F J Pallarés

University of Murcia, Murcia, Murcia, Spain

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Publications (87)128.35 Total impact

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    ABSTRACT: Genetic susceptibility or resistance to diseases is currently drawing increasing attention. This work describes two different breeding herds showing signs of periweaning failure-to-thrive syndrome (PFTS), an emergent swine disease. The disease was diagnosed based on clinical picture and confirmed by histopathology. The possibility of main infectious pathogens was ruled out by immunohistochemistry and PCR. In a simple approach, sires of the affected piglets have been determined using microsatellite paternity analysis, including a healthy group in each case. In each of the two farms, a single boar was found to have sired 45-50 per cent sick animals. Removal of this sire from two farms resulted in a significant decrease in the prevalence of the disease among the offspring, in accordance with other two cases diagnosed, although without including a control group. Since the analysed animals belonged to three different genetic lines, these findings point to the existence of individual genetic susceptibility to this syndrome. British Veterinary Association.
    03/2015; DOI:10.1136/vr.102748
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    ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215–06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-α and IFN-α in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-γ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.
    Veterinary Research 03/2015; 46(1). DOI:10.1186/s13567-015-0161-8 · 3.38 Impact Factor
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    ABSTRACT: This work was undertaken to evaluate whether a real-time quantitative polymerase chain reaction (qPCR) is as an adequate method for detection and quantification of human-specific DNA elements (Alu gene) in tissues and blood samples of pigs in which human stem cells were engrafted. Real-time qPCR quantification was performed with the use of previously described primers. The human DNA was mixed with different quantities of porcine DNA. The primer concentration and specificity, the qPCR efficiency, the quantification variations due to different porcine DNA concentrations, and the dissociation curve produced by the assay were evaluated. The qPCR proved to be specific, robust, with a reproducible and specific bimodal melting curve. High porcine DNA concentration produced subquantification, especially with low human DNA quantity. However, the assay proved to be useful for the detection of chimeric piglets produced by human cells injected in utero, because the effect caused by the porcine DNA interference was corrected in quantification of human DNA from piglets. Copyright © 2015 Elsevier Inc. All rights reserved.
    Transplantation Proceedings 01/2015; 47(1):132-5. DOI:10.1016/j.transproceed.2014.11.016 · 0.95 Impact Factor
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    ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5-week-old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock-infected and used as controls. The animals were euthanized at 3, 7 and 35 days post-infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes–macrophages. BE-infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV-infected cells in the tissues.
    Transboundary and Emerging Diseases 11/2014; DOI:10.1111/tbed.12272 · 3.12 Impact Factor
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    ABSTRACT: The objective of this study was to determine the effect of crude glycerin (Gly) added to nursery pig diet on nutrient digestibility, the digestive and metabolic status, intestinal morphology and intestinal cytokine expression. A total of 18 male piglets (weaned at 23 days) were used. There were three dietary treatments that differed in the inclusion level of Gly (0, 9 and 18%). On day 14 of the experiment, the animals were weighed and plasma samples were collected before slaughtering. In addition urine, digesta content and intestinal tissues were sampled post mortem. No differences were observed among the tested diets as regards the coefficients of apparent ileal digestibility of DM and CP. The concentration of lactic acid decreased linearly (P<0.05) in the jejunum and ileum segments as the level of Gly increased, although the concentrations of volatile fatty acids in the cecum and colon were not affected. The plasma concentrations of glucose, fructosamine and IGF-1 were not affected. However, urinary glycerol concentrations increased (P<0.01) with increasing levels of Gly. In general, there were no differences in villus height, crypt depth, villus/crypt ratio or number of lymphocytes in the intestinal segments