Elucidating the Onset of Cross-Protective Immunity after Intranasal Vaccination with the Attenuated African Swine Fever Vaccine Candidate BA71ΔCD2.

African swine fever (ASF) is a deadly disease of swine currently causing a worldwide pandemic, leading to severe economic consequences for the porcine industry. The control of disease spread is hampered by the limitation of available effective vaccines. Live attenuated vaccines (LAVs) are currently the most advanced vaccine prototypes, providing strong protection against ASF. However, the significant advances achieved using LAVs must be complemented with further studies to analyze vaccine-induced immunity. Here, we characterized the onset of cross-protective immunity triggered by the LAV candidate BA71ΔCD2. Intranasally vaccinated pigs were challenged with the virulent Georgia 2007/1 strain at days 3, 7 and 12 postvaccination. Only the animals vaccinated 12 days before the challenge had effectively controlled infection progression, showing low virus loads, minor clinical signs and a lack of the unbalanced inflammatory response characteristic of severe disease. Contrarily, the animals vaccinated 3 or 7 days before the challenge just showed a minor delay in disease progression. An analysis of the humoral response and whole blood transcriptome signatures demonstrated that the control of infection was associated with the presence of virus-specific IgG and a cytotoxic response before the challenge. These results contribute to our understanding of protective immunity induced by LAV-based vaccines, encouraging their use in emergency responses in ASF-affected areas.

Cross-protection against African swine fever virus upon intranasal vaccination is associated with an adaptive-innate immune crosstalk.

African swine fever virus (ASFV) is causing a worldwide pandemic affecting the porcine industry and leading to important global economic consequences. The virus causes a highly lethal hemorrhagic disease in wild boars and domestic pigs. Lack of effective vaccines hampers the control of virus spread, thus increasing the pressure on the scientific community for urgent solutions. However, knowledge on the immune components associated with protection is very limited. Here we characterized the in vitro recall response induced by immune cells from pigs intranasally vaccinated with the BA71ΔCD2 deletion mutant virus. Vaccination conferred dose-dependent cross-protection associated with both ASFV-specific antibodies and IFNγ-secreting cells. Importantly, bulk and single-cell transcriptomics of blood and lymph node cells from vaccinated pigs revealed a positive feedback from adaptive to innate immunity. Indeed, activation of Th1 and cytotoxic T cells was concomitant with a rapid IFNγ-dependent triggering of an inflammatory response characterized by TNF-producing macrophages, as well as CXCL10-expressing lymphocytes and cross-presenting dendritic cells. Altogether, this study provides a detailed phenotypic characterization of the immune cell subsets involved in cross-protection against ASFV, and highlights key functional immune mechanisms to be considered for the development of an effective ASF vaccine.

DNA Vaccines in Pigs: From Immunization to Antigen Identification.

DNA vaccination is one of the most fascinating vaccine strategies currently in development. Two of the main advantages of DNA immunization rely on its simplicity and flexibility, being ideal to dissect both the immune mechanisms and the antigens involved in protection against a given pathogen. Here we describe several strategies used to enhance the immune responses induced and the protection afforded by experimental DNA vaccines tested in swine and provide very basic protocols describing the generation and in vivo application of a prototypic DNA vaccine. The future will say the last word regarding the definitive implementation of DNA vaccination in the field.

Uptake and Survival of African Swine Fever Virus in Mealworm (Tenebrio molitor) and Black Soldier Fly (Hermetia illucens) Larvae.

