WO1999002184A1 - Agent pathogene du syndrome d'avortement et de mortalite chez les truies (sams), compositions de vaccins, anticorps et procede correspondants - Google Patents

Agent pathogene du syndrome d'avortement et de mortalite chez les truies (sams), compositions de vaccins, anticorps et procede correspondants Download PDF

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WO1999002184A1
WO1999002184A1 PCT/US1998/014351 US9814351W WO9902184A1 WO 1999002184 A1 WO1999002184 A1 WO 1999002184A1 US 9814351 W US9814351 W US 9814351W WO 9902184 A1 WO9902184 A1 WO 9902184A1
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virus
causative agent
mortality syndrome
sow abortion
sams
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PCT/US1998/014351
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Han Soo Joo
Jinho Shin
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Regents Of The University Of Minnesota
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates to the isolation, characterization and utilization of the causative agent of Sow Abortion and Mortality Syndrome (SAMS).
  • SAMS Sow Abortion and Mortality Syndrome
  • PRRS porcine reproductive and respiratory syndrome
  • SAMS has been defined as acute onset, high mortality (>5%) in sows, and a high abortion (> 10%) among sows of all gestational stages and parities. These problems were observed in at least 138 herds in 13 Midwestern states of the United States and in Canada. Zimmerman. Major outbreaks of SAMS occurred on many farms in southern Iowa during the last quarter of 1996. While PRRS virus infection has been routinely diagnosed in samples from affected farms, the etiology of SAMS is not fully understood. Since many of the outbreaks have occurred in farms where PRRS virus vaccine was used, a PRRS virus variant and/or a combination of different pathogens has been suggested as the cause. A previously unidentified agent has not been ruled out for the etiology.
  • the present disclosure is directed to isolation and characterization of a causative agent of Sow Abortion and Mortality Syndrome (SAMS), a method for isolating the agent, a method for testing antibody response to the agent, an antibody reactive with the agent, and a composition for immunizing or vaccinating an animal against the agent.
  • SAMS Sow Abortion and Mortality Syndrome
  • Antibodies raised against SAMS are useful in the diagnosis, prevention, and treatment of SAMS.
  • An antigenic composition comprising all or a portion of the SAMS isolate is useful for immunizing sows to induce anti- SAMS antibodies in the immunized animals and to prevent or treat SAMS.
  • FIG. 1 illustrates the percent of aborted gilts and sows between January 1994 and December 1997 in a Minnesota farm affected with SAMS
  • FIG. 2 illustrates the farrowing rates, returned to service rates and death rates of breeding animals between January 1994 and December 1997 in the Minnesota farm of FIG. 1;
  • FIG. 3 is an electron micrograph from a section of swine testicle cells postinoculation with SAMS isolate
  • FIG. 4 is a cDNA sequence from the C8 fragment of a SAMS isolate.
  • FIG. 5 is a Western irnmunoblot of VR 2580 proteins using pig sera obtained before and after injection with SAMS agent vaccines.
  • the present invention is directed to an isolated causative agent of Sow Abortion and Mortality Syndrome (SAMS).
  • SAMS Sow Abortion and Mortality Syndrome
  • the isolated agent can be included in an immunizing composition for simulating antibody production in an animal against the agent, a vaccine composition for preventing or treating SAMS, and antibodies raised against the SAMS isolate can be used for preventing, treating, detecting or identifying the presence of the agent in a tissue sample.
  • the term "isolate,” when referring to the SAMS causative agent means that the agent is separated from an affected animal in a form suitable for identification or for use in an immunizing or therapeutic composition, with or without further purification.
  • the isolated causative agent of SAMS has been identified, characterized, and deposited with the American Type Culture Collection on July 29, 1997 as ATCC accession no. VR 2580.
  • an "immunizing composition” is a composition which when administered to an animal stimulates production of antibodies that react with the causative agent of SAMS as well as with the antigenic component of the composition.
  • An immunizing composition of the invention can also be used as a vaccine for treatment or prevention of SAMS.
  • the SAMS isolate may or may not be purified prior to use.
  • the isolated agent can be inactivated using known methods including, for example, heat, ether, formaline, ⁇ -propyl lactone, or attenuated by ultra-violet light, serial passaging, etc. to render the agent non- pathogenic.