between the different treatments. Nevertheless, the control treatment produced a higher level of goblet cells in the ileum than either of the Gly treatments (P<0.01), while in the jejunum, the number of IgA-secreting cells in the 9% Gly group was higher (P<0.01) than in the control group. There were no differences among the experimental treatments concerning the gene expression of IL-10, IL-12 p40 and TNF-α. Gene expressions of TGF-β, IL-12 p35, IFN-γ and IFN-α were remained unaffected or increased, depending on the intestinal segment and level of Gly addition. In conclusion, the inclusion of Gly at 9 and 18% to the nursery pig diet did not affect nutrient digestibility or plasma metabolites but increased the levels of urinary glycerol, suggesting that metabolic pathways of glycerol utilization became saturated when high levels of Gly are used. In addition, the intestinal cell structure and intestinal cytokine expression might be affected when Gly is included in the feed.
    Livestock Science 09/2014; 167. DOI:10.1016/j.livsci.2014.05.013 · 1.10 Impact Factor
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    ABSTRACT: Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases of swine. PRRS virus (PRRSV) infection in the pig is characterized by a weak or absent host innate immune response. The underlying mechanisms of PRRSV pathogenesis are still unclear. The analysis of transcript levels represents an alternative to immunoassays for the detection of cytokines that sometimes are difficult to detect due to their low amounts. This study sets out to determine the differences in pathogenesis and the immune response between lung, tonsil, tracheobronchial lymph node (Tb-LN) and retropharyngeal LN (Rf-LN) of PRRSV 2982 strain infected pigs. PRRSV strain 2982 avoided the onset of an effective innate immune response, especially in PRRSV main target (lung) and reservoir (tonsil) organs. PRRSV lead to an impaired expression of IFN-α and TNF-α gene expression, which finally induced a weak and delayed adaptive immune response trough an inefficient IL-12 and IFN-γ expression. Finally, PRRSV replication favoured the expression of the anti-inflammatory IL-10 cytokine in infected pigs.
    Veterinary Immunology and Immunopathology 07/2014; 160(1-2). DOI:10.1016/j.vetimm.2014.03.008 · 1.75 Impact Factor
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    ABSTRACT: This study reports the performance of the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay for M. bovis in a cattle herd with high prevalence of paratuberculosis (PTB). A total of 58/350 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ, a breeding cow that seroconverted to PTB and showed signs compatible with a wasting disease. Infection status was determined by post mortem diagnostic tests that included histopathology examination and mycobacterial cultures coupled with PCR identification for M. bovis and M. avium subsp. paratuberculosis (MAP). In 7/58 animals primary tuberculosis (TB) lesions, affecting only the retropharyngeal and/or mediastinal lymph nodes, were found; 3/7 animals were found SIT positive. PTB was confirmed in 35/58 animals, of which 30 had seroconverted and 14 had typical clinical signs. 45/58 animals were IFN-γ+ using the most stringent criterion (cut-off point≥0.05); however, IFN-γ test was only positive in 33 animals when using a higher threshold (cut-off point≥0.1). Three animals co-infected also showed extensive TB and diffuse PTB lesions. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of IFN-γ+ animals with no visible TB lesion could be due, in part, to some protection conferred by prior infection with MAP
    Veterinary Microbiology 06/2014; 171(1-2). DOI:10.1016/j.vetmic.2014.03.035 · 2.73 Impact Factor
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    Journal of Comparative Pathology 01/2014; 150(1):81. DOI:10.1016/j.jcpa.2013.11.023 · 1.10 Impact Factor
  • Journal of Comparative Pathology 01/2014; 150(1). DOI:10.1016/j.jcpa.2013.11.102 · 1.10 Impact Factor
  • Journal of Comparative Pathology 01/2014; 150(1):100. DOI:10.1016/j.jcpa.2013.11.101 · 1.10 Impact Factor
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    ABSTRACT: The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-β activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-β. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-β activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-β-treated MΦ (p<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-β stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information not only important to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.
    Virus Research 11/2013; 179. DOI:10.1016/j.virusres.2013.08.009 · 2.83 Impact Factor
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    ABSTRACT: This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.