Insect production offers a sustainable source of nutrients for livestock. This comes with a risk for transmission of pathogens from the insects into the livestock sector, including viruses causing serious diseases, such as African swine fever virus (ASFV), classical swine fever virus and foot-and-mouth disease virus. ASFV is known to survive for a long time within animal meat and byproducts. Therefore, we conducted experimental exposure studies of insects to ASFV using larvae of two key insect species produced for food and feed, the mealworm; Tenebrio molitor, and the black soldier fly, Hermetia illucens. The larvae were exposed to ASFV POL/2015/Podlaskie, via oral uptake of serum or spleen material from ASFV-infected pigs. Using qPCR, the amounts of viral DNA present immediately after exposure varied from ~104.7 to 107.2 genome copies per insect. ASFV DNA was detectable in the larvae of H. illucens for up to 3 days post exposure and in T. molitor larvae for up to 9 days post exposure. To assess the presence of infectious virus within the larvae and with this, the risk of virus transmission via oral consumption, pigs were fed cakes containing larvae exposed to ASFV. Pigs that consumed 50 T. molitor or 50 H. illucens virus-exposed larvae did not become infected with ASFV. Thus, it appears, that in our experimental setting, the risk of ASFV transmission via consumption of unprocessed insect larvae, used as feed, is low.

Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

Deletion Mutants of the Attenuated Recombinant ASF Virus, BA71ΔCD2, Show Decreased Vaccine Efficacy.

African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71∆CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding ß-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71∆CD2 as a template: the single mutant BA71∆CD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71∆CD2f, BA71∆CD2DP96R and BA71∆CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71∆CD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future.

M448R and MGF505-7R: Two African Swine Fever Virus Antigens Commonly Recognized by ASFV-Specific T-Cells and with Protective Potential.

African swine fever (ASF) is today's number one threat for the global swine industry. Neither commercial vaccine nor treatment is available against ASF and, thus far, only live attenuated viruses (LAV) have provided robust protection against lethal ASF virus (ASFV) challenge infections. Identification of ASFV proteins inducing protective immune responses is one of the major challenges to develop safer and efficient subunit vaccines. Immunopeptidomic studies recently performed in our laboratory allowed identifying ASFV antigens recognized by ASFV-specific CD8+ T-cells. Here, we used data from the SLAI-peptide repertoire presented by a single set of ASFV-infected porcine alveolar macrophages to generate a complex DNA vaccine composed by 15 plasmids encoding the individual peptide-bearing ORFs. DNA vaccine priming improved the protection afforded by a suboptimal dose of the BA71ΔCD2 LAV given as booster vaccination, against Georgia2007/1 lethal challenge. Interestingly, M448R was the only protein promiscuously recognized by the induced ASFV-specific T-cells. Furthermore, priming pigs with DNA plasmids encoding M488R and MGF505-7R, a CD8+ T-cell antigen previously described, confirmed these two proteins as T-cell antigens with protective potential. These studies might be useful to pave the road for designing safe and more efficient vaccine formulations in the future.

Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach.

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

Live Attenuated African Swine Fever Viruses as Ideal Tools to Dissect the Mechanisms Involved in Cross-Protection.

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.

Fecal microbiota transplantation from warthog to pig confirms the influence of the gut microbiota on African swine fever susceptibility.

African swine fever virus (ASFV) is the causative agent of a devastating hemorrhagic disease (ASF) that affects both domestic pigs and wild boars. Conversely, ASFV circulates in a subclinical manner in African wild pigs, including warthogs, the natural reservoir for ASFV. Together with genetic differences, other factors might be involved in the differential susceptibility to ASF observed among Eurasian suids (Sus scrofa) and African warthogs (Phacochoerus africanus). Preliminary evidence obtained in our laboratory and others, seems to confirm the effect that environmental factors might have on ASF infection. Thus, domestic pigs raised in specific pathogen-free (SPF) facilities were extremely susceptible to highly attenuated ASFV strains that were innocuous to genetically identical domestic pigs grown on conventional farms. Since gut microbiota plays important roles in maintaining intestinal homeostasis, regulating immune system maturation and the functionality of the innate/adaptive immune responses, we decided to examine whether warthog fecal microbiota transplantation (FMT) to domestic pigs affects host susceptibility to ASFV. The present work demonstrates that warthog FMT is not harmful for domestic weaned piglets, while it modifies their gut microbiota; and that FMT from warthogs to pigs confers partial protection against attenuated ASFV strains. Future work is needed to elucidate the protective mechanisms exerted by warthog FMT.