  • the inactivated or attenuated SAMS agent can then be combined with a suitable physiological carrier, for example, physiological saline, ringers, lactated ringers phosphate buffered saline, etc. to form a composition for administration to an animal.
  • Immune stimulants or adjuvants can also be added to the composition to enhance the immune response.
  • Suitable adjuvants include, for example, emulsif ⁇ ers, Quil A, mineral oil, aluminum hydroxide, aluminum phosphate, etc.
  • a dose of about 1-10 ml, preferably about 2-5 ml of a composition including between 10 4'5 — 10 7"5 TCID 50 can be administered to provide an immunizing or therapeutic effect.
  • the composition can be administered in two doses at least two weeks apart. Thereafter at least one annual dose can be administered .
  • the vaccine composition can be administered through known routes including oral, intranasal, subcutaneous, intramuscular, intradermal, etc.
  • Detection of the SAMS causative agent can be performed by virus isolation on swine testicle (ST), Crandell feline kidney (CRFK), or other cells disclosed herein, and reacting the infected cells with antibodies directed against the SAMS isolate.
  • Bound antigen antibody complexes can be analyzed qualitatively or quantitatively by known methods, including direct or indirect staining techniques using, for example, chromatic or fluorescent compounds.
  • Antibodies against the SAMS isolate can be polyclonal or monoclonal antibodies.
  • Polyclonal antibodies can be prepared by inoculating an animal with an immunizing composition including the SAMS isolate and harvesting the antibodies produced by the immunized animal. Known methods can be used to prepare monoclonal antibodies.
  • reproductive performance on a farm with SAMS were analyzed to compare the data reported for farms with typical PRRS infections.
  • Three major differences in the reproductive abnormalities were identified; sow abortions regardless gestational ages, high percents of return to service, and no increases in the numbers of stillborn and mummified pigs. Most interestingly, there were no major increases in the numbers of stillborn and mummified pigs. Following the initial severe abortion problem, there were three minor but repeated incidents of abortion during a 2-year period. Reduced severity indicated that an immunity to the agent may have developed in the herd.
  • 21 of 53 abortions 39.6% were recorded in gilts. This could further support an involvement of immunity, since the farm regularly introduced new gilts from a breeding company.
  • the isolates appear to have a wide range of in vitro cell susceptibility, and are sensitive to ether or heat treatment.
  • the characteristics indicate that the agent is an enveloped virus containing DNA.
  • the infectivity of the SAMS isolates was not reduced in the presence of antisera to common swine viruses. This indicates that SAMS agent is not related to a common swine virus.
  • the electron microscopic observation indicates a similar morphology and size to a mycoplasma virus Gourlay RN et al., "Further studies on the morphology and composition of Mycoplasmatales virus- laidlawii 2," J. Gen. Virol. 18:127- 133 (1973).
  • the virus ⁇ like particles were located within a structure which could be a mycoplasma organism.
  • enhanced growth characteristics in the presence of M. hyopneumoniae and 92%o nucleotide sequence similarity to M. hyopneumoniae 23 S rRNA gene were observed. These results suggest that VR 2580 may be a M. hyopneumoniae associated virus.
  • Serological methods including ELISA and western immunoblotting appear to be specific to VR 2580 proteins for the sera from vaccinated pigs.
  • the clinical signs and reproductive parameters of SAMS are different from those of typical PRRS, and the agent appears to be causing transplacental infection and pathogenicity in the fetuses.
  • the agent is a new and previously unidentified agent in swine.
  • a 1 ,200-sow farm in Minnesota experiencing severe abortions was the first case of SAMS investigated.
  • a PRRS modified live virus vaccine (ResPRRS, Boehringer Ingelheim Animal Health, Inc., St. Joseph, MO) was first used for all breeding stock on this farm. Production data between January 1994 and December 1997 recorded on the PigCHAMP computerized record system were used to analyze reproductive abnormalities.
  • Four units, approximately 4,000 sows each, of a large corporate swine farm in Iowa which had a similar abortion problem were also investigated between April and July of 1997.
  • the Iowa farm also used a postfarrowing PRRS vaccination program in affected units.