    Biomedical Microdevices 07/2013; 15(6). DOI:10.1007/s10544-013-9790-8 · 2.77 Impact Factor
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    ABSTRACT: A 12-year-old, male, fox terrier dog presented with an abnormal gait of the left pelvic limb. Computed tomography revealed a large, homogeneous, hypoattenuating, noncontrast enhancing mass within the left epaxial muscles that invaded the L5-6 vertebral canal and caused spinal cord compression. Imaging findings were consistent with an infiltrative lipoma. The mass was removed and a left hemilaminectomy was performed in the affected area. Histopathology confirmed the mass to be an infiltrative lipoma. The dog recovered and regained neurologic function within 2 weeks. Computed tomography assisted preoperative planning by characterizing the shape, size, and location of the mass.
    Veterinary Radiology &amp Ultrasound 04/2013; 54(4). DOI:10.1111/vru.12038 · 1.26 Impact Factor
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    ABSTRACT: Objective-To characterize the kinetics of interleukin (IL)-4, IL-5, and IL-13 secretion in peripheral blood and lymph node mononuclear cells isolated from porcine circovirus type 2 (PCV2)-vaccinated pigs after cells were challenged with PCV2 open reading frame 2 antigen. Animals-10 pigs. Procedures-5 pigs were vaccinated with a PCV2 vaccine and received a booster dose 3 weeks later. They were kept together with a similar group of 5 nonvaccinated pigs that served as controls. One week after the second vaccination, peripheral blood mononuclear cells (PBMCs) and excised retropharyngeal lymph node mononuclear cells (LNMCs) were isolated and cultured. Cells were then challenged by exposure to PCV2 open reading frame 2 and evaluated at 2, 12, 24, and 48 hours to determine the expression of IL-4, IL-5, and IL-13 via quantitative PCR assay. Changes in gene expression were analyzed relative to the results from analysis of the sample at 0 hours (calibrator). Results-All ILs were upregulated differently in LNMCs and PBMCs from vaccinated pigs. Lymph node mononuclear cells from vaccinated animals produced significantly more IL-4 mRNA than did PBMCs at 2, 12, and 48 hours (relative change: 2.8 vs -3.6, 13.0 vs 3.6, and 9.8 vs 1.8, respectively) and more IL-5 mRNA at 2, 12, 24, and 48 hours (relative change: 1. 2 vs -4.8, 2.2 vs 0.2, 3.2 vs -1.9, and 4.0 vs -3.6, respectively). Interleukin-13 mRNA reached its highest concentration at 24 hours but was 11.9-fold higher in PBMCs than in LNMCs. Conclusions and Clinical Relevance-Results supported the importance of IL-4, IL-5, and IL-13 in pigs, suggesting that PBMCs and LNMCs express cytokines in a tissue-specific manner.
    American Journal of Veterinary Research 01/2013; 74(1):110-4. DOI:10.2460/ajvr.74.1.110 · 1.21 Impact Factor
  • Journal of Comparative Pathology 01/2013; 148(1):76. DOI:10.1016/j.jcpa.2012.11.120 · 1.10 Impact Factor
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    ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages andinducing apoptosis of immune cells. An imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin-10, in PRRS may impair the immune response of the lung. Pulmonary macrophage subpopulations have a range of susceptibilities to different PRRSV strains and different capacities to express cytokines. Infection with PRRSV decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. PRRSV infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (CD163) and induce the synthesis of anti-inflammatory mediators. The balance between pro- and anti-inflammatory cytokines modulates the expression of CD163, which may affect the pathogenicity and replication of the virus in different tissues. With the emergence of highly pathogenic PRRSV, there is a need for more information on the immunopathogenesis of different strains of PRRS, particularly to develop more effective vaccines.
    The Veterinary Journal 12/2012; 195(2). DOI:10.1016/j.tvjl.2012.11.012 · 2.17 Impact Factor
  • 12/2012; 28. DOI:10.6018/j/188771
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    ABSTRACT: Metabolic and cardiovascular diseases (CVDs) have risen to alarming proportions, and there is a need for therapeutic and preventive measures. The polyphenol resveratrol (RES) protects against CVDs, but in vivo molecular mechanisms responsible for protection are not yet understood. Peripheral blood mononuclear cells (PBMNCs) are involved in the development of atherosclerosis and metabolic disorders. The identification of PBMNCs genes responding to dietary compounds might help to understand the mechanisms underlying the effects of polyphenols. We determined gene expression differences between PBMNCs from pigs fed a high-fat diet manifesting a mild increase of