a generalised disease is unknown. Therefore, this study evaluated the organic distribution of these agents in free-range pig carcasses whole condemned due to generalised TBL.A total of 37 totally condemned animals were analysed, and samples of lymph nodes and organs were obtained (n = 262) and subjected to histopathological and microbiological examination. In addition, T. pyogenes and streptococci species were further characterised by PFGE analysis. Two different patterns were evidenced with lack or occasional lesions in superficial inguinal (SILN) and popliteal (PLN) lymph nodes and advanced lesions in submandibular (SLN) (35/36) and gastrohepatic (GHLN) (33/35) lymph nodes (stages III and IV). Early stage granulomas (stage I and II) prevailed in lungs (16/20), liver (14/31) and spleen (7/18). The microbiological analysis revealed that MTC, detected by qPCR, was present in 31 out of 37 animals and 90 (90/262) samples. In 26 out of the 31 pigs, MTC was detected from two or more organs. SLN (24/31) and GHLN (19/31) were the MTC+ organs most frequently detected, with 29 out of 31 MTC+ pigs detected as positive in one or both samples, which points out that both lymph nodes must be included in the sampling of surveillance programs. Other pathogens, such as T. pyogenes and Streptococcus spp., were also involved in generalised lymphadenitis, being frequently isolated from SLN and other organs, such as liver (T. pyogenes), tonsils or lung (Streptococcus spp.). A wide genetic diversity among streptococci was observed, showing the ubiquitous character of these pathogens, however, the isolation of a single clone of T. pyogenes from different organic locations from animals with generalised TBL was a common finding of this study, highlighting that the role of this pathogen in porcine lymphadenitis may be underestimated. These results should be considered in future studies on the pathogenesis and control of porcine lymphadenitis.
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Highly virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains have increasingly overwhelmed Asia and Europe in recent years. This study aims to compare the clinical signs, gross and microscopic findings as well as the expression of CD163 within live pulmonary alveolar macrophages (PAMs) from bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with two PRRSV strains of different virulence. Pigs were infected with either a subtype 1 PRRSV-1 3249 strain or a subtype 3 PRRSV-1 Lena strain and consecutively euthanized at 1, 3, 6, 8 and 13 days post-inoculation. Clinical signs were reported daily and BALF and lung tissue samples were collected at the different time-points and accordingly processed for their analysis. Pigs infected with Lena strain exhibited greater clinical signs as well as gross and microscopic lung scores compared to 3249-infected pigs. A decreased frequency of PAMs from BALF was observed early in pigs infected with Lena strain. Moreover, the frequency and median fluorescence intensity (MFI) of CD163 within PAMs were much lower in Lena-infected pigs than in 3249-infected pigs. This downregulation in CD163 was also observed in lung sections after the assessment of macrophages expressing CD163 by means of immunohistochemistry. This outcome may result from the effect of PRRSV replication, PRRSV-induced inflammation, the influx of immature macrophages to restore lung homeostasis and/or the evidence of CD163low cells after CD163+ cells decrease in BALF.
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Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Bronconeumonía/veterinaria , Macrófagos Alveolares/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Receptores de Superficie Celular/genética , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Líquido del Lavado Bronquioalveolar/citología , Bronconeumonía/virología , Regulación hacia Abajo , Femenino , Pulmón/citología , Pulmón/virología , Macrófagos Alveolares/virología , Masculino , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Receptores de Superficie Celular/inmunología , Porcinos , VirulenciaRESUMEN
The emergence of virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV), causing atypical and severe outbreaks, has been notified worldwide. This study assesses the expression, distribution and kinetics of PRRSV N-protein, CD163 and CD107a in the lung and tonsil from experimentally-infected piglets with three different PRRSV-1 strains: a virulent PRRSV-1 subtype 3 strain (SU1-bel) and two low-virulent subtype 1 strains, Lelystad virus (LV) and 215-06. SU1-bel replicated more efficiently in the lungs and tonsils. The number of CD163+ cells decreased in both tissues from all infected groups at 7 dpi, followed by an increase at the end of the study, highlighting a negative correlation with the number of N-protein+-infected cells. A significant increase in CD107a was observed in all infected groups at 35 dpi but no differences were observed among them. Whereas the initial decrease of CD163+ cells appears to be associated to virus replication and cell death, the later recovery of the CD163+ population may be due to either the induction of CD163 in immature cells, the recruitment of CD163+ cells in the area of infection, or both. These results highlight the ability of macrophage subpopulations in infected animals to recover and restore their potential biological functions at one-month post-infection, with the greatest improvement observed in SU1-bel-infected animals.
