Sow Vaccination with a Protein Fragment against Virulent Glaesserella (Haemophilus) parasuis Modulates Immunity Traits in Their Offspring.

Glaesserella (Haemophilus) parasuis, an early colonizer of the nasal cavity in piglets, is a highly heterogeneous species, comprising both commensal and virulent strains. Virulent G. parasuis strains can cause fibrinous polyserositis called Glässer's disease. Colostrum is a source of passive immunity for young piglets. When vaccinating sows, protective antibodies are transferred to their offspring through the colostrum. Here, sow vaccination was performed with a protein fragment, F4, from the outer membrane trimeric autotransporters VtaAs exclusively found in virulent G. parasuis. Piglets were allowed to suckle for 3 weeks, following which a challenge with two virulent strains of G. parasuis was performed. A group of nonvaccinated sows and their piglets were included as a control. Antibodies against F4 were confirmed using ELISA in the vaccinated sows and their offspring before the G. parasuis challenge. Compared to the control group, F4-vaccination also resulted in an increased level of serum TGF-ß both in vaccinated sows and in their offspring at early time points of life. After the challenge, a lower body temperature and a higher weight were observed in the group of piglets from vaccinated sows. One piglet from the non-vaccinated group succumbed to the infection, but no other significant differences in clinical signs were noticed. At necropsy, performed 2 weeks after the virulent challenge, the level of surfactant protein D (SP-D) in bronchoalveolar lavage was higher in the piglets from vaccinated sows. Vaccination did not inhibit the nasal colonization of the piglets by the challenge strains.

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.

Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs.

The present study has evaluated the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the Rift Valley Fever virus (RVFV) glycoproteins Gn and Gc in lambs. Three groups of six to seven lambs were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 10(5) TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. Together, the data suggest that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. Further optimization of this vaccine approach in lambs is warranted.

Immunization with DNA vaccines containing porcine reproductive and respiratory syndrome virus open reading frames 5, 6, and 7 may be related to the exacerbation of clinical disease after an experimental challenge.

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<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.

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.

Effect of Cholesterol and Equex-STM Addition to an Egg Yolk Extender on Pure Spanish Stallion Cryopreserved Sperm.

Cholesterol and Equex-STM are frequently added to different commercial and experimental extenders improving postthawing sperm quality. Doses of 125-150 mM of cholesterol from pig liver and 0.5-0.7% of Equex-STM were evaluated in a standard eggyolk extender (Martin et al., 1979). Six ejaculates per stallion from six pure Spanish stallions (6-8 years old) were collected in Martin's extender (B) and different mixtures of 125 mM-0.5% (I), 125 mM-0.7% (II), 150 mM-0.5% (III), and 150 mM-0.7% (IV) were added to original Martin's extender. Samples were frozen in 0.5 mL straws (100 × 10(6) spermatozoa) and thawed (21 s., 37°C water bath). After thawing the following parameters were evaluated: viability (V), motility (computer assisted sperm analysis, CASA; % nonprogressive NP; % progressive MP), hipoosmotic swelling test (HOST), acrosome integrity (A), fluorescence test (FL), and resistance test (RT). Sperm quality was significantly affected by stallion (in the parameters V, VI, NP, MP, HOST, A, FL, and RT), extraction (VI, NP, MP, HOST, A, and FL), and the different combinations of Equex-STM-cholesterol (FL). We concluded that 0.5% of Equex-STM mixed with 125 mM of cholesterol has obtained better sperm quality results than those of original Martin's extender, showing a simple and economic improvement of this home-made practical seminal extender.

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.

Malignant pheochromocytoma in a pig.

Endocrine tumors are rarely observed in pigs, and pheochromocytomas have been only punctually described. The current report describes a white and firm, 15-cm in diameter, neoplastic mass located in the adrenal gland with metastasis to regional lymph nodes in a 2.5-year-old sow. The masses had marked desmoplasia that supported a population of polygonal-to-spindle-shaped neoplastic cells arranged into cords and packets within a delicate fibrovascular stroma. Immunohistochemical staining of the tumor was positive for chromogranin and negative for neurofilament protein in adrenal and lymph node masses, which was characteristic of a malignant pheochromocytoma.

Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV.

The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation, that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). Our results confirm the role of CCL20 to increase antibody-mediated responses. At the same time suggest the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs previously reported. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN. Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge. The levels of IFN-alpha detected in pigs immunized with pE2 and principally in non-vaccinated challenged animals can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed. Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV. To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the immune induction and protection against virus challenge in swine infection model.

Experimental infection of young adult European breed sheep with Rift Valley fever virus field isolates.

The increasing interest in Rift Valley fever virus (RVFV) and its potential impact on naive animal populations deserve revisiting experimental reproduction of RVFV infection, particularly in those animal breeds for which no data about their susceptibility to RVFV infection have ever been recorded. In this study we show the susceptibility of 9-10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and virus shedding (variable in length depending on the RVFV isolate used) were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in adult lambs and no other clinical symptoms were observed, with the exception of corneal opacity ("blue eye") found in 3 out of 16 subcutaneously inoculated sheep. In conclusion, adult sheep from this European breed are readily infected with RVFV without apparent clinical manifestations.