Porcine Circovirus Antigens in PK-15 Cell Line
`(ATCC CCL-33) and Evidence of Antibodies to
`Circovirus in Canadian Pigs
`
`Gilles C. Dulac and Ahmad Afshar
`
`ABSTRACT
`
`Permanent infection of a PK-15
`(ATCC CCL-33) cell
`line by the
`porcine circovirus was demonstrated.
`This virus appears to be common in
`the Canadian swine population as
`indicated by the presence of antibodies
`detected by indirect immunofluores-
`cence or immunoperoxidase tests in
`the serum of culled sows and sows in
`commercial herds. One source of the
`PK-15 cell line was found to be free of
`circovirus antigen.
`
`RESUME
`
`Les auteurs ont demontre la pre-
`sence d'une infection permanente de la
`lignee cellulaire PK-15 (ATCC-CCL-
`33) avec le circovirus porcin. Ce virus
`semble repandu dans la population
`porcine canadienne si l'on en juge par
`les anticorps decelis par les epreuves
`d'immunofluorescence et d'immuno-
`peroxydase indirectes chez une popu-
`lation de truies envoyees a l'abattoir.
`Un lot de cellules PK-15, exemptes de
`circovirus, a ete identifie.
`
`INTRODUCTION
`
`Porcine circovirus (PCV) is a
`nonenveloped virus of 17 ± 3 nm in
`diameter containing covalently closed,
`circular, single stranded DNA (1). It
`was originally detected as a noncyto-
`pathic contaminant of PK-15 cell lines
`(2). In slaughter pigs, in two districts
`of the Federal Republic of Germany,
`where the virus was demonstrated,
`antibodies were found in 77-95% of
`
`the sera tested (3). However, PCV did
`not cause clinical disease in one day
`old or in nine month old pigs (3).
`The purpose of this paper is to
`record the presence of PCV antigens in
`a PK-15 subline and to report the
`occurrence of a PCV antigen-free PK-
`15 subline. Also, the presence of PCV
`antibodies is documented in conven-
`tionally raised swine in Canada. The
`inconvenience of using a PCV antigen-
`carrier PK-15 subline is discussed in
`regard to applying immunofluores-
`cence and immunoperoxidase tests in
`routine diagnosis.
`
`MATERIALS AND METHODS
`
`CELL CULTURE
`The 132nd subculture of the PK-15
`cell subline (CCL-33), found to
`contain PCV antigen, was obtained
`from the American Type Culture
`Collection (ATCC) in 1978. The cells
`were subcultured twice weekly and
`seeded at a concentration of 67,000
`cells/cm2 and grown in plastic flasks
`Ruther-
`(Falcon-Becton-Dickinson,
`ford, New Jersey) with minimum
`Earle's
`salts
`essential medium in
`(MEM-E), supplemented with 10%
`fetal bovine serum (FBS), free of
`bovine virus diarrhea virus, 200 mM
`glutamine, 10 mM nonessential amino
`acids, 110 mg/ L Na pyruvate, 100
`units/ mL of penicillin and 100 jig/ mL
`of streptomycin.
`The PCV-free PK-15 subline and
`the swine testicle (ST) subline were
`obtained through the courtesy of Dr.
`Gene Erickson, National Veterinary
`Services Laboratory (NVSL), Ames,
`Iowa. These latter two lines were
`
`subcultured as described for the
`contaminated PK-15 subline.
`
`REFERENCE ANTISERA
`A rabbit circovirus antiserum was
`graciously provided by Dr. Ilse
`Tischer, Robert Koch Institute,
`Berlin, Federal Republic of Germany.
`The porcine antiparvovirus serum was
`prepared in our laboratory as des-
`cribed previously (4).
`
`TEST SERA
`Three different groups of sera were
`used to verify the presence of PCV
`antibodies in Canadian swine. Group I
`consisted of 18 samples which were
`submitted from a commercial swine
`herd by Dr. Susy Carman, Veterinary
`Laboratory Services, Guelph, On-
`tario, for exclusion of antibodies to
`hog cholera/ African swine fever and
`Aujeszky's disease. Group II was
`made up of 120 sera from culled sows
`at slaughterhouses for a
`collected
`Canadian pseudorabies survey, and
`group III comprised 27 sera from the
`Specific Pathogen Free
`Institute's
`(SPF) swine herd.
`
`INDIRECT IMMUNOFLUORESCENCE
`As substrate for immunofluores-
`cence, 50,000 cells/cm2 were seeded
`onto Lab Tek (Miles Laboratory,
`Naperville, Illinois) slides and incu-
`bated at 370C in a humid atmosphere
`of 95% air and 5% CO2. After 24 h, the
`cells were fixed in acetone at 40C and
`stained. The rabbit circovirus anti-
`serum was used as primary reagent for
`identification of PCV antigens in the
`PK-15 cells. It was diluted in twofold
`steps from 1:10 to 1:320 and allowed to
`react for 30 min at 370 C. The primary
`
`Agriculture Canada, Animal Diseases Research Institute, NEPEAN, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9.
`Submitted December 9, 1988.
