Ivan Díaz - Academia.edu (original) (raw)

Papers by Ivan Díaz

Frontiers in Immunology, 2020

In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, st... more In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1 + CD14 − MHC-II + CD172a −/lo CD1 − CD163 − DEC205 + CD11R3 lo CD11R1 + CD33 + CD80/86 +. They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4 + CD8 + T cells, but were deficient in phagocyting inactivated Staphylococcus aureus (S. aureus). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1 + CD14 − MHC-II + C D172a + CD1 + CD163 −/lo DEC205 lo CD11R3 + CD11R1 + CD33 + CD80/86 + ; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4 + CD8 − /CD4 + CD8 + T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14 + population was identified with the phenotype CADM1 + CD14 + MHC-II + CD172a + CD1 + CD163 + DEC205 − CD11R3 + CD11R1 + CD33 −/lo CD80/86 +. They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14 + population was efficient in phagocyting S. aureus but showed less maturation upon TLR ligand stimulation than cDC1 or cDC2. The alternative methods of DC derivation including GM-CSF and/or IL-4 produced mostly CADM1 − cells that did

Transboundary and Emerging Diseases

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic offers a unique opportu... more The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic offers a unique opportunity to study the introduction and evolution of a pathogen into a completely naïve human population. We identified and analysed the amino acid mutations that gained prominence worldwide in the early months of the pandemic. Eight mutations have been identified along the viral genome, mostly located in conserved segments of the structural proteins and showing low variability among coronavirus, which indicated that they might have a functional impact. At the moment of writing this paper, these mutations present a varied success in the SARS-CoV-2 virus population; ranging from a change in the spike protein that becomes absolutely prevalent, two mutations in the nucleocapsid protein showing frequencies around 25%, to a mutation in the matrix protein that nearly fades out after reaching a frequency of 20%.

BMC Veterinary Research

Background Diarrhoea is a major cause of death in neonate pigs and most of the viruses that cause... more Background Diarrhoea is a major cause of death in neonate pigs and most of the viruses that cause it are RNA viruses. Next Generation Sequencing (NGS) deeply characterize the genetic diversity among rapidly mutating virus populations at the interspecific as well as the intraspecific level. The diversity of RNA viruses present in faeces of neonatal piglets suffering from diarrhoea in 47 farms, plus 4 samples from non-diarrhoeic piglets has been evaluated by NGS. Samples were selected among the cases submitted to the Veterinary Diagnostic Laboratories of Infectious Diseases of the Universitat Autònoma de Barcelona (Barcelona, Spain) and Universidad de León (León, Spain). Results The analyses identified the presence of 12 virus species corresponding to 8 genera of RNA viruses. Most samples were co-infected by several viruses. Kobuvirus and Rotavirus were more commonly reported, with Sapovirus, Astrovirus 3, 4 and 5, Enterovirus G, Porcine epidemic diarrhoea virus, Pasivirus and Posavir...

Veterinary Record

BackgroundSows in breeding herds are often mass vaccinated against porcine reproductive and respi... more BackgroundSows in breeding herds are often mass vaccinated against porcine reproductive and respiratory syndrome (PRRS) every few months using modified live vaccines (MLV). Field veterinarians repeatedly report that multiple vaccinated sows test negative in ELISA. Obviously, this creates uncertainty when assessing the compliance of vaccination and the status of sows.MethodsIn the present study, four commercial ELISAs were used to assess the serological PRRS status in gilts and sows of three farms that were PRRS MLV vaccinated every four months. Animals were tested before vaccination (BV) and postvaccination (PV). Total and neutralising antibodies and cell-mediated responses were also measured in animals that yielded negative results in all ELISAs.ResultsThe proportion of seronegative animals BV varied depending on the farm and the ELISA used. When samples were analysed using only one ELISA, a substantial number of negative results obtained BV remained as negative afterwards. Five an...

