Veterinary Microbiology, 13 (1987) 371--381 371 Elsevier Science Publishers B.V., A m s t e r d a m - Printed in The Netherlands
THE PROPAGATION OF FELINE PANLEUKOPENIA VIRUS IN MICRO- CARRIER CELL CULTURE AND USE OF THE INACTIVATED VIRUS IN THE PROTECTION OF MINK AGAINST VIRAL ENTERITIS
ESTEBAN RIVERA, KARLoAXEL KARLSSON and RUNE BERGMAN
The National Veterinary Institute, Division of Vaccine Research, Box 7073, S-750 07 Uppsala (Sweden)
(Accepted for publication 17 July 1986)
ABSTRACT
Rivera, E., Karlsson, K.-A. and Bergman, R., 1987. The propagation of feline panleu- kopenia virus in microcarrier cell culture and use of the inactivated virus in the pro- tection of mink against viral enteritis. Vet. Microbiol., 13: 371--381.
Using microcarrier cell culture for the production of virus antigen, a formalin-in- activated feline panleukopenia virus vaccine was evaluated for protection of mink against specific mink enteritis virus infection. The vaccine showed a good immunogenic effect in mink when used either alone or in combination with Clostridium botulinum type C-toxoid and/or Pseudomonas aeruginosa vaccine. A single vaccination induced persis- tent immune responses for periods of at least 1 year, as evaluated by ELISA and challenge tests. Neither immunological interference between vaccine constituents nor adverse reactions were observed.
INTRODUCTION
Mink enteritis virus (MEV) is a parvovirus which causes an acute disease of mink (Johnson et al., 1974). The virus is closely related antigenically to feline panleukopenia virus (FPLV) and to canine parvovirus (CPV), as demonstrated by conventional serological tests (Burger and Gotham, 1963), restriction enzyme analysis (McMaster, 1981) and use of mono- clonal antibodies (Parrish et al., 1982). Because of this close relationship, it has been possible to immunise mink effectively against specific MEV infection using FPLV vaccines (Wills, 1956) and conversely, cats against FPLV-infection with MEV vaccine (Burger and Gorham, 1963). Sub- sequent combinations of MEV vaccine with Clostrium botulinum type C-toxoid (Winans and Marty, 1968) and/or Pseudomonas aeruginosa vaccine (Elsheik, 1985) have been successfully tested without any adverse effect on the potency or safety of the vaccine. Although the efficacy of FPLV vaccines in protecting mink has been established (Gorham, 1965; Farell, 1972), no serological evaluations or studies on the duration of immunity
have previously been reported. The present paper describes the propaga- tion of FPLV in microcarrier culture using a feline cell line, and evalua- tion of the inactivated FPLV vaccine in protecting mink against virus ente- ritis infection.
MATERIALS AND METHODS
Virus strains
A feline panleukopenia virus (FPLV), isolated from tissues of naturally infected cats, was adapted and propagated in a permanent cat cell line. The virus was identified and characterised as FPLV by serological tests including serum neutralisation, immunofluorescence, haemagglutination inhibition, and ELISA as well as by buoyant density in CsC1 gradients and by electron microscopy. A parvovirus was isolated from mink during an acute outbreak of mink enteritis and its relationship to FPLV was con- firmed as described above. Stocks of both virus strains were prepared as described below, and held a t - 2 0 ° C.
Cell cultures
An embryonated feline lung (EFL) cell line from the National Veterinary Institute, Uppsala, Sweden, was utilised to propagate virus. Cells were grown in roller bottles (400 cm 2) using Eagle's minimum essential medium (MEM) supplemented with 2 nmol-L-glutamine, 8% fetal calf serum (FCS) and antibiotics.
Virus propagation in microcarrier cell culture
When cell monolayers in roller bottles were confluent, cells were tryp- sinized and resuspended in growth media at a concentration of 4--5 X 106 cells m1-1. For each litre of culture, 250 ml of this cell suspension, and 3 g microcarrier beads (Biopsilon-Nunc Denmark) were incubated in spinner flasks. The cells were first allowed to attach to the microcarriers for at least 18--20 h at 37°C with occasional stirring. Cultures were then virus-infected using 10 ml FPLV suspension at a titre of 5 X 104 TCIDs0 m1-1. After I h of virus absorption, 750 ml of extra medium was added. Spinner flasks were incubated at 37°C with continuous stirring at a speed just sufficient to keep all of the microcarriers in suspension. Virus pro- pagation was determined daily by haemagglutination (HA) of swine ery- throcytes at 4°C {Konishi et al., 1975).