Differential expression of porcine microRNAs in African swine fever virus infected pigs: a proof-of-concept study.

BACKGROUND: African swine fever (ASF) is a re-expanding devastating viral disease currently threatening the pig industry worldwide. MicroRNAs are a class of 17-25 nucleotide non- coding RNAs that have been shown to have critical functions in a wide variety of biological processes, such as cell differentiation, cell cycle regulation, carcinogenesis, apoptosis, regulation of immunity as well as in viral infections by cleavage or translational repression of mRNAs. Nevertheless, there is no information about miRNA expression in an ASFV infection. METHODS: In this proof-of-concept study, we have analyzed miRNAs expressed in spleen and submandibular lymph node of experimentally infected pigs with a virulent (E75) or its derived attenuated (E75CV1) ASFV strain, as well as, at different times post-infection with the virulent strain, by high throughput sequencing of small RNA libraries. RESULTS: Spleen presented a more differential expression pattern than lymph nodes in an ASFV infection. Of the most abundant miRNAs, 12 were differentially expressed in both tissues at two different times in infected animals with the virulent strain. Of these, miR-451, miR-145-5p, miR-181a and miR-122 presented up-regulation at late times post-infection while miR-92a, miR-23a, miR-92b-3p, miR-126-5p, miR-126-3p, miR-30d, miR-23b and miR-92c showed down-regulation. Of the 8 differentially expressed miRNAs identified at the same time post-infection in infected animals with the virulent strain compared with animals infected with its attenuated strain, miR-126-5p, miR-92c, miR-92a, miR-30e-5p and miR-500a-5p presented up-regulation whereas miR-125b, miR-451 and miR-125a were down-regulated. All these miRNAs have been shown to be associated with cellular genes involved in pathways related to the immune response, virus-host interactions as well as with several viral genes. CONCLUSION: The study of miRNA expression will contribute to a better understanding of African swine fever virus pathogenesis, essential in the development of any disease control strategy.

BA71ΔCD2: a New Recombinant Live Attenuated African Swine Fever Virus with Cross-Protective Capabilities.

African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). The lack of available vaccines makes its control difficult; thus, African swine fever virus (ASFV) represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against ASFV. Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, although little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates and have hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found that the deletion of the viral CD2v (EP402R) gene highly attenuated the virulent BA71 strain in vivo Inoculation of pigs with the deletion mutant virus BA71ΔCD2 conferred protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose dependent, and the cross-protection observed in vivo correlated with the ability of BA71ΔCD2 to induce specific CD8+ T cells capable of recognizing both BA71 and E75 viruses in vitro Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in areas where the virus is endemic and where multiple viruses are circulating.IMPORTANCE African swine fever virus (ASFV) remains enzootic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being in Georgia in 2007. Since then, ASFV has spread toward neighboring countries, reaching the European Union's east border in 2014. The lack of available vaccines against ASFV makes its control difficult; so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but they have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71ΔCD2. Inoculation of BA71ΔCD2 protected pigs not only against experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in Eastern Europe.

DNA Vaccines: Experiences in the Swine Model.

DNA vaccination is one of the most fascinating vaccine-strategies currently in development. Two of the main advantages of DNA immunization rely on its simplicity and flexibility, being ideal to dissect both the immune mechanisms and the antigens involved in protection against a given pathogen. Here, we describe several strategies used to enhance the immune responses induced and the protection afforded by experimental DNA vaccines tested in swine and provide with very basic protocol describing the generation and in vivo application of a prototypic DNA vaccine. Only time will tell the last word regarding the definitive implementation of DNA vaccination in the field.

Live attenuated African swine fever viruses as ideal tools to dissect the mechanisms involved in viral pathogenesis and immune protection.