  • FIGs. 1 and 2 Abortion, farrowing, return to service and death rates of bred females in the Minnesota farm between January 1994 and December 1997 are shown in FIGs. 1 and 2.
  • acute clinical signs including 130 abortions were first observed.
  • Affected gilts and sows had inappetence and depression for 1-3 days before abortion.
  • Percent of bred females returning to service in October, November and December 1995 were 8.6%, 49.7% and 43.6%, respectively.
  • Farrowing rates in the last 3 months of 1995 were 80.2%, 46.3% and 56.6%, respectively (Fig. 2).
  • Death rates of gilts and sows during those 3 months were 12.6, 8.4 and 2.0%, respectively.
  • MA-104 cell line MA-104 cell line
  • Kim HS et al. "Enhanced replication of PRRS virus in a homogeneous subpopulation of MA-104 cell line," Arch. Virol.133 :477-483 (1993), was used for PRRS virus isolation.
  • swine testicle (ST), Crandell feline kidney (CRFK), pig kidney (PK-15), rabbit kidney (RK-13) and baby hamster kidney (BHK-21) cell lines were used. These cell lines were initially obtained from the cytogenetic laboratory, College of Veterinary Medicine, University of Minnesota.
  • CPE cytopathic effect
  • Tissue samples were prepared by homogenizing and diluting 5-10 times in MEM. After centrifugation at 3,000 rpm for 20 min, each supernatant was inoculated onto 1-day old ST cell monolayers, adsorbed at 37° C for 1 hr, and medium was added. Serum samples were inoculated similarly to ST cell monolayers. The monolayers were examined daily for CPE for 5-7 days. Samples with negative CPE were passed once on fresh ST cell monolayers and examined for another 5-7 days. After the second passage, samples with no CPE were considered as isolation negative.
  • a cytopathic agent was first isolated from 7 tissue pools and 3 sera of 10 weak-born pigs from the Minnesota farm (Table 2). The isolation was made using ST cell monolayers. Infected cell monolayers showed circular empty areas with swollen cells around the edge, and then the monolayers became lytic after 5-7 days of infection. The isolates could readily be passed on ST cell cultures up to 12 times.
  • a similar cytopathic agent was isolated from serum samples collected from 9 10-week old nursery pigs and 10 aborted sows from 4 different units of the Iowa farm (Table 2). The cytopathic isolates were tentatively designated as SAMS agent. Isolate MN-3 was deposited at the American Type Culture Collection 12301 Parklawn Dr., Rockville, MD 20852 and designated as ATCC VR 2580.
  • Nucleic acid type was examined by the inhibitory effect of 5-iodo-2'-deoxyuridine (IUDR) on replication of DNA viruses, Yoon IJ et al., "Isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome," J. Vet. Diagn. Invest. 4:139-143 (1992).
  • Infected CRFK cell monolayers were maintained for 5 days with medium containing 100 ⁇ g/ml of IUDR.
  • Infectivity titers of the culture fluids with RJDR were determined using 96-well microplates, and the results were compared to those without IUDR.
  • Pseudorabies virus (PRV - Shope strain) was included in the test as a reference DNA virus.
  • PRV - Shope strain Pseudorabies virus
  • ether sensitivity equal volumes of each isolate and anesthetic grade ether were mixed vigorously and left at 4° C for 1 hr. Id. After centrifugation at 5,000 rpm for 15-20 rnin, the aqueous layer was removed, and the remaining samples were titrated for infectivity. Heat stability was examined by heating aliquots of the isolates at 56° C for 10, 20 or 30 rnin, and infectivity titers were measured.
  • Infectivity titers of the clarified supernatant for each isolate were examined following filtration through 0.45, 0.20, 0.10 or 0.05 ⁇ m membrane filters (Millipore Products Division, Belford, MA). Red blood cells (RBC) of guinea pig, mouse, sheep and chicken were collected, and hemagglutination ability of the isolates was tested at room temperature using routine methods in U-bottom 96-well microplates.
  • RBC Red blood cells
  • Serologic characterization of the isolates was carried out by serum neutralization (SN) test.
  • SAMS isolates were incubated for 1 hr at 37° C with each of antisera to different swine viruses, and infectivity titers were measured.
