Clinical signs usually appear first in a small number of growing pigs which show non-specific signs of depression, sleepiness, and reluctance to get up or to eat.
- Younger piglets may appear chilled, shiver and huddle together.
- Initially affected pigs may appear to be constipated but this generally changes to a yellow-grey diarrhoea as the disease progresses.
- A constant early sign, which persists throughout the disease until just before death, is a high fever, over 42ºC (107ºF)
- As the disease progresses the affected pigs become very thin and weak and develop a staggering walk.
- Affected pigs die in 10-20 days. Some pigs go into convulsions before death.
- Affected pigs may be ill for up to 30 days before they die.
Classical swine fever (CSF) is a highly contagious, often fatal, notifiable disease which is responsible for significant losses in the swine industry worldwide (1, http://www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2014). The etiological agent is an enveloped RNA virus (CSF virus [CSFV]) that belongs to the Pestivirus genus of the family Flaviviridae (2). Vaccination of pigs with lapinized Chinese vaccines is still practiced in some regions of the world in which the virus is enzootic, in order to prevent and control the spread of the disease.
Swine Fever Vaccine For Pig ,such vaccines are not safe enough and do not allow differentiation of infected from vaccinated animals (DIVA). Attenuated CSFV vaccines can potentially influence CSFV evolution through recombination with wild viruses, suggesting that it is necessary to avoid their excessive use (3).
Despite their accepted advantages, these vaccines have one major disadvantage: serological DIVA is impossible (4). Subunit recombinant marker vaccines derived from viral proteins have been considered safer and cheaper alternatives against CSFV (5).
The E2 glycoprotein is the major antigen that induces neutralizing and protective antibodies in CSFV-infected pigs. This glycoprotein is exposed as a homodimer on the outer surface of the virus and mediates the viral entry into the target cells (6, 7). Several marker vaccines based on E2 have been generated so far for the induction of a protective immune response against CSFV (8,–12). However, the structural complexity of E2 has demonstrated the necessity to produce this glycoprotein in superior production cell systems in order to enhance its immunogenicity and protective capacity (12). Recently, we developed a new marker subunit vaccine based on E2-CSFV, which is produced in the mammary glands of genetically transformed goats.
This formulation has consistently shown an early and elevated protective activity in vaccinated swine (13, 14). Despite these characteristics, the new goat milk-derived marker vaccine has a complex downstream process, which involves separation of fat milk and whey, as well as an additional immobilized-metal affinity chromatography (IMAC) step (15).
Here, we demonstrate that the protective capacity of the goat milk-derived E2 vaccine remains invariable when the formulation is generated by using the E2 antigen present within the whey, without the necessity of purifying the glycoprotein by a chromatography step. A simple and effective viral inactivation step was also considered in the proposed process, which has improved the vaccine biosafety. This is a significant advantage toward the commercial introduction of the new vaccine due to the potential reduction in antigen production costs and simplicity of the separation and formulation processes.
Go to:MATERIALS AND METHODS
E2-CSFV antigen production.
The E2 antigen was produced in the mammary glands of goats adenovirally transduced with Ad-E2 as previously described (15). The studies involving experimentation with goats were in accordance with guidelines and recommendations from the Guide for the Care and Use of Laboratory Animals (current edition) and policies from the Chilean Biosafety Manual from Fondecyt-Conicyt. The experimental protocols were drafted by the authors and approved by the Ethics Committee of the Universidad de Concepción, Chile. In all cases, supervision of veterinary authorities from the School of Biological Sciences, Universidad de Concepción, Chile, was guaranteed.
Briefly, the adenoviral vector Ad-E2 contains the extracellular domain of E2-CSFV glycoprotein with a hexahistidine tag in the C′ terminus preceded by the tissue plasminogen signal peptide. The Ad-E2 vector was infused through the nipple channel at a concentration of 1 × 109 gene transfer units (GTU)/ml until the udder volume was replenished. Four female goats (Saanen), 1.5 years old and in the second month of lactation, were used in the experiment. Twenty-four hours after adenovirus instillation, udders were extensively milked to remove the infused solution. A further udder washing step at 24 h postinoculation, consisting of phosphate saline buffer instillation, was carried out prior to milk collection.
Milk sampling started 48 h after adenovirus infusion, and it was maintained for the subsequent 20 days. The E2 expression levels obtained in the samples of whey were quantified by enzyme-linked immunosorbent assay (ELISA) as described elsewhere (12).
Viral inactivation and milk processing.Swine Fever Vaccine For Pig
Fresh milk samples collected each day were first titrated for the presence of the E2 adenoviral vector. Titration experiments were conducted in three independent experiments as previously described (16), based on the infection capacity of viable vectors in the HEK-293 cell line, and expressed as gene transfer units (GTU). The photosensitizer agent methylene blue was then utilized at a concentration of 50 μM for inactivation assays, which were conducted with overnight stirring at 4°C.
Samples were subjected to white light irradiation for 1 h at a dose of 6,000 lx, using a slide projector with a 360-W Apollo Orizon EYB 71 lamp as light source. After treatment, the milk samples were retitrated to analyze the adenoviral vector reduction. In addition, the E2 antigen integrity was evaluated by Western blotting.