African swine fever virus (ASFV) is the causal agent of African swine fever, a hemorrhagic and often lethal porcine disease causing enormous economical losses in affected countries. Endemic for decades in most of the sub-Saharan countries and Sardinia, the risk of ASFV-endemicity in Europe has increased since its last introduction into Europe in 2007. Live attenuated viruses have been demonstrated to induce very efficient protective immune responses, albeit most of the time protection was circumscribed to homologous ASFV challenges. However, their use in the field is still far from a reality, mainly due to safety concerns. In this study we compared the course of the in vivo infection caused by two homologous ASFV strains: the virulent E75 and the cell cultured adapted strain E75CV1, obtained from adapting E75 to grow in the CV1 cell-line. Interestingly, the kinetics of both viruses not only differed on the clinical signs that they caused and in the virus loads found, but also in the immunological pathways activated throughout the infections. Furthermore, E75CV1 confirmed its protective potential against the homologous E75 virus challenge and allowed the demonstration of poor cross-protection against BA71, thus defining it as heterologous. The in vitro specificity of the CD8(+) T-cells present at the time of lethal challenge showed a clear activation against the homologous virus (E75) but not against BA71. These findings will be of utility for a better understanding of ASFV pathogenesis and for the rational designing of safe and efficient vaccines against this virus.

Expression library immunization can confer protection against lethal challenge with African swine fever virus.

UNLABELLED: African swine fever is one of the most devastating pig diseases, against which there is no vaccine available. Recent work from our laboratory has demonstrated the protective potential of DNA vaccines encoding three African swine fever viral antigens (p54, p30, and the hemagglutinin extracellular domain) fused to ubiquitin. Partial protection was afforded in the absence of detectable antibodies prior to virus challenge, and survival correlated with the presence of a large number of hemagglutinin-specific CD8(+) T cells in blood. Aiming to demonstrate the presence of additional CD8(+) T-cell determinants with protective potential, an expression library containing more than 4,000 individual plasmid clones was constructed, each one randomly containing a Sau3AI restriction fragment of the viral genome (p54, p30, and hemagglutinin open reading frames [ORFs] excluded) fused to ubiquitin. Immunization of farm pigs with the expression library yielded 60% protection against lethal challenge with the virulent E75 strain. These results were further confirmed by using specific-pathogen-free pigs after challenging them with 10(4) hemadsorbing units (HAU) of the cell culture-adapted strain E75CV1. On this occasion, 50% of the vaccinated pigs survived the lethal challenge, and 2 out of the 8 immunized pigs showed no viremia or viral excretion at any time postinfection. In all cases, protection was afforded in the absence of detectable specific antibodies prior to challenge and correlated with the detection of specific T-cell responses at the time of sacrifice. In summary, our results clearly demonstrate the presence of additional protective determinants within the African swine fever virus (ASFV) genome and open up the possibility for their future identification. IMPORTANCE: African swine fever is a highly contagious disease of domestic and wild pigs that is endemic in many sub-Saharan countries, where it causes important economic losses and is currently in continuous expansion across Europe. Unfortunately, there is no treatment nor an available vaccine. Early attempts using attenuated vaccines demonstrated their potential to protect pigs against experimental infection. However, their use in the field remains controversial due to safety issues. Although inactive and subunit vaccines did not confer solid protection against experimental ASFV infection, our DNA vaccination results have generated new expectations, confirming the key role of T-cell responses in protection and the existence of multiple ASFV antigens with protective potential, more of which are currently being identified. Thus, the future might bring complex and safe formulations containing more than a single viral determinant to obtain broadly protective vaccines. We believe that obtaining the optimal vaccine formulation it is just a matter of time, investment, and willingness.

Standardization of pathological investigations in the framework of experimental ASFV infections.