Harvesting o f the virus
When cytopathic effects became evident and the HA titre of virus in supernatant fluids reached 1024--2048, the virus was harvested by allowing
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the microcarriers to sediment and removing the supernatant fluids: Cell' bound virus antigen was extracted by treating the microcarrier with 0.2 M glycine buffer (pH 9.0) as described by Hallauer and Kronauer (1965). After the addition of fresh medium, the cells were further cultured at 37°C with continuous stirring. Virus was harvested daffy as long as the HA titre in the supernatant fluids remained > 512. Both infected super- natant and cell-extract fluids were combined, then clarified by centrifuga- tion at 800 × g and stored at -20°C.
Vaccine preparation
FPLV yields were thawed and passed through a Gelman Acrodisc 45 filter, and the HA titre of the filtrate was determined. Formalin was then added to a final 0.2% v/v concentration, and the treated fluids were stirred continuously at 37°C for 5 days. Aliquots of this treated FPLV were then tested for any residual live virus by infection of EFL and checking these infected cells for active virus by immunofluorescent technique (see below) after four passages at intervals of 4 days.
Inactivated viral fluids were diluted in 0.01 M phosphate buffered saline (PBS), pH 7.2 to contain 16, 64, 256 and 1024 HAU in 1-ml aliquots, based on the pre-inactivation HA titres. These fluids were mixed with 5% v/v of Alhydrogel (3% AL (OH)a; Superfors A/S Denmark). Vaccines were also tested in combination with P. aeruginosa and/or Cl. botulinum toxoid. Each vaccine const i tuent was diluted so that the final dose was 1 ml in all cases.
Animals
Young mink, 8--10 weeks old, and free from detectable ant ibody to FPLV were used to test the potency and safety of the vaccines. Mink were divided into groups of five animals for each vaccine to be tested and were injected subcutaneously with 1 ml vaccine. Vaccinated and unvaccinated mink were housed together in isolation during the course of the experiment. Blood samples from both vaccinated and control animals were taken at various intervals for up to 1 year after vaccination. The fur of the vaccinated minks was frequently examined for colour changes at the vaccination site.
Challenge test and virus isolation
Following vaccination, groups of 4 mink receiving uni- or multivalent vaccines were challenged, together with 4 control mink, at intervals of 3 weeks, 6, 9 and 12 months post vaccination. Mink were challenged per os with 2 ml of MEV-faecal suspension containing approximately 60 000 HAU of virus, and the dosage was repeated the following day. Stool samples were collected daily for 7 days commencing 2 days after the second chal- lenge dose. Faeces were assayed as follows: samples were diluted 1:10
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in 0.15 M PBS (pH 6.8), with antibiotics and were allowed to stand for 30 min at room temperature. They were then centrifuged at 8.000 × g for 30 min, after which the supernatant fluid was vigorously shaken with 30% v/v chloroform for 2--3 min and centrifuged again as described above. This step removed lipids and clarified the faecal fluids. The presence of virus in the supernatant fluids was monitored by HA, ELISA and IF staining of EFL cells inoculated with faeces extract.
Haemagglutination test (HA) and haemagglutination inhibition test (HI)
The HA test was performed on U-type microplates at 4°C using a 0.5% swine red blood cell (RBC) suspension as described by Konishi et al. (1975). The HI test was performed according to Johnson {1967}, using V-type microplates and 16 HA units of FPLV or MEV. Before assay, sera were heat-inactivated at 56°C for 30--60 min and successively absorbed with 25% kaolin suspension (in PBS, pH 7.3) and 50% swine RBC suspension. After absorption, the final serum dilution was 1:10.