  • the antisera used (Table 5) had been produced in our laboratory by experimental inoculation of pigs or rabbits with PRRS virus, PRV, encephalomyocarditis virus
  • Infectivity titers of SAMS agent were decreased or not detected when the isolates or PRV were cultured in the medium containing IUDR, or treated with ether or heating at 56° C for 10, 20 or 30 rnin (Table 3). Infectivity titers following filtration through filters of different pore sizes are shown in Table 4. Infectivity was not detected or markedly decreased following filtration through 0.1 or 0.05 ⁇ m filters.
  • the VR 2580 (10 45 TCID 5u 0.1 ml) was inoculated onto each cell monolayer and incubated for 7 days with examination of a CPE. The inoculated cultures were frozen and thawed twice and passed once on each cell line to confirm replication.
  • Mycoplasma hyopneumoniae J strain, ATCC, Rockville, MD
  • BM-cyclin mycoplasma growth inhibitor
  • Mycoplasma hyopneumoniae was cultured using Friis medium.
  • MARC-145 cells in RPMI-1640 medium containing Mycoplasma hyopneumoniae or BM-cyclin were inoculated with VR 2580, and infectivity titers were measured after 7 days postinoculation.
  • CPE was evident on CRFK, MARC-145 cell lines and primary cultures of PAM, swine lung cells and BM cells.
  • Table 6 shows an association of VR 2580 growth in the presence of M. hyopneumoniae or BM-cyclin in the medium. Replication of VR 2580 was enhanced in the medium containing M. hyopneumoniae, while the growth was inhibited in the medium containing BM-cyclin.
  • Infected ST cell monolayers with approximately 50% CPE were scraped, and the cells were pelleted by centrifugation. These cells were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, washed and postfixed in 1% osmium in 0.1 M phosphate buffered solution. Pellets were embedded, cut on an ultramicrotome, and examined under an electron microscope. The longest and shortest diameters of 30 particles were measured from an enlargement, and the average diameter was calculated.
  • the infected cells contained particles with virus-like morphology under an electron microscope (Fig. 3).
  • the particles were located within a structure (1.6 x 2.4 ⁇ m). The structure was smaller than size of the cells and appeared to have no membrane.
  • the virus-like particles were enveloped and mostly spherical with a mean diameter of about 95 run (range 72 - 114 nm). They were negatively stained but a densely stained spot was present in the center of the particles.
  • Example 5 Differential display (DD) RT-PCR, PCR cloning and sequence similarity search
  • a sequential 2-step DD approach was employed. First, a total of 80 primer pairs were tested with RNA samples from VR 2580 infected and control PAM, and 15 primer pairs produced discrete signals from infected PAM were selected. Second, the selected 15 primer pairs were tested with the cellular RNA from VR 2580 infected PAM, RNA from VR 2580 infected PAM culture supernatants, and cellular RNA from VR 2580 infected CRFK cells.
  • RNA of the cytopathic agent is present in PAM culture supernatants and CRFK cells as well as in infected PAM, one specific primer pair should generate the equal band size from all of the 3 different samples.
  • Three different amplified DNA fragments (G8, Gl 8, and C8) from RNA of VR 2580 were obtained. These fragments were subsequently isolated, cloned and sequenced. Nucleotide sequence data obtained from the G8, G18, and C8 fragments displaced approximately 92% sequence similar to Mycoplasma hyopneumoniae 23 S rRNA gene, extending the position from bp 661 to bp 1439 (779-bp in length).
  • nucleotide sequences from C8 fragment showed 98.8%) sequence similar to the 5' UTR region of PTI-1 alpha gene (human prostatic cancer inducing oncogene), extending the position from 305 to bp 637 (333-bp in length).
  • the C8 fragment is shown in FIG. 4 as SEQ. ID. NO. 1.
  • This cDNA fragment, or regions thereof, can be used as a probe for detecting the presence of SAMS using known technology.
  • One-day old ST cell monolayer was inoculated with VR 2580, and 3-5 days later when about 50% CPE were evident, the monolayers were scraped into the medium.
  • a pool of the medium was frozen and thawed twice, centrifuged at 4,000 rpm for 20 min, and the supernatants were collected.