African swine fever is still one of the major viral diseases of swine for which a commercial vaccine is lacking. For the design and development of such preventive products, researchers involved in African swine fever virus (ASFV) vaccinology need standardized challenge protocols and well characterized clinical, pathological and immunological responses of inbreed and outbreed pigs to different viral strains and vaccine-like products. The different approaches used should be assessed by immunologist, virologist and pathologist expertise. The main objectives of this guideline are to (1) briefly contextualize the clinical and pathological ASFV presentations focusing on points that are critical for pathogenesis, (2) provide recommendations concerning the analysis of clinical, gross and microscopic observations and (3) standardize the pathological report, the terminology employed and the evaluation of the severity of the lesions between the ASFV research groups for comparing inter-group data. The presented guidelines establish new approaches to integrate such relevant pathological data with virological and immunological testing, giving support to the global interpretation of the findings in the future experiments of ASFV-related vaccinology and immunology.

DNA vaccination partially protects against African swine fever virus lethal challenge in the absence of antibodies.

The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8(+) T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8(+) T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease.

Ingestion of deoxynivalenol (DON) contaminated feed alters the pig vaccinal immune responses.

Deoxynivalenol (DON), a mycotoxin produced by some Fusarium species, is a frequent contaminant of cereals. This toxin is known to modulate the immune function but only few studies have investigated the effect of DON on the vaccinal immune response. In the present experiment, 24 pigs received for 9 weeks either control feed or feed naturally contaminated with 2.2-2.5 mgDON/kg feed. At days 4 and 15 of the experiment, the animals were subcutaneously immunized with ovalbumin. Consumption of DON-contaminated diet does not have a major effect on the hematological and biochemical blood parameters. By contrast, ingestion of DON significantly affects the global and the specific immune response of the pigs. In the serum, DON increases the concentration of total IgA and, in vaccinated animals, DON also increases the concentration of ovalbumin-specific IgA and IgG. DON does not modulate lymphocytes proliferation after mitogenic stimulation but the toxin had a biphasic effect on lymphocyte proliferation after antigenic stimulation (up-regulation at day 21 and down-regulation at day 35-49). Because cytokines play a key role in immunity, the expression levels of TGF-beta, IFN-gamma, IL-4 and IL-6 were measured, by RT-PCR in the spleen, the ileum and the mesenteric lymph node of the animals at the end of the experiment. In the mesenteric lymph node, a significantly lower expression of both TGF-beta and IFN-gamma mRNA expression levels is observed in animals feed with DON when compared with control piglets. Taken together, our data indicate that DON alters the vaccinal immune response. These results may have implications for humans and animals consuming DON-contaminated food or feed as breakdown in vaccinal immunity may lead to the occurrence of disease even in properly vaccinated populations.

Taxonomy and significance of black aspergilli.

Members of Aspergillus section Nigri (formerly A. niger group) are distributed worldwide and are regarded as common food spoilage fungi. Some of them are widely used and studied for industrial purposes. They are common sources of extracellular enzymes and organic acids to be used in food processing and are also used in the production of traditional foods, especially in the Orient. Products produced by strains of Aspergillus niger hold the GRAS (Generally Recognised As Safe) status from the FDA. However some species in Aspergillus section Nigri can produce ochratoxin A, a nephrotoxic mycotoxin. In spite of their industrial importance, the taxonomy of black aspergilli ( Aspergillus section Nigri ) is not clear and many attempts have been made in order to find suitable taxonomic criteria. The aim of this paper is to provide an overview of the significance of black aspergilli focusing on all the approaches made in the taxonomy of this group of fungi. Some species, such as A. carbonarius and uniseriate species can be easily recognised. In the A. niger aggregate, although speciation at molecular level has been proposed, no morphological differences can be observed and species identification will therefore remain problematic. Phylogenetic analyses of ITS and 5.8S rDNA gene region of representative black Aspergillus species and a simple key to the most common species that can be easily distinguished by morphological criteria are also included.