Porcine Health Management

Background: The development of the innate and adaptive immune responses to Porcine reproductive a... more Background: The development of the innate and adaptive immune responses to Porcine reproductive and respiratory syndrome virus (PRRSV) after vaccination of 1 day-old pigs with a PRRSV-1 based modified live virus (MLV) vaccine by intramuscular (IM) and intranasal (IN) routes was characterised, before and after challenge with a heterologous PRRSV-1 isolate at 18 weeks post-vaccination. Twenty-five PRRSV-seronegative piglets were used. At 1 day of age, pigs were administered with a single dose of vaccine via the IM (n = 10) or the IN route (n = 10). Control group (n = 5) received saline solution. After vaccination, pigs were bled at days 3, 7, 28, 56, 83, 113 and 125. Levels of cytokines IL-10, IL-8, IFN-α (measured by ELISA tests of serum), TNF-α and IFN-γ (measured by ELISA and ELISPOT, respectively, from stimulated peripheral blood mononuclear cells), and serum neutralising antibodies (NA) to the vaccine strain, were measured. Results: The induction of IL-10 was rare, indicating that IL-10 mediated immunomodulation/immune dysfunction was not a feature of this vaccine or of the challenge virus. IL-8 was detected in only two pigs following vaccination, but in the majority of pigs after challenge, indicating that their ability to produce an innate immune response was not impaired. TNF-α was not detected in any vaccinated pigs until day 83. After challenge, only a minority of pigs produced TNF-α. IFN-α was detected in all vaccinated pigs following vaccination, indicating the potential for development of an effective Th1 adaptive immune response. IFN-γ-secreting cells were detected in all vaccinated pigs after vaccination. NA to the vaccine strain were first detected at day 56 in pigs vaccinated by both routes, and remained at similar levels until challenge. After challenge, a boost in NA was observed. The efficacy of the vaccine was demonstrated by reduction of viraemia and nasal shedding after challenge. Conclusions: The administration of a PRRSV-1 based MLV vaccine to 1 day-old piglets was able to induce an immune response characterised by: (1) undetectable or low levels of IL-10, IL-8 and TNF-α, (2) an increase in IFN-α expression within the first seven days, (3) a gradual increase in the number of antigen-specific IFN-γ-secreting cells, and (4) induction of detectable NA. After challenge with a heterologous strain, there was a rapid boost in NA titres, indicating a priming effect of the vaccine.

Veterinary microbiology, 2018

This study was aimed to evaluate the effect of Porcine circovirus 2 (PCV2) sow vaccination on cel... more This study was aimed to evaluate the effect of Porcine circovirus 2 (PCV2) sow vaccination on cell-mediated immune responses in sows and their progeny. At 7 weeks before farrowing, fifteen PCV2 PCR negative pregnant sows with medium-low antibody values were selected and randomly distributed in two groups according to the antibody levels. Seven sows were vaccinated with a commercial PCV2 vaccine and eight were injected with phosphate-buffered saline at 6 and 3 weeks before farrowing. Blood samples were taken from sows and their piglets (n = 90) during the study duration. PCV2 DNA and antibodies were tested in sera, and cytokine (IFN-α, IFN-γ, IL-12p40, TNF-α, IL-1β, IL-8, IL-4, IL-6 and IL-10) levels were assessed in supernatant from cultured peripheral blood mononuclear cells. All sows and piglets were negative by PCV2 PCR throughout the study. Significantly higher PCV2 antibody levels were detected in vaccinated sows after vaccination and in their offspring after colostrum ingestio...

Preventive veterinary medicine, 2017

In the present study, the transmission parameters of swine influenza virus (SIV) and porcine repr... more In the present study, the transmission parameters of swine influenza virus (SIV) and porcine reproductive and respiratory virus (PRRSV) have been calculated using the basic reproductive rate (R) parameter in two commercial pig farms (F1 and F2). In order to do this, a serological (PRRSV genotype 1 and SIV) and virological (SIV) follow-up of a batch of animals was carried out weekly from 3 weeks of age until the age of slaughter on each farm. Results of the analysis for SIV and PRRSV showed different transmission profiles depending on the farm, the pathogen, and time of transmission. In F1, transmission of both viruses was detected throughout the sampling. The Rt (R for a given period of time) value for SIV ranged from 1.5 [0.9-2.3] to 3.6 [2.3-4.9] from farrowing to the beginning of the fattening period, and the Rt value for PRRSV was 3.3 [2.9-4.3] to 3.5 [2.8-4.1] from farrowing until the slaughter age. These results indicated that both viruses were transmitted enzootically in that...