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Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Regulación de la Expresión Génica/inmunología , Pulmón/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Tonsila Palatina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Receptores de Superficie Celular/genética , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/inmunología , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Receptores de Superficie Celular/inmunología , Porcinos , Virulencia/inmunologíaRESUMEN
The emergence of "highly pathogenic" isolates of porcine reproductive and respiratory syndrome virus (HP-PRRSV) has raised new concerns about PRRS control. Cells from the porcine monocyte-macrophage lineage represent the target for this virus, which replicates mainly in the lung, and especially in HP-PRRSV strains, also in lymphoid organs, such as the thymus. This study aimed at evaluating the impact of two PRRSV strains of different virulence on thymic macrophages as well as after heterologous vaccination. After experimental infection with PR11 and PR40 PRRSV1 subtype 1 strains (low and high virulent, respectively) samples from thymus were analysed by histopathology and immunohistochemistry for PRRSV N protein, TUNEL, CD172a, CD163, CD107a and BA4D5 expression. Mortality was similar in both infected groups, but lung lesions and thymus atrophy were more intense in PR40 group. Animals died at 10-14 dpi after PR11 or PR40 infection showed the most severe histopathological lesions, with a strong inflammatory response of the stroma and extensive cell death phenomena in the cortex. These animals presented an increase in the number of N protein, CD172a, CD163 and BA4D5 positive cells in the stroma and the cortex together with a decrease in the number of CD107a positive cells. Our results highlight the recruitment of macrophages in the thymus, the increase in the expression of CD163 and the regulation of the host cytotoxic activity by macrophages. However, no marked differences were observed between PR11- and PR40-infected animals. Heterologous vaccination restrained virus spread and lesions extent in the thymus of PR40-infected animals.
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Macrófagos/virología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Timo/virología , Animales , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Pulmón/patología , Macrófagos/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Receptores de Superficie Celular/genética , Porcinos , Timo/inmunología , Carga Viral , Vacunas Virales/uso terapéutico , VirulenciaRESUMEN
Unlike mice and humans, porcine γδ T cells represent a prominent subset of T cells in blood and secondary lymphatic organs. GATA-3, T-bet and Eomesodermin (Eomes) are transcription factors with crucial functions in T-cell development and functional differentiation, but their expression has not been investigated in porcine γδ T cells so far. We analyzed the expression of these transcription factors in γδ thymocytes, mature γδ T cells from blood, spleen, lymph nodes, and lung tissue as well as in vitro stimulated γδ T cells on the protein level by flow cytometry. GATA-3 was present in more than 80% of all γδ-thymocytes. Extra-thymic CD2- γδ T cells expressed high levels of GATA-3 in all investigated organs and had a CD8α-/dimCD27+perforin- phenotype. T-bet expression was mainly found in a subset of CD2+ γδ T cells with an opposing CD8αhighCD27dim/-perforin+ phenotype. Eomes+ γδ T cells were also found within CD2+ γδ T cells but were heterogeneous in regard to expression of CD8α, CD27, and perforin. Eomes+ γδ T cells frequently co-expressed T-bet and dominated in the spleen. During aging, CD2-GATA-3+ γδ T cells strongly prevailed in young pigs up to an age of about 2 years but declined in older animals where CD2+T-bet+ γδ T cells became more prominent. Despite high GATA-3 expression levels, IL-4 production could not be found in γδ T cells by intracellular cytokine staining. Experiments with sorted and ConA + IL-2 + IL-12 + IL-18-stimulated CD2- γδ T cells showed that proliferating cells start expressing CD2 and T-bet, produce IFN-γ, but retain GATA-3 expression. In summary, our data suggest a role for GATA-3 in the development of γδ-thymocytes and in the function of peripheral CD2-CD8α-/dimCD27+perforin- γδ T cells. In contrast, T-bet expression appears to be restricted to terminal differentiation stages of CD2+ γδ T cells, frequently coinciding with perforin expression. The functional relevance of high GATA-3 expression levels in extra-thymic CD2- γδ T cells awaits further clarification. However, their unique phenotype suggests that they represent a thymus-derived separate lineage of γδ T cells in the pig for which currently no direct counterpart in rodents or humans has been described.