cholesterol and pigs fed a high-fat diet containing low doses of RES. Although the consumption of RES did not modify the levels of cholesterol, microarray analyses indicated that some of the differentially expressed genes, collagens (COL1A, COL3A), lipoprotein lipase (LPL) and fatty-acid binding proteins (FABPs) involved in CVDs and lipid metabolism were up-regulated by the high-fat diet and down-regulated by RES. Reverse transcriptase polymerase chain reaction confirmed that RES and RES-containing grape extract prevented the induction of FABP4 in PBMNCs in female pigs fed a high-fat diet. Low micromolar concentrations of RES and its metabolite dihydroresveratrol exerted a minor but significant reducing effect on the induction of FABP4 expression in human macrophages treated with oxidized low-density lipoprotein. Our results show that the consumption of low doses of RES modulates the expression of genes related to lipid metabolism and metabolic disorders that are affected by a high-fat diet and suggest that some of the circulating RES metabolites may contribute to these effects.
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    ABSTRACT: Despite the numerous studies carried out, the mechanisms used by porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) to impair the host immune response are not yet clear. The aim of this study was to determine the expression of IL-12, IL-10, IFN-α and IFN-γ in lymphoid organs of PRRSV experimentally-infected pigs. Twenty eight piglets were inoculated with PRRSV field isolate 2982 and killed in batches of four at 3, 7, 10, 14, 17, 21 and 24 days post-inoculation (dpi). Control animals were mock-inoculated and killed at the end of the study. Samples from mediastinal and retropharyngeal lymph nodes and tonsil were collected and fixed for histopathological and immunohistochemical analyses. PRRSV antigen was mainly detected in the cytoplasm of macrophages, displaying a bimodal expression with a first peak at 3-7dpi and a second peak at 14dpi. The expression of IFN-α showed an early enhancement at 3dpi, and both IL-12 and IFN-γ displayed a similar trend in all the lymphoid organs analysed, showing an increase at 3-7dpi and at 14-17dpi. On the other hand, the expression of IL-10 was lower than the one observed for the other cytokines. The expression of IL-10 compared with the higher expression of IL-12, IFN-α and IFN-γ detected in this study, indicates that other mechanisms besides the expression of IL-10 play a role in the inducement of an erratic host immune response. Taking into account the enhanced expression of IFNs together with the detection of PRRSV antigen until the end of the study in the examined lymphoid organs, further studies are being conducted to rule out a down-regulation in IFN signalling pathway.
    Veterinary Immunology and Immunopathology 07/2012; 149(3-4):262-71. DOI:10.1016/j.vetimm.2012.07.011 · 1.75 Impact Factor
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    ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) can persist in different organs of infected pigs, which suggests a failure in the immune response. Antigen-presenting cells (APCs) play a pivotal role in the induction of effective T- and B-cell responses. In this study, we investigated the changes in the different APC subpopulations and T- and B-cell counts in the tonsil, retropharyngeal and mediastinal lymph nodes of pigs experimentally infected with a European PRRSV field isolate. Our results demonstrated that the expression of S100, SWC3, HLA-DR molecule and CD3 was diminished in the studied organs throughout the study, observing a significant negative correlation between viral antigen and HLA-DR expression in both retropharyngeal and mediastinal lymph nodes. In contrast, λ-light chains showed an increase during the study. Taking all into account, after PRRSV infection, no enhancement in the number of APCs and T cells was observed, suggesting an impairment of the immune function which may allow the persistence of PRRSV into the organism.
    Transboundary and Emerging Diseases 07/2012; 60(5). DOI:10.1111/j.1865-1682.2012.01363.x · 3.12 Impact Factor

Publication Stats

792 Citations
128.35 Total Impact Points

Institutions

  • 1999–2015
    • University of Murcia
      • • Department of Anatomy and Compared Pathological Anatomy
      • • Department of Animal Medicine and Surgery
      • • Facultad de Veterinaria
      Murcia, Murcia, Spain
  • 2013
    • Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario
      Murcia, Murcia, Spain
  • 2002–2004
    • Iowa State University
      • • Department of Veterinary Diagnostic and Production Animal Medicine
      • • Department of Veterinary Microbiology and Preventive Medicine
      • • Veterinary Diagnostic Laboratory (VDL)
      Ames, IA, United States