Veterinary Research, 2016

Little is known about the host factor in the response to PRRSV vaccination. For this purpose, pig... more Little is known about the host factor in the response to PRRSV vaccination. For this purpose, piglets were immunized with a commercial PRRSV-live vaccine and classified as high responders (HR) or low responders (LR) as regards to the frequencies of virus-specific IFN-γ-secreting cells. Six weeks post vaccination, PBMCs isolated from three individuals with the most extreme responses in each HR and LR groups and 3 unvaccinated controls, were either stimulated with phytohaemagglutinin, challenged with the vaccine or mock treated for 24 h, prior conducting transcriptional studies, gene ontology and pathway analyses. The LR group had very low neutralizing antibody levels and showed a higher number of down-regulated transcripts compared with the HR group (FDR < 0.2, P < 0.001). Down-regulated genes encoded chemoattractants, proinflammatory cytokines and the interferon-inducible GBP family, and showed enrichment in wounding (FDR < 3.6E-13), inflammation (FDR < 8E-12), defence (FDR < 8.7E-09) and immunity (FDR < 7.6E-08), suggesting immune response impairment. In the HR group, down-regulated genes were involved in protein transport (FDR < 4.77E-03), locomotory behavior (FDR < 5.47E-3), regulation of protein localization (FDR < 1.02E-02), and regulation of TNF superfamily member 15 and miR181. In contrast, the HR group presented up-regulated transcripts associated with wounding (FDR < 4.95). Moreover, IFN-γ was predicted to be an inhibited upstream regulator since IFN-γ pathways were associated with higher number of down-regulated genes in the LR (n = 40) than the HR (n = 10). Divergent responses to PRRSV-vaccination may be the result of the genetic background of the host.

PloS one, 2016

H-index is the most commonly applied tool to evaluate scientific productivity. In this study, the... more H-index is the most commonly applied tool to evaluate scientific productivity. In this study, the use of the H-index to evaluate scientific production in swine veterinary medicine was explored. A database of 137 pig infectious agents was constructed, including its taxonomic division, zoonotic potential, status as emerging pathogen and whether it was OIE-listed. The H-index and the total number of citations were calculated for those pathogens, the location of the affiliation of the first author of each paper included in the H-index core was registered and, for the ten pathogens with the highest H-index, evolution over time was measured. H-index values were compared to the M quotient, A-index, G-index, HG-index and the G/H ratio. H-indices were found to be severely affected by search accuracy and the database was hand curated. Swine pathogen H-indexes were highly dispersed ranging from 0 to 106 and were generally higher for pathogens causing endemic diseases in large pig producing cou...

Viruses, Jan 11, 2013

This work describes peptides from non-structural proteins (nsp) of porcine reproductive and respi... more 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, the...

PLoS ONE, 2010

A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to est... more A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to establish a new model of infection in minipigs. The 20-week monitoring period showed a marked Th1 response and poor humoral response for the whole infection. A detailed histopathological analysis was performed after slicing the formalin-fixed whole lungs of each animal. All lesions were recorded and classified according to their microscopic aspect, their relationship with the intralobular connective network and their degree of maturity in order to obtain a dissemination ratio (DR) between recent and old lesions. CFU counts and evolution of the DR with time showed that the proposed model correlated with a contained infection, decreasing from week 9 onwards. These findings suggest that the infection induces an initial Th1 response, which is followed by local fibrosis and encapsulation of the granulomas, thereby decreasing the onset of new lesions. Two therapeutic strategies were applied in order to understand how they could influence the model. Thus, chemotherapy with isoniazid alone helped to decrease the total number of lesions, despite the increase in DR after week 9, with similar kinetics to those of the control group, whereas addition of a therapeutic M. tuberculosis fragment-based vaccine after chemotherapy increased the Th1 and humoral responses, as well as the number of lesions, but decreased the DR. By providing a local pulmonary structure similar to that in humans, the mini-pig model highlights new aspects that could be key to a better understanding tuberculosis infection control in humans.