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Factor de Transcripción GATA3/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Porcinos/inmunología , Proteínas de Dominio T Box/biosíntesis , Subgrupos de Linfocitos T/inmunología , Animales , Factor de Transcripción GATA3/inmunología , Fenotipo , Proteínas de Dominio T Box/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Porcine reproductive and respiratory syndrome viruses (PRRSV) present a wide phenotypic and genetic diversity. Experimental infections have demonstrated viral replication, including highly pathogenic strains (HP-PRRSV), in primary lymphoid organs such as the thymus. However, studies of the bone marrow are scarce but necessary to help elucidate the immunobiology of PRRSV strains of differing virulence. In this study, whereas viral RNA was detected within the bone marrow of animals experimentally infected with both low virulent Lelystad (LV) and 215-06 PRRSV-1 strains and with the highly virulent SU1-bel strain, PRRSV positive cells were only occasionally detected in one SU1-bel infected animal. PRRSV RNA levels were associated to circulating virus with the highest levels detected in LV-infected pigs. At 3 dpi, a decrease in the proportion of haematopoietic tissue and number of erythroid cells in all infected groups was associated with an increase in TUNEL or cleaved caspase 3 labelling and higher counts of myeloid cells compared to control. The expression of IL-1α and IL-6 was elevated at the beginning of the infection in all infected animals. The expression of TNF-α was increased at the end of the study in all infected groups with respect to control. Different PRRSV-1 strains induced, presummably by indirect mechanisms and independently of viral load and strain virulence, moderate and sustained hypoplasia of erythroid cells and myeloid cell hyperplasia at early stages of infection. These changes were paralleled by a peak in the local expression of IL-1α, IL-6 and TNF-α in all infected groups.
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Síndrome Respiratorio y de la Reproducción Porcina/patología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Animales , Médula Ósea/patología , Médula Ósea/virología , Citocinas/inmunología , Células Eritroides/patología , Células Eritroides/virología , Hiperplasia/patología , Hiperplasia/veterinaria , Hiperplasia/virología , Masculino , Células Mieloides/patología , Células Mieloides/virología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Porcinos , Carga Viral , Virulencia , Replicación ViralRESUMEN
UNLABELLED: Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67(+)) CD8(+) T cells with an early effector phenotype (perforin(+) CD27(+)) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4(+) and CD8(+) T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4(+) and CD8(+) T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4(+) and CD8(+) memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs. IMPORTANCE: Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Reacciones Cruzadas/inmunología , Subtipo H1N2 del Virus de la Influenza A/inmunología , Pulmón/inmunología , Infecciones por Orthomyxoviridae/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Linfocitos T CD4-Positivos/virología , Vacunas contra la Influenza/inmunología , Interferón gamma/inmunología , Interleucina-2/inmunología , Pulmón/virología , Porcinos , Enfermedades de los Porcinos/virología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two distinct viruses, type 1 (PRRSV-1) and type 2 (PRRSV-2). Atrophy of the thymus in PRRSV-2 infected piglets has been associated with a loss of thymocytes. The present study aimed to evaluate the impact of PRRSV-1 strains of differing virulence on the thymus of infected piglets by analysing the histomorphometry, the presence of apoptotic cells and cells producing cytokines. Thymic samples were taken from animals experimentally infected (with LV, SU1-bel, and 215-06 strains) or mock inoculated animals at 3, 7 and 35days post-infection (dpi) and processed for histopathological and immunohistochemical analyses. PRRSV antigen was detected in the thymus from 3dpi until the end of the study in all virus-infected animals with the highest numbers of infected cells detected in SU1-bel group. The histomorphometry analysis and counts of CD3(+) thymocytes in the thymic cortex displayed significant differences between strains at different time-points (p≤0.011), with SU1-bel group showing the most severe changes at 7dpi. Cell death displayed statistically significant increase in the cortex of all infected animals, with SU1-bel group showing the highest rate at 3 and 7dpi. The number of cells immunostained against IL-1α, TNF-α and IL-10 were predominantly detected in the medulla (p≤0.01). An increase in the number of TNF-α and IL-10 positive cells was observed in LV and SU-1bel groups. Our results demonstrate that different PRRSV-1 strains induced depletion of the thymic cortex due to apoptosis of thymocytes and that the most severe depletion was associated with the highly virulent SU1-bel strain.