Afterward, the milk was processed by 4-fold serial dilutions in a milk-separating buffer (10 mM Tris-HCl, 10 mM CaCl2, pH 8.0) and chilled on ice for 30 min. The mix was separated by centrifugation at 10,000 rpm for 30 min at 4°C. The fatty layer was discarded, and the serum milk containing the E2 antigen was separated from the casein precipitate. Three filtration steps were carried out using 0.8- and 0.4-μm membranes (Sartorius, Germany) for goat whey clarification. The total amount of proteins in serum samples was determined by the bicinchoninic acid method (Pierce, USA).
The E2 antigen, goat whey derived without purification, was dialyzed against 20 volumes of phosphate buffer (10 mM NaH2PO4, pH 7.4) and was sterilized by filtration (0.2-μm filter; Sartorius, Germany). The oil-based adjuvant Montanide 888 (SEPPIC, France) was mixed in sterile mineral oil (Sigma, USA) at a ratio of 1:9. A water-in-oil emulsion was produced in and Ultra-Turrax T25 basic homogenizer (IKA Works Inc., USA) at a proportion of 40% oil phase and 60% aqueous phase.
Starting from goat milk samples containing E2 at a concentration of 1 g per liter, different dilutions for immunizations were considered. Serum samples clarified as described above were diluted to contain 100, 50, and 25 μg of E2 in 2-ml formulation doses. The preparations were stored in nonpyrogenic 50-ml centrifuge tubes (Corning, USA) with 7 doses (14 ml) each. As a placebo, formulation samples of goat whey diluted in phosphate buffer, without the E2 antigen, were emulsified and stored under the same conditions.
Swine immunization trial and challenge.
The experiments with pigs were conducted in accordance with guidelines and recommendations from the Guide for the Care and Use of Laboratory Animals (current edition) and policies from the Cuban Society of Laboratory Animal Science (SCCAL). The experimental protocols were drafted by the authors and approved by the Animal Welfare Commission of the Center for Genetic Engineering and Biotechnology. In all cases, supervision of veterinary authorities from the Institute for Veterinary Medicine, Havana, Cuba, was guaranteed. Pigs were housed in individual rooms, and appropriate feeding, water supply, and health monitoring were permanently provided. Animals that were euthanized were humanly handled.
Crossbred Dubroc/Yorkshire swine 6 weeks old, weighing about 20 kg, serologically negative for CSFV, and belonging to a nonvaccinated and CSF-free herd were used in the experiment. Thirty animals were divided into five groups. All groups, of six animals each, were housed in separate experimental rooms and were handled according to international guidelines for experimentation with animals.
The groups were immunized with formulations of clarified whey containing E2 antigen at 100 μg (group A), at 50 μg (group B), and at 25 μg (group C). As a positive control, animals were injected with 25 μg of E2 purified from whey as described previously (15). Animals from the placebo group were immunized with goat whey in Montanide 888 at a dilution equivalent to that for group A.
Animals were vaccinated 3 weeks apart and challenged 1 week after the last immunization. Challenges were conducted by intramuscular injection of 105 50% pig lethal doses (PLD50) of homologous CSFV Margarita strain. All animals were euthanized at 2 weeks postchallenge (p.c.).
Reactogenicity, clinical study, and detection of CSFV-neutralizing antibodies.
Ten days after immunizations, local reactogenicity associated with the inoculation site, rectal temperature, and food intake variability were closely monitored. Then, after challenge, parameters such as CSF clinical signs, depression, fever, and food intake variability were scored daily. Serum samples for neutralizing peroxidase-linked assay (NPLA) were taken at day 0 before the first immunization and each week until challenge. Samples for NPLA were also taken weekly after challenge and at the time of euthanasia.
After sacrifice, animals were subjected to an exhaustive necropsy in which the presence of pathological lesions in different organs and tissues was evaluated. Examination of gross pathological lesions on spleen, kidneys, tonsils, small intestine, and brain was conducted postmortem in all animals. For observation of lymphocyte perivascular infiltration, brain samples were fixed in 10% formalin, embedded in paraffin, cut, and stained with hematoxylin-eosin by standard procedures.
Heparinized blood samples were collected on days 0, 2, 4, 6, 8, 10, 12, and 14 p.c. for viral isolation assay. Each sample was inoculated in 8 wells from 96-well microplates (Costar, USA) containing monolayers of PK-15 cells growing in Dulbecco modified Eagle medium (DMEM) and 5% fetal bovine serum (free of pestivirus and pestivirus antibodies). After 1 h of incubation at 37°C in 5% CO2, the monolayers were washed twice, the medium was replaced, and plates were incubated for 3 days. Plates were washed twice with phosphate-buffered saline (PBS), fixed, and stored at 4°C until the immunoperoxidase monolayer assay.
Immunodetection of viral antigen was performed with the 1G6 anti-E2-CSFV monoclonal antibody followed by a goat anti-mouse–horseradish peroxidase-conjugated IgG (Sigma, USA) as the secondary antibody. Those wells containing at least one spot were considered positive. The viremia level was expressed on a scale from 1 to 8 according to the number of positive wells, regardless of the number of spots per well.
article source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248785/