Natural Immunity of Sheep and Lambs Against the Schmallenberg Virus Infection.

Since the first reports of the Schmallenberg disease (SBD) outbreaks in late 2011, the disease has spread across Europe, affecting cattle and sheep farms. While Schmallenberg virus (SBV) causes a mild clinical disease in adults, infection of pregnant females may lead to the production of typical congenital malformations (CMFs) in their offspring. It is speculated that the immunity acquired after a SBV infection is effective in preventing further infections. However, this has not been proven in naturally infected sheep, especially if they are pregnant when reinfected. The aim of this study was to monitor the natural immunity in SBV-infected sheep. Twenty-four ewes from the only Spanish farm with a SBV OIE-notified outbreak were sampled. Subsequently, nine pregnant ewes were inoculated with SBV infectious plasma under controlled conditions. Six of them were euthanized before delivery, and their fetuses were inspected for lesions indicative for the SBV infection. The three remaining ewes were allowed to deliver one lamb each. Inoculation of the lambs was scheduled at approx. 3 months after birth. All samples were analyzed for viral RNA by RT-PCR, and for antibodies by an indirect ELISA and a virus neutralization test (VNT). The majority of the 24 ewes showed a serological reaction against SBV. The three ewes that were allowed to lamb down demonstrated variable degrees of seroconversion which corresponded to the levels of immune reaction observed in their lambs. Moreover, no viral RNA was detected, no lesions were observed in the fetuses, and no clinical signs were detected in the inoculated animals. These findings suggest that the immunity acquired by sheep following a natural SBV infection could be sufficient to stop SBV reinfection. However, vaccination could be a valuable tool to control SBV infections and associated economic losses as it affords a more uniform and predictable protection at the flock/herd level.

A genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) improves clinical, pathological and virological outcomes in postweaning multisystemic wasting syndrome affected farms.

The present study describes the effects of a commercially available genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) on clinical, pathological and virological features in three multi-site farms suffering from postweaning multisystemic wasting syndrome (PMWS). The vaccine product was able to reduce clinical signs, PCV2 viral load in lymphoid organs and/or sera, and overall mortality in nurseries and fattening units. This is the first time in which is shown that a PCV2 vaccine is able to decrease specifically PMWS-associated mortality. Another novelty of this study is the assessment of PMWS-like histological lesions in a large number of vaccinated and non-vaccinated pigs under field conditions.

In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2.

The aim of this study was to describe the generation of a PCV2 (porcine circovirus type 2) infectious clone (pIC-PCV2) and its infectivity under in vitro and in vivo conditions. The constructed pIC-PCV2 contained the whole PCV2 genome from a German isolate together with a partial duplication of 467 bp. PK-15 cells were transfected with pIC-PCV2 and an indirect immune fluorescence assay (IFA) was performed 7 days post-transfection. The PCV2 Cap gene was expressed in approximately 20 % of the cultured cells, and only the recombination product, and not pIC-PCV2, was subsequently detected by PCR and Southern blot. This result indicated that infection by pIC-PCV2 delivered genomic PCV2 DNA specifically into susceptible cells and led to the expression of a functional virus genome. Eighteen 30- to 40-day-old conventional pigs were distributed into three groups. Group 1 pigs (n=6) were inoculated intranasally (i.n.) with a Spanish isolate of PCV2 propagated in cell culture; pigs from group 2 (n=6) were inoculated with pIC-PCV2 intramuscularly (i.m.), and the last group of pigs (n=6) was inoculated with pIC-PCV2 intraperitoneally (i.p.). All pigs remained clinically healthy during the whole experimental period (35 days). Pigs that received pIC-PCV2 i.p. and i.m., as well as those PCV2 i.n. inoculated, became infected based on an in situ hybridization (ISH), PCR, TaqMan PCR and serological results. The results of this study confirm that cloned PCV2 genomic DNA is infectious both in vitro and in vivo, and is able to cause PMWS-like lesions in i.p. and i.m. experimentally inoculated pigs.

Experimental inoculation of conventional pigs with tissue homogenates from pigs with post-weaning multisystemic wasting syndrome.