Viral Immunology, 2013

Pigs were immunized with DNA plasmids containing different open reading frames (ORFs) of a porcin... more Pigs were immunized with DNA plasmids containing different open reading frames (ORFs) of a porcine reproductive and respiratory syndrome virus (PRRSV) genotype I strain. One group was injected with three inoculations of ORF7, a second group was immunized with three inoculations of plasmids containing ORF5 and ORF6, and a third group was kept as controls. Later, +21 days after the last inoculation, animals were challenged with the homologous strain. After the challenge, PRRSV-specific interferon (IFN)-γ-secreting cells and anti-PRRSV IgG antibodies developed faster in DNA vaccinated pigs (p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). However, DNA-immunized pigs showed an exacerbation of the disease compared to the unvaccinated challenged pigs. The data suggest that previous immunization with DNA vaccines against glycoprotein 5 and/or matrix protein of PRRSV, as well as nucleoprotein but to a lesser degree, could result in an exacerbation of the clinical course in terms of fever upon challenge.

Veterinary Immunology and Immunopathology, 2009

Veterinary Immunology and Immunopathology, 2005

ELISPOT and ELISA were standardised for pig interferon-g (IFN-g), interleukin-10 (IL-10) and inte... more ELISPOT and ELISA were standardised for pig interferon-g (IFN-g), interleukin-10 (IL-10) and interleukin-4 (IL-4) with the aim to study the evolution of the immune response in conventional pigs from birth to 6 months of age and also to compare results of both techniques. Five pigs were bled at 1, 6, 9, 12 and 22 weeks of age and peripheral blood mononuclear cells (PBMC) were stimulated with phytohemagglutinin. The frequencies of cytokine secreting cells (CSC) and the levels of secreted cytokines were compared. For IFN-g the mean of CSC increased with age (p < 0.05) from an average of 486/10 6 PBMC at first week of age to 1256/10 6 PBMC at 22 weeks of age. No correlation was found between the number of IFN-g CSC and the cytokine levels obtained by ELISA. For IL-10, frequencies of CSC did not increase with age of pigs, having a low of 315/10 6 PBMC at first week of age and a high of 1485/10 6 PBMC at six weeks. Comparison of ELISA and ELISPOT results for IL-10 showed a certain degree of correlation (r = 0.74; p < 0.05). Spontaneous secretion was observed in unstimulated cultures. For IL-4, frequencies of CSC were low (50-70/10 6 PBMC). In this case, comparison of ELISA and ELISPOT could not be done because cytokine levels in culture supernatants were often below the detection limit of the IL-4 ELISA. All these values can serve as a reference for future studies and also, our observations suggest that ELISPOT and ELISA should be carefully interpreted and do not necessarily correlate.

Vaccine, 2009

In silico prediction and ex vivo evaluation of potential T-cell epitopes in glycoproteins 4 and 5... more In silico prediction and ex vivo evaluation of potential T-cell epitopes in glycoproteins 4 and 5 and nucleocapsid protein of genotype-I (European) of porcine reproductive and respiratory syndrome virus

The Veterinary Journal, 2012

Porcine circovirus type 2 (PCV2) negatively modulates the immune response in vitro. The objective... more Porcine circovirus type 2 (PCV2) negatively modulates the immune response in vitro. The objective of this study was to investigate if PCV2 interferes with the development of the immune response to Aujeszky&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease virus (ADV) vaccine, using an in vivo experimental subclinical model. Pigs were divided into four groups: (group CC) not infected with PCV2 and not vaccinated against ADV; (group IC) infected with PCV2 but not vaccinated against ADV; (group CV) not infected with PCV2 but vaccinated against ADV, and (group IV) infected with PCV2 and vaccinated against ADV. Pigs in groups IC and IV were inoculated intranasally with PCV2 and 14 days later, pigs in the CV and IV groups were vaccinated IM with a gE(-)tk(-) attenuated ADV vaccine. Clinical signs and weight gains were recorded from days 0 to 35 post-PCV2 inoculation (PI), at which point the pigs were euthanased and examined post-mortem. Throughout the experiment the PCV2 load was quantified in serum, antibodies to PCV2 and ADV were determined and antigen-specific cellular responses against both viruses were measured using an interferon-γ ELISPOT. PCV2 inoculated animals developed subclinical infection and had lower weight gain relative to non-infected controls. No differences were observed between the CV and IV groups in terms of the humoral or cellular immune responses to vaccination against Aujeszky&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease, suggesting that subclinical infection with PCV2 does not alter the response to this vaccine.

The Veterinary Journal, 2011

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porci... more The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.