  • the suspension was made up to 70%) saturation (472 gm/liter) with ammonium sulphate in an ice bath with stirring. This was further stirred at 4° C for 2 hr, and then centrifuged at 10,000 rpm for 60 min at 4° C.
  • the resulting supernatant was discarded, and the precipitate was dissolved in a small volume of phosphate buffered saline (PBS, pH 7.2) and dialyzed using a dialysis membrane tubing (molecular weight (MW) cut-off 3,500) against 500 volumes of PBS with 2 changes overnight at 4° C.
  • the dialyzed precipitate (22 ml) was then layered over a two-step sucrose gradient consisting of 5 ml of 10% (w/w) sucrose layered over 5 ml of 30% sucrose in distilled water in polyallomer tube (25 x 89 mm).
  • Antibody response to SAMS isolates were tested by an enzyme linked immunosorbent assay (ELISA) and SN methods.
  • An ELISA antigen was prepared by concentration and purification as previously described. The enriched antigen was mixed with Triton X-100 at a final concentration of 0.25%, and solubilized by stirring for 3-5 hr at 4° C. The solubilized preparation was used as SAMS isolate ELISA antigen.
  • Antigen at an optimal dilution in 0.05 M carbonate buffer, pH 9.6 was added to a 96-well ELISA microplate (100 ⁇ l/well) (Immunolon I, flat bottomed; Dynatech Laboratories, Inc., Alexandria, VA). The plates were sealed and kept at 4° C until use.
  • Biotin labeled IgG fraction of goat anti-swine IgG at an optimal dilution in PBS was added to each well (50 ⁇ l/well), and allowed to react for 30 min at 37° C. After washing 3 times, peroxidase labelled streptoavidin (50 ⁇ l well; KPL, MD 1 :5,000 in PBS) was added and incubated for 30 min at 37° C. A freshly-prepared substrate solution (50 ⁇ l/well; 10 mg o-phenylenediamine, 50 ⁇ l of 30% hydrogen peroxide in 24 ml citrate- phosphate buffer, pH 5.0) was added to each well.
  • Optical density (OD) at 490 nm was measured by a micro ELISA auto-reader (Dynatech Laboratories, Inc., Alexandria, VA).
  • test sera were inactivated at 56° C 30 minutes and were serially diluted 2 fold in 96-well microplates.
  • the VR 2580 diluted to be 10-100 TCID 50 was added to each well.
  • CRFK cell suspension (1- 10 x 10 5 cells/ml) were added as an indicator cell. The results were read at 6-7 days after incubation when a complete CPE was evident in the wells with 10 or 100 TCID.
  • VR 2580 proteins along with molecular markers separated in the gels were electrophoretically transferred to 0.45 - ⁇ m nitrocellulose membrane (NCM) (Bio-Rad, Richmond, CA) using a mini-transblot electrophoretic transfer cell, Towbin H et al., "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications," Proc. Natl. Acad. Sci. U.S.A. 76:4350- 4354 (1979).
  • NCM nitrocellulose membrane
  • Transfer was carried out at 4° C for 60 min at 80 V and for 10 min at 100 V in transfer buffer (pH 8.3) consisting of 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol.
  • transfer buffer pH 8.3
  • the NCM was cut into 1-cm strips after transfer. The nonspecific binding sites on the strips were blocked for 2 hrs at 37° C with 3% BSA in Tris buffered saline (TBS; 20 mM Tris and 500 mM NaCl, pH 7.5).
  • Serum samples collected from pigs before and after inoculation with SAMS isolate vaccines were diluted 1 :50 in 1% BSA in TBS.
  • Sows B3 and A4 showed fetal retention problem. Sow B3 farrowed 2 liveborns and 1 stillborn. Seven additional stillboms were pulled out at least 12 hours after the initiation of the farrowing. The pulled out stillboms showed autolysis of the internal organs, and the deaths were judged to occur several days before farrowing. The sow farrowed 2 mummified fetuses 3 and 4 days post- farrowing, respectively. Sow A4 farrowed 2 liveborns. At least 6 hours later, 1 liveborn and 2 stillboms with advanced deaths were pulled out. The SAMS agent was recovered from 6 of 10 and 1 of 4 fetuses of Sows B3 and A4, respectively.
  • a SAMS agent vaccine composition was prepared using infected ST cell culture fluids or detergent cell extracts.