This report describes the experimental inoculation of conventional pigs with a tissue homogenate obtained from two pigs affected with postweaning multisystemic wasting syndrome (PMWS). Eight 2-month-old pigs were inoculated by the intranasal route, and two pigs were left as uninfected controls. Clinical signs, rectal temperatures and body weights were recorded. Pigs were necropsied at days 14 or 21 post-inoculation, and tissue samples were taken for histopathology and porcine circovirus (PCV) in-situ hybridization. Although only mild clinical signs of disease were observed, lesions of PMWS were seen, and PCV was shown to have been successfully transmitted to six of the eight pigs. Seroconversion of all inoculated pigs to PCV-2, but not to PCV-1, was also detected, suggesting that the PCV nucleic acid detected by in-situ hybridization in inoculated pigs corresponded to PCV-2. In conclusion, this report shows that PCV-2 is transmissible to pigs, and the inoculation of tissue homogenates containing the virus results in the development of PMWS-like lesions.

Efficacy of an inactivated vaccine for prevention of reproductive failure induced by porcine reproductive and respiratory syndrome virus.

This report describes the results of experiments with an inactivated oily vaccine containing per dose about 10(5.5) median tissue culture infectious dose (TCID50) of the Spanish strain of porcine reproductive and respiratory syndrome (PRRS) virus grown in porcine alveolar macrophages (PAMs). In order to evaluate the efficacy of the vaccine, two experimental infection routes were tested in sows; subsequent intranasal (i.n.) and intravenous (i.v.) (out of a total of 93 piglets born to 7 sows, 16% were mummified, 18.2% were weak and died within 48 h of birth, 37% were stillborn, 5.3% died between 2 and 7 days of age, 22.5% lived for more than one week) and intranasal alone (out of a total of 65 piglets born to 5 sows, 0% were mummified, 22.5% were weak and died within 48 h of birth, 40% were stillborn, 4.6% lived for more than one week). I.N. alone was selected to evaluate the efficacy of the vaccine because this is the natural route of infection. A number of experiments were conducted to test the immunogenicity of the vaccine. In general, after challenge with the homologous strain, protection in vaccinated sows was high (at least 70% of the piglets were born alive and healthy), whereas protection in unvaccinated sows was low (only 10% of the piglets were born alive and healthy). Vaccinated animals devoid of antibodies by immunoperoxidase monolayer assay (IPMA) at the time of challenge were still protected at experimental infection.

Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 proteins in protection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tübingen isolates and between 61-64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.

Characterization of transmissible gastroenteritis coronavirus S protein expression products in avirulent S. typhimurium delta cya delta crp: persistence, stability and immune response in swine.

The spike protein from transmissible gastroenteritis virus (TGEV) was expressed in attenuated S. typhimurium delta cya delta crp delta asd chi 3987. Three partially overlapping fragments of TGEV S gene, encoding the amino-terminal, intermediate, and carboxy-terminal end of the protein, as well as the full length gene were inserted into the asd+ plasmid pYA292 to generate recombinant plasmids pYATS-1, pYATS-2, pYATS-3, and pYATS-4, respectively, which were transformed into S. typhimurium chi 3987. Recombinant S. typhimurium chi 3987 (pYATS-1) and chi 3987 (pYATS-4) expressing constitutively a 53 kDa amino-terminal fragment of the S protein and the full length protein (144 kDa), respectively, showed high stability. After 50 generations in vitro 60% and 20% of the bacteria transformed with pYATS-1 and pYATS-4, respectively, expressed the S-protein antigen. Since S. typhimurium chi 3987 (pYATS-1) showed a better level of expression and stability in vitro, this recombinant strain was selected as a potential bivalent vector to induce both immunity to Salmonella and TGEV in swine. In order to study colonization of swine tissues by S. typhimurium delta cya delta crp, a gene conferring resistance to rifampicin was cloned into the chromosome of S. typhimurium chi 3987, generating chi 4509 strain. Both S. typhimurium chi 4509 (pYA292) and chi 4509 (pYATS-1) colonized the ileum of orally inoculated swine with clearance of bacteria between days 10-20 post-infection. The expression of the amino-terminal fragment of the S protein diminished the ability of S. typhimurium chi 4509 (pYATS-1) to colonize deep tissues. The recombinant strain S. typhimurium chi 3987 (pYATS-1) induced TGEV specific antibodies in both serum and saliva of orally inoculated swine.