`
`Can J Vet Res 1989; 53: 431-433
`
`431
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`serum was removed by washing three
`times (5 min each) in 0.01 M phos-
`phate buffered saline (PBS), contain-
`ing 0.05% (v/ v) Tween 20 and adjusted
`to pH 7.6. The cells were then covered
`with a commercial goat antirabbit
`(Cappel Laboratory, Technology
`Court, Malvern, Pennsylvania) affin-
`ity purified antiserum conjugated to
`fluorescein isothiocyanate (FITC), for
`an additional 30 min. The conjugate
`was washed as above and the slides
`were mounted with a 90% (v/v)
`glycerine and 10% (v/v) 0.1 M Tris
`HCI buffer, pH 8.6. The Tris buffer
`contained 5% (w/v) n-propyl gallate
`as an antioxidant which reduced the
`quenching of fluorescence under
`ultraviolet light (5). The specimens
`were examined with a Zeiss fluores-
`cent microscope, Universal Model,
`illuminated with a HBO-50 mercury
`burner.
`Cells permanently infected with the
`circovirus were used to detect anti-
`bodies to circovirus in swine sera. The
`cells, grown on Lab Tek slides and
`otherwise processed as described
`above were first treated for 10 min
`with a solution of 10% (v/v) normal
`rabbit serum (NRS) in PBS. This
`preliminary step reduced the subse-
`quent nonspecific background stain-
`ing with the rabbit antiswine IgG
`conjugate. Secondly, the solution of
`NRS was discarded and without
`washing, the cells were covered with a
`1:10 dilution of swine serum in PBS.
`Thirdly, the cells were flooded with a
`commercial rabbit antiswine FITC
`conjugate (Cappel Laboratory) for an
`additional 30 min and then treated,
`mounted and examined as
`stated
`previously. Porcine circovirus-free
`PK-15 cells were used as control.
`
`INDIRECT IMMUNOPEROXIDASE
`A modification of previously des-
`cribed immunoperoxidase assay
`procedures (6,7,8) was used as follows.
`Freshly trypsinized PK-15 cells,
`suspended in MEM(E) with 10% FBS
`and 50 ,.g gentamycin/mL were
`dispensed in 96 wells of tissue culture
`microtiter plates (Falcon). Each well
`was seeded with approximately 50,000
`cells. The plates were incubated at
`37°C in a humidified atmosphere of
`5% CO2 and 95% air for 48 h by which
`time a cell monolayer was formed. The
`microtiter plates were voided of their
`
`432
`
`Fig. 1. Monolayer of PK-15 cells contaminated
`with porcine circovirus stained by indirect
`immunofluorescence. (X160). Note the few
`individual fluorescing cells among the majority
`of nonreacting cells.
`
`Fig. 2. Monolayer as in Fig. 1, stained by
`indirect immunoperoxidase. (X160). Note the
`five individually stained cells among the
`majority of unstained cells.
`
`inversion. The
`medium by gentle
`monolayers were rinsed briefly with
`warm PBS, drained and fixed with
`20% acetone, in PBS containing 0.02%
`bovine serum albumin (BSA), for 10
`min at room temperature (RT). The
`monolayers were dried under a bench
`lamp (300C) for 3 h and were used on
`the same day or stored in sealed plastic
`bags at -70° C for subsequent tests.
`Prior to their use, the stored plates
`were warmed to RT and were rinsed
`with PBS. Swine field sera were tested
`at a 1:10 dilution in PBS containing
`0.05% (v/v) Tween 20 and 2.95%
`(w/v) NaCl (PBSTN). The wells were
`thoroughly drained of serum and
`rinsed with PBS containing 0.05%
`Tween 20 (PBST), three times for 2
`min each at RT. The wells were filled
`with 50,iL of a 1:1000 dilution of
`peroxidase (HRP)-
`horseradish
`labelled rabbit antiswine IgG (heavy
`and light chains) (Cappel) in PBSTN.
`After 15 min incubation at 30°C, the
`wells were again emptied and rinsed in
`PBST, then filled with 50 ,iL of sub-
`solution, which was slowly
`strate
`added to 50 mL of 0.05 M sodium
`acetate buffer, pH 5.0 and 0.2 mL of
`3% (v/v) H202. The substrate solution
`was freshly prepared by dissolving
`
`10 mg of 3-amino-9-ethylcarbazol
`3.0 mL of
`N,N-
`in
`(AEC)
`dimethylformamide (Aldrich Chemi-
`cal Co., Milwaukee, Wisconsin). The
`enzymatic reaction was stopped by
`emptying the wells and rinsing them
`with PBS and finally with tap water.
`Readings were done with an inverted
`light microscope. Serum samples were
`considered negative if no foci of brown
`colored infected cells were observed.
`
`RESULTS
`
`staining was
`Intense fluorescent
`observed in less than 1% of the cells
`treated with the rabbit
`circovirus
`antiserum (Fig. 1). Staining was not
`detected in cells treated with porcine
`parvovirus antiserum or normal
`rabbit and swine serum. The PK-15
`and ST cell lines obtained from Dr.