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Atrofia/virología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Timocitos/virología , Timo/virología , Animales , Antígenos Virales/metabolismo , Atrofia/fisiopatología , Muerte Celular , Citocinas/metabolismo , Linfocitos/citología , Linfocitos/virología , Síndrome Respiratorio y de la Reproducción Porcina/fisiopatología , Especificidad de la Especie , Porcinos , Timocitos/citología , Timo/patologíaRESUMEN
The transcription factors GATA-3, T-bet and Eomesodermin play important roles in T-cell development, differentiation and memory formation. However, their expression has not been studied in great detail in porcine T cells. We report on protein expression at the single cell-level of these transcription factors in thymocytes and mature αß T cells. GATA-3 expression was found in γδ(-) thymocytes, with decreasing expression from the CD4(-)CD8α(-) stage towards single-positive stages. Extra-thymic CD4(+) T cells but not CD8ß(+) T cells expressed low levels of GATA-3, which decreased with age. CD4(+) and CD8ß(+) T-bet(+) cells mainly displayed a CD8α(+)CD27(-) and perforin(+)CD27(dim/-) phenotype, respectively and had the capacity for IFN-γ production; indicative of an effector/effector memory phenotype. Eomesodermin(+) αß T cells had mixed phenotypes in regard to CD8α, CD27 and perforin expression. In conclusion, our data so far support the hitherto reported roles for GATA-3 in T-cell development and T-bet for Th1 effector-differentiation, but question the role of Eomesodermin for memory formation of porcine T-cells.
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Factor de Transcripción GATA3/metabolismo , Proteínas de Dominio T Box/metabolismo , Linfocitos T/metabolismo , Animales , Células Cultivadas , Femenino , Factor de Transcripción GATA3/genética , Expresión Génica , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Sus scrofa , Proteínas de Dominio T Box/genéticaRESUMEN
In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)(+) and N-protein(-) moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein(+) moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein(-) moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3(+) regulatory T cells present within CD4(+) T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4(+) T cells showed differences in regard to proliferation and frequency of Foxp3(+) T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.
Asunto(s)
Factores de Transcripción Forkhead/genética , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Animales , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Monocitos/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Porcinos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Porcine reproductive and respiratory syndrome viruses (PRRSV) show high genetic differences both among and within genotypes. Recently, several highly pathogenic PRRSV (HP-PRRSV) strains have been described. This study compares and characterizes the production of cytokines by pulmonary macrophages in pigs experimentally infected with four different PRRSV-1 strains: two low-virulent strains, Lelystad (LV) and a British field strain (215-06); a HP strain (SU1-bel) from Belarus and the attenuated vaccine strain DV (Porcilis(®) PRRS). Animals were clinically monitored and post-mortem examinations were performed at 3, 7 and 35 days post-infection (dpi). Lung samples were processed for histopathological and immunohistochemical studies by using specific antibodies against PRRSV, IL1-α, IL-6, TNF-α, IL-10 and IFN-γ. SU1-bel infected animals presented the highest mean scores for clinical observations, gross and microscopic lesions as well as for PRRSV expression compared with the other infected groups (p≤0.027). These animals displayed the highest expression of IL1-α at 7dpi, together with the highest score for lung pathology, whereas LV, 215-06 and DV inoculated animals only showed a transient enhancement in some of these cytokines. SU1-bel-infected pigs showed a positive correlation between the amount of PRRSV antigen and IL-1α expression (r=0.645, p<0.001). The highest expression of IL-10 was detected in 215-06-infected animals (p≤0.004), with a positive correlation with the numbers of virus-infected cells (r=0.375, p≤0.013). In conclusion, the HP-PRRSV SU1-bel strain replicated more efficiently in the lung of infected animals and induced a higher expression of IL-1α than the other PRRSV-1-infected groups, which may have played a key role in the onset of the clinical signs and interstitial pneumonia.