Immunofluorescent test (IF)
The indirect method was utilised both for identification of viral isolates from stool samples and for detecting residual live virus in vaccine batches. Rabbit anti-FPLV or MEV serum was prepared by repeated intramuscular injections of purified virus emulsified in Freund's complete adjuvant (Rivera and Sundquist, 1984). FITC¢onjugated swine anti-rabbit IgG serum was obtained commercially (Dakopatts a/s Denmark). Freshly trypsinized EFL cells were inoculated and cultured in Leighton tubes (with cover slips) at 37°C for 72--96 h. After incubation, the cells on the cover slips were washed once with 0.15 M NaC1 (saline) and then fixed by immersing the specimens in a 2 0 % acetone bath (PBS, pH 7.4, 200 mg BSA 1 -~) at room temperature for 5 min, after which the preparations were drained. Cover slips were incubated with specific antiserum (1:50) in a humid cham- ber at 37°C for 30 min, then washed twice with wash fluid (0.15 M NaC1, 0.05% Tween 80) and once with saline. After draining, cover slips were stained with FITC~onjugated swine anti-rabbit IgG (1:100), incubated, washed and drained as above, and mounted using buffered glycerol.
Microserum neutralization test (MSN)
This test was performed as described by Joo et al. (1975) with some modifications. Freshly trypsinized cells were used instead of 24-h-old cul- tures. No treatment of the cells with alkaline buffer was found necessary. Since MEV showed better HA patterns than FPLV, MEV was the virus of choice for detect ion of endpoints by HA.
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ELISA
The procedure, reagents and material utilised in the ELISA have been described by Rivera and Sundquist (1984). The double ant ibody sandwich method (Voller et al., 1976) was utilised for detecting virus in the super- natant fluids of stool samples, whereas the competit ive method was em- ployed when serum conversion of vaccinated mink was evaluated. Serum samples (diluted 1:100) taken before and after vaccination were incubated in FPLV-coated wells with cat anti-FPLV IgG peroxidase conjugate. The difference in substrate degradation between the conjugate alone and the sample + conjugate was determined after measuring each in a spectrophoto- meter at 492 nm (Fig. 1).
1.8
1.6
C l I ~
"~1,2
~>'1.0
0.8
% 0.5
0.2 ~ , i i i 2 8 32 52
Weeks a f fe r vaccinafion
Fig. 1. Compet i t ive ELISA. Kinetics of the an t ibody response to FPLV in mink serum af ter a single vaccinat ion. Groups were vaccinated wi th a one -componen t vaccine, FPLV (~ D); a two-componen t vaccine, FPLV + P. aeruginosa (o o); a three-com- ponen t vaccine, FPLV + P. aeruginosa + Cl. b o t u l i n u m toxo id (m m), or were not vaccinated (o o ).
Histopathology
Experimentally infected mink showing clinical symptoms of mink enter- itis were killed, and pieces of their intestine were fixed in 10% neutral buffered formaldehyde solution. The histological sections were then exam- ined for the presence of ballooned cells (Krunajevic, 1970).
R E S U L T S
Virus propagation in microcarrier cell culture
The results presented in Table I show that FPLV grew well both in cells cultured in roller bott les and on microcarrier beads. However, some differ- ences regarding the stage of cell culture in relation to time of virus infec-
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TABLE I
Yields of FPLV obtained from microcarrier cell cultures and roller bottle monolayers
Days after Cell culture Test inoculation
HA a ELISA b
R.B. c 28 0.333 3 MC d, 1 1 2 I° 0.721
MC, 101 2 '0 0.669
R.B. 29 0.593 4 MC, 1 1 29 1.010
MC, 10 l 211 1.580
R.B. 21° 1.544 5 MC, 1 1 24 0.142
MC, 10 1 211 1.887
R.B. 2 l° 1.110 6 MC, 1 1 <2 0.051
MC, 10 1 29 0.732
R.B. 2 s 0.367 7 MC, 1 1 <2 0.062
MC, 10 l 2 ~ 0.298
aliA = haemagglutination titres expressed in log2. bAbsorbance value at 492 nm. CR.B. = roller bottle monlayers. dMC = microcarrier cell culture.