`Erickson were negative when treated
`similarly with the rabbit circovirus
`antiserum. Immunoperoxidase stain-
`ing revealed a similar number of single
`cells showing dark brown pigments
`indicative of specific staining (Fig. 2).
`The prevalence of PCV antibodies
`in the swine sera tested was as follows.
`Ten out of the 18 sera (55%) from
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`group I had antibodies detectable by
`indirect immunofluorescence. Thirty-
`two sera of the 120 from group II
`(26%) were positive by immunofluo-
`rescence. The immunoperoxidase test
`detected antibodies in five sera which
`had been classified negative by
`immunofluorescence. Sera of the 27
`adult breeding swine of the SPF herd
`(Group III) at ADRI were negative by
`indirect immunofluorescence.
`
`DISCUSSION
`
`The presence of circovirus antigens
`was first suspected at the 142nd
`subculture ofthe PK-1 5 cells, nine years
`after they were received from ATCC.
`Retrospective tests have shown, how-
`that circovirus antigens were
`ever,
`present at the 134th subculture, i.e. two
`subcultures from the original lot from
`ATCC. It is believed, therefore, that the
`PCV antigens were present in the cells
`Similar
`when they were received.
`conclusions were reached in Germany
`by Tischer (2) on two different sub-
`clones of PK-15 cells. The origin of the
`PCV contaminant in the PK-15 cell line
`is not known. The virus may have been
`present in the original swine tissue.
`Alternatively, the PCV could have been
`introduced into the PK-15 line by
`contaminated serum or other additives.
`The fact that the PK-15 (NVSL) subline
`remained free of PCV suggests that the
`bovine and swine sera, used in the
`growth medium, are an unlikely source
`of contamination.
`The presence of circovirus contami-
`nation was not suspected during the
`nine years that the PK-15 cell line was
`used because the PCV did not cause any
`cytopathic effect in the infected cell line.
`Similarly, it was not detected with any
`ofthe control sera used over the years in
`immunofluorescence tests because all
`these sera were prepared in pigs from
`the Institute's SPF herd which was
`negative for PCV. The PCV was
`discovered only when field sera were
`tested for antibodies to other viral
`agents. Then it was found that
`approximately half of the field sera
`reacted with the PCV antigen in the
`cells infected with hog cholera and
`Aujeszky's disease viruses as well as the
`"noninfected cells".
`The type of immunofluorescence or
`cells
`in
`immunoperoxidase staining
`
`infected with circovirus is fairly typical
`and an experienced microscopist is
`differentiate a circovirus
`likely to
`contamination from infection caused
`by hog cholera or Aujeszky's disease
`virus. On morphology alone, PCV
`antigens may be difficult to differen-
`tiate from the antigens appearing in a
`parvovirus infection, unless differen-
`tiating tests using monospecific antisera
`are used.
`studies
`Although no quantitative
`were made, the presence of PCV
`antigens did not appear to have
`interfered with the production of
`viruses such as hog cholera and
`did,
`It
`Aujeszky's disease viruses.
`undoubtedly, contaminate virus stocks
`prepared in this PK-15 subline. This
`contaminating antigen may have
`affected the quality of diagnostic
`reagents intended to be monospecific.
`For that reason alone, the contami-
`nated cell line should be replaced by a
`noncontaminated line or cloned to
`eliminate the circovirus contaminant.
`Elimination of the circovirus contami-
`nant by cloning was accomplished by
`Tischer (personal communication).
`The small proportion of cells express-
`ing the circovirus antigens undoubtedly
`facilitated the elimination of the PCV
`infection by cloning.
`Under the culture conditions des-
`cribed in this paper, 15 to 20 cells
`harboring PCV antigens were visible in
`most microscopic fields seen with a lOx
`objective. This number of antigen-
`containing cells was adequate for
`indirect immunofluorescence or immu-
`noperoxidase assay and remained
`constant between the 134th to the
`142nd subcultures.
`The indirect immunofluorescence
`and the immunoperoxidase tests were
`adequate to detect the contaminant in
`the cell line. However, for antibody
`detection, immunoperoxidase was
`slightly more sensitive than immuno-
`fluorescence. The immunoperoxidase
`test also had the advantage of being
`plates
`in microtiter plastic
`feasible
`rather than Lab Tek slides. This
`allowed the processing of a greater
`number of specimens at reduced cost.
`The limited serological tests per-
`formed with sera collected from
`conventionally raised swine indicated
`that the PCV infection may be common
`in Canada. However, no links have yet
`been made between PCV and disease in
`
`swine. Experimentally, PCV was not
`pathogenic for one day and nine month
`old pigs (3). Because multiplication of
`PCV depends on the cellular DNA
`(10), pathological
`synthesis in cells
`changes may occur in actively growing
`organs such as fetal tissues. It may be
`appropriate to examine tissues of
`aborted fetuses for the presence of PCV
`antigen as well as parvovirus antigens
`(9).
`
`ACKNOWLEDGMENTS
`
`We thank Mr. R. McLaurin and Mr.
`Ian Cheney for their technical assist-
`ance in immunofluorescence and
`immunoperoxidase tests, respectively
`and Mrs. Diane Mercier for maintain-
`ing the cell lines.
`
`REFERENCES
`
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