Asunto(s)
Citocinas/biosíntesis , Interleucina-1alfa/biosíntesis , Pulmón/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Animales , Antígenos Virales/sangre , Pulmón/patología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos , Regulación hacia ArribaRESUMEN
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a major infectious pathogen in pigs leading to huge economical losses worldwide. PRRSV is able to escape from host immunity and causes transient infections. In the present study, expression of different apoptotic markers and its connection with PRRSV were assessed in tonsil and mediastinal lymph node from PRRSV-infected pigs. Cleaved caspase (CCasp)8, CCasp9, Fas, Daxx, CCasp3 and PRRSV expression were analyzed by immunohistochemistry. An up-regulation of CCasp8, Fas and CCasp3 expression in lymphocytes and macrophages from both organs was found during PRRSV infection, indicating the activation of the extrinsic-mediated pathway of apoptosis. Moreover, Daxx expression was also enhanced in macrophages of both organs, suggesting a simultaneous caspase-independent pathway of apoptosis. A correlation between the expression of the different apoptotic markers and IL-10, IL-6 and TGF-ß but not with PRRSV antigen was found in our study, which supports the hypothesis of an indirect mechanism in PRRSV-induced apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Caspasas/genética , Caspasas/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/virología , Linfocitos/metabolismo , Linfocitos/virología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Nucleares/genética , Tonsila Palatina/metabolismo , Tonsila Palatina/virología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Transducción de Señal , Porcinos , Factor de Crecimiento Transformador beta/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine industry. The causal agent, PRRS-virus (PRRSV), is able to evade the host immune response and survive in the organism causing transient infections. Despite all scientific efforts, there are still some gaps in the knowledge of the pathogenesis of this disease. Antigen presenting cells (APCs), as initiators of the immune response, are located in the first line of defense against microorganisms, and are responsible for antigen recognition, processing and presentation. Dendritic cells (DCs) are the main type of APC involved in antigen presentation and they are susceptible to PRRSV infection. Thus, PRRSV replication in DCs may trigger off different mechanisms to impair the onset of a host effective immune response against the virus. On the one side, PRRSV may impair the basic functions of DCs by regulating the expression of major histocompatibility complex class II and CD80/86. Other strategy followed by the virus is the induction of cell death of APCs by apoptosis, necrosis or both of them. The impairment and/or cell death of APCs could lead to a failure in the onset of an efficient immune response, as long as cells could not properly activate T cells. Future aspects to take into account are also discussed in this review.
RESUMEN
This work describes peptides from non-structural proteins (nsp) of porcine reproductive and respiratory syndrome virus (PRRSV) predicted as potential T cell epitopes by bioinfornatics and tested for their ability to induce IFN-γ and IL-10 responses. Pigs immunized with either genotype 1 or genotype 2 PRRSV attenuated vaccines (n=5/group) and unvaccinated pigs (n = 4) were used to test the peptides. Swine leukocyte antigen haplotype of each pig was also determined. Pigs were initially screened for IFN-γ responses (ELISPOT) and three peptides were identified; two of them in non-conserved segments of nsp2 and nsp5 and the other in a conserved region of nsp5 peptide. Then, peptides were screened for IL-10 inducing properties. Six peptides were found to induce IL-10 release in PBMC and some of them were also able to inhibit IFN-γ responses on PHA-stimulated cells. Interestingly, the IFN-γ low responder pigs against PRRSV were mostly homozygous for their SLA haplotypes. In conclusion, these results indicate that nsp of PRRSV contain T-cell epitopes inducing IFN-γ responses as well as IL-10 inducing segments with inhibitory capabilities.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Péptidos/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/inmunología , Secuencia de Aminoácidos , Animales , Biología Computacional , Epítopos de Linfocito T/inmunología , Haplotipos , Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Péptidos/química , PorcinosRESUMEN
Transforming growth factor ß (TGFß) is an immunomodulatory cytokine which is able to modulate the host immune response eliciting an inefficient response against pathogens. In this sense, the role of this cytokine in porcine reproductive and respiratory syndrome (PRRS) has been poorly studied and the reported results are contradictory. Thus, in the present study, the expression of TGFß was analysed both at tissue (lymphoid organs and lung) and serum level to study its correlation with the expression of PRRS virus (PRRSV). To carry out this study, 32 pigs were inoculated with the European PRRSV field isolate 2982 and sequentially killed from 0 dpi to the end of the study (24 dpi). Blood and tissue samples were collected to determine the expression of PRRSV and TGFß. PRRSV was detected in inoculated animals from 3 dpi until the end of the study, however TGFß was not detected in sera from inoculated animals. Contrary, an increase of TGFß antigen was observed both in the lymphoid organs and in the lung of PRRSV-inoculated pigs when compared with control group. Since TGFß play a role as an immunomodulatory cytokine of the immune response and also in the differentiation of regulatory T cells (Tregs), the upregulation of the TGFß at tissue level may play a role in the impairment of the host immune response observed during PRRS, being observed a significant correlation between PRRSV and TGFß expression at lung level.