t ion and the yie lds o f virus o b t a i n e d were no t ed . Using rol ler bo t t l e s , high- er y ie lds o f virus were o b t a i n e d w h e n cells were inocu la t ed i m m e d i a t e l y a f t e r t ryps in i sa t ion ; whereas in t he mic roca r r i e r sys t em, the bes t resul ts were o b t a i n e d w h e n cells were inocu la t ed a f t e r i ncuba t i on wi th beads fo r 18 - -24 h. Harves t ing o f virus was bes t carr ied o u t 3 - -4 days a f t e r virus inocu la t ion , usual ly over 3- -4 consecu t ive days (or fo r as long as the H A t i t re r ema ined high). Scaling up the mic roca r r i e r sy s t em f r o m 1 to 10 1 a l lowed harves t ing o f virus for a longer pe r iod (Table I) . The changes in p H wi th 10-1 scale p r o d u c t i o n were m i n i ma l f r o m day to d a y and the cells r e m a i n e d a t t a c h e d to the beads fo r longer pe r iods c o m p a r e d wi th 1-1 scale p r o d u c t i o n .
Vaccines
A c o m p a r a t i v e s t udy o f vaccines con ta in ing d i f f e ren t a m o u n t s of FPLV- HAU showed t h a t all f ou r dosage ra tes eva lua ted were associa ted w i th a good serological r e sponse a t 6 m o n t h s pos t vacc ina t ion (Table II) . I m m u - n i ty a t 12 m o n t h s was c o n f i r m e d b y chal lenge tes t s and assessed b y appear - ance or n o t o f clinical signs and faecal viral exc re t i on (Table I I I ) . Combina -
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TABLE I I
M e a n s o f a n t i b o d y t i t r e s a g a i n s t F P L V a n d M E V in m i n k m e a s u r e d b y H I a n d M S N , 6 m o n t h s a f t e r F P L V v a c c i n a t i o n
V a c c i n e A n t i b o d y t i t r e a
H I M S N
F P L V M E V M E V
C o n t r o l < 1 : 1 0 < 1 : 1 0 < 1 : 1 0 H A 2 '0 1 : 3 5 2 1 : 5 1 2 1 : 2 8 8
H A 2 ' 1 : 4 4 8 1 : 2 8 8 1 : 3 2 0 H A 26 1 : 2 2 4 1 : 4 0 0 1 : 4 9 5
H A 2 ' 1 : 5 6 0 1 : 2 9 0 1 : 3 2 0
a T i t r e m e a n o f 5 m i n k .
TABLE III
Faecal concentrat ions of virus af ter challenge of mink wi th MEV, 1 year after vaccination with in- act ivated FPLV
tions of FPLV vaccine containing 64 HAU with P. aeruginosa and/or Cl. botulinum toxoid (Fig. 1) did not affect the potency of the FPL antigens utilised, as shown by resistance to challenge of vaccinated mink with a pathogen MEV at 6 or 12 months post vaccination. No adverse reactions at the site of vaccination were noted; small swellings of the skin after vacci- nation usually disappeared within 2 weeks.
Challenge test and virus isolation
Assays for virus replication and faecal shedding following challenge were performed daily for 7 days. However, to eliminate the possibility of
378
isolating the challenge virus (administered per os) rather than replicating virus, no samples were taken during the 2-day period following infection. Although a good general correlation between faecal HA test and ELISA was obtained (Table III), there was occasional disparity between the results of the two tests. The use of IF techniques in such cases confirmed a higher specificity of ELISA versus the HA test. Usually, challenged control mink shed virus for 3--4 days starting 3--4 days after infection. Diarrhoea and anorexia were observed, young mink showing more severe clinical symptoms than older mink. Histological studies of intestine from clinically ill mink revealed the presence of ballooned cells. In contrast, vaccinated mink show- ed no clinical signs of disease, and showed no evidence of faecal viral shedd- ing following challenge.
Serological tests
Table II shows that 6 months following vaccination, high levels of HI and SN ant ibody were still present in mink vaccinated with between 24 and 21° HAU of inactivated virus. Antibodies could still be detected 1 year after vaccination in all groups by competit ive ELISA technique, and im- muni ty at this time was also confirmed by challenge test. However, no ant ibody titres were demonstrated by HI or MSN at this time in any vacci- nated mink. Contact control mink remained ELISA, HI and MSN ant ibody negative throughout the experiment. Kinetic studies of ant ibody response to FPLV in mink immunized with combined vaccines demonstrated a rapid immune response after vaccination (Fig. 1). Two weeks after inocu- lation, antibodies were detected by competit ive ELISA in all immunized mink. The highest ant ibody levels were measured at 8 weeks post vaccination and antibodies were still detectable by competit ive ELISA at least 1 year after vaccination.