  • One-day old ST cell monolayer was inoculated with VR 2580, and 3-5 days later when about 50% CPE were evident, the flasks were frozen and thawed twice, centrifuged at 4,000 rpm for 20 min, and the supernatant was used.
  • a detergent extracted antigen was prepared as previously described in Joo HS et al., "Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum.," Am. J. Vet. Res. 45:2096-2098 (1984). Briefly, infected ST cell monolayers were scraped into the medium and sedimented by centrifugation.
  • Pelleted cells were suspended in a 0.05M tris aminomethane 0.025 M EDTA buffer containing 0.5% Triton X-100 at a volume of 4-8 times that of the packed cells. The mixture was stirred for 90 min at 4° C and was centrifuged at 10,000 x g for 1 hour. The extract (1 ml) was mixed with infected culture fluid (9 ml).
  • each antigen was mixed with water-in-oil adjuvant.
  • the adjuvant consisted of 3 components: a light mineral oil (Drakeol 6 VR, Penreco, Butler, PA) and 2 emulsifiers, Span 85 and Tween-85 (Sigma chemical, St. Louis, MO).
  • the emulsion was prepared by mixing the oil, Span 85, Tween-85 and antigen in an 18:1 :1 :20 ratio and sonicating the mixture for 2-4 minutes.
  • Ten 4-week old healthy pigs were purchased from a farm with no history of reproductive failure. They were housed in an isolation room, and pig numbers 1-4, and 5-8 were injected intramuscularly with 1 ml each of the culture fluid and detergent extracted vaccines, respectively. The remaining 2 pigs served as controls. A booster injection was made 2 weeks after the first injection. Weekly blood samples were obtained from each animal, and antibody response was measured by western immunoblot, ELISA and SN methods.
  • lane 1 illustrates molecular markers of 102, 78, 49.5, 34.2 and 28.3 kD.
  • Lane 2, 3, 4 and 5 are taken at 0, 2, 3, 4 and 5 weeks post vaccination of pig no. 1 and lanes 6, 7, 8 and 9 are taken at 0, 2, 3, 4 and 5 weeks post vaccination of pig no. 5.
  • At least 5 VR 2580 proteins with molecular masses of approximately 57, 79, 98, 105 and 117 kD were identified. Antibodies specific for the 57, 79 and 98 kD proteins were first detected 2 weeks after the first vaccination.
  • Antibodies specific for the 105 and 117 kD proteins were detected in 7 of 8 vaccinated pigs 3 weeks after the second vaccination. Antibody response to the 57 kD protein was the most obvious and first detected in 2 weeks after the first vaccination. Antibody responses of vaccinated pigs by ELISA and SN methods are summarized in Table 9. In ELISA, OD values of >60 were considered positive. The first positive OD readings were observed in pigs 2 weeks after the first vaccination, ant the highest OD readings were observed in pigs between 2 and 3 weeks after the second vaccination. In contrast, the SN antibody levels were low in the vaccinated pigs.
  • Antibodies prepared by immunizing an animal with an immunizing composition of the invention can be used in a kit for detecting or identifying SAMS.

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Abstract

Des signes cliniques graves du syndrome d'avortement et de mortalité des truies (SAMS) de cause inconnue ont été observés dans des fermes d'élevages porcins du Midwest des Etats-Unis. Un virus cytopathogène jusqu'à présent non identifié a été isolé à partir de 7 échantillons de tissus et de 3 échantillons de sérum de 10 porcelets différents nés dans un état faible dans une ferme et à partir de 19 sérums de 4 individus différents dans une autre ferme. La réplications des isolats a pu être considérablement réduite dans un milieu contenant un inhibiteur d'ADN et complètement inhibée après un traitement à l'éther ou après un traitement avec chauffage à 56 °C. Cette invention propose un antigène permettant de préparer un agent immunisant ou un vaccin contre ce syndrome et des anticorps permettant de détecter, de traiter ou de prévenir ce syndrome.
PCT/US1998/014351 1997-07-10 1998-07-10 Agent pathogene du syndrome d'avortement et de mortalite chez les truies (sams), compositions de vaccins, anticorps et procede correspondants WO1999002184A1 (fr)

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