DISCUSSION
Several authors have utilised conventional serological assays to demon- strate the antigenically close relationships among MEV, FPLV and CPV. However, further studies using restriction enzyme analysis and monoclonal antibodies have shown a greater similarity between the genomes of FPLV and MEV than between either of these two viruses and CPV (McMaster, 1981; Tratschin et al., 1982; Parrish et al., 1982). Since the genome of FPLV differs from the genome of MEV by only one restriction site (Trat- sehin et al., 1982), with little apparent differences in surface antigens, FPLV can be utilised in the immunisation of mink against specific MEV infection. Although the efficacy of vaccines to control MEV infection has been proven (Winans and Marty, 1968; Ackerman et al., 1971), no reports concerning serological evaluation, dose response or duration of
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immunity can be found in the literature. The present report contributes such data.
The introduction of microcarrier cell culture systems (Van Hemert et al., 1969; Van Wezel, 1973; Van Wezel et al., 1979) has allowed the cultivation of large quantities of cells and virus in one-unit systems and has considerably reduced problems of production. Although the yields of virus harvested from roller bottles and microcarrier culture are com- parable, microcarrier systems have the advantage of requiring less personnel, less labour, and less space for equipment. The virus produced showed good immunogenic effect when evaluated either as a monovalent antigen or in combination with P. aeruginosa and/or Cl. botul inum type C-toxoid vac- cines. Neither immunological interference between antigens nor adverse reactions were observed. In the present work vaccines containing 16--64 HAU elicited strong immunity for periods of at least 1 year as demonstrated by ELISA and by experimental challenge. To date, more than 4 million doses of uni- and multivalent vaccine have been used in the field with no adverse results. That low concentrations of inactivated virus can still elicit serological responses and protection against infectious challenge virus one year post-vaccination seems rather unusual. By contrast, Smith and Johnson {1986), studying the serological responses of dogs after immunization with vaccines containing large amounts of inactivated canine parvovirus, found that HI titres usually disappear 3--4 months after vaccination.
The apparent discrepancies regarding the antigen dose/serological re- sponses observed after vaccinating mink and dogs with inactivated parvo- virus vaccines might be explained by one of the following hypotheses. (i) Our results and those reported by Elsheik {1985) following a single vaccination of mink with P. aeruginosa vaccine suggest that in contrast to dogs, mink would have an already highly developed immune system at the time of the vaccination (8--10 weeks}. This hypothesis is supported by the demonstration that high titres of antibodies against both P. aeru- ginosa and FPLV are already present 2 weeks after vaccination (Fig. 1). (ii) FPLV vaccine inactivated with only 0.2% v/v formalin might still con- tain very low numbers of inactivated virus particles (not detectable by IF tests} and such small amounts of live virus in inactivated vaccines might not be sufficient to cause symptoms of the disease, but could still potentiate the vaccine's effects. Alternatively, even if live virus is initially present in the vaccine, it might be neutralized in vivo by a rapid immune response of the mink (Fig. 1) before reaching levels high enough to provoke the disease. (iii) Various quantities (v/v) of formalin have been reported nec- essary for parvovirus inactivation {Smith and Johnson, 1986; Rivera et al., 1986). Since formalin binds to all proteins the amount of formalin needed to inactivate a virus may be directly proportional to the total protein concentration in the virus yields, thereby explaining the variation in the amounts of formalin reported in the literature.
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In conclusion, the microcarrier cell culture provides a simple technique for the large-scale production of parvoviruses without any loss in immuno- genic properties. Furthermore, MEV vaccines can be combined with Cl. botulinum toxoid or P. aeruginosa vaccine without altering the properties of any antigen in the combined vaccine.
ACKNOWLEDGEMENTS
We thank Anne-Marie Wikmark and Ingegerd Andur~n for expert technical assistance, and Ulla MalmstrSm and Carina Bohlin for typing this manus- cript.
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