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Quantitative Assay of Turkey Herpesvirus in Vaccines Against Marek’s Disease of Chickens1,2
Quantitative Assay of Turkey Herpesvirus in Vaccines Against Marek's Disease of Chickens1,2 E . F . KALETA AND O. SIEGMANN
Institute of Poultry Diseases, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-3000 Hannover, Federal Republic of Germany (Received for publication March 11, 1974)
POULTRY SCIENCE 53: 2153-2158, 1974
INTRODUCTION HE degree of protection provided by turkey herpesvirus (HTV) against the development of clinically overt Marek's disease (MD) in chickens depends among others on the dose of virus inoculated right after hatch (Calnek and Smith, 1972; Eidson et al., 1972, Okazaki et al., 1971; Purchase et al., 1971). Therefore, legal authorities in our country require a minimum threshold level of infectious virus in vaccines. Consequently, manufacturers and independent research laboratories need for the control of the viral content in cell-associated and lyophilized vaccines a valid in vitro system for quantitative assays of HVT (Burmester et al, 1970). As far as the mode of preservation of viral infectivity is concerned, three types of HVT vaccines are presently available in the Federal Republic of Germany. These are (i) frozen cell-associated vaccine, (ii) lyophilized vaccine prepared from sonically disrupted infectious cells, and (iii) lyophilized vaccine
T
1. Partially supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. 2. Based on a paper presented at the Symposium of the International Association for Biological Standardisation, Lyon, France, 28-30 August, 1973.
prepared from whole intact infectious cells. It was the aim of this study to investigate the influence of some factors on the growth of chicken embryo fibroblasts and on the plaque formation of HVT in order to establish an in vitro assay system which is equally sensitive and reproducible in detecting infectious HVT in all types of commercially available vaccines. MATERIALS AND METHODS If not otherwise stated, the following materials and basic methods were used for production and maintenance of cell cultures and for plaque counts: Media. Eagle's basal medium (BME) containing Earle's salts was used in most of the experiments. In addition, minimum essential medium (MEM), Dulbecco's modification of MEM (DMEM) and medium 199 (M 199) were employed. These media were supplemented with 10% tryptose phosphate broth (TPB) in some cases. All media were obtained from Flow Laboratories, Bonn. Buffers. Sodium bicarbonate (NaHCO,) was generally used as buffer. In addition, N-2-hydroxyethylpiperazin-N'-ethanesulfon-
2153
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ABSTRACT The type of the cell culture medium and the type of the buffers used in this study do not seem to be very important factors for quantitative assays of herpesvirus of turkeys (HVT) in vaccines against Marek's disease of chickens. A dense monolayer should be used in order to gain high plaque counts induced by infectious HVT. This is especially important for assays of cell-free virus. The low proportion of cell-free virus in cell-associated vaccines allows to neglect this viral fraction. The most important factor was in our hands the interval of time between inoculation of the cultures and the plaque count. Our data indicate that plaques should be counted on the 5th day post inoculation.
2154
E. F. KALETA AND O. SIEGMANN
ic acid (HEPES) and tris-(hydroxymethyl)amino-methane (TRIS) were employed.
HVT Vaccines. All types of vaccines were obtained either directly from the manufacturers or sent to us by veterinarians for the control of the viral content. In addition to these vaccines, own preparations of HVT (strain FC 126) were employed in some cases (21). Titration. Tenfold dilutions in BME were prepared from both types of cell-associated vaccines and were inoculated into the growth medium using a volume of 0.2 ml. per plate. The growth medium was removed 24 hours later and replaced by maintenance medium. The latter had the same composition as the growth medium, except the fetal calf serum (FCS) content was reduced from 5 to 2%. Maintenance medium was changed every other day. Tenfold dilutions of lyophilized cell-free vaccine virus were inoculated onto drained cultures to facilitate viral adsorption (45 min.) to the cells. A volume of 0.05 ml. per plate was used (Adldinger and Calnek, 1971; Biilow and Lorenz, 1973; Patrascu and Calnek, 1972). Thereafter 4 ml. of maintenance medium was added and changed every other day. At least two plates per dilution were used for all titrations. Plaques were counted 5 days after inoculation of the cultures by the aid of an inverted microscope at a 60-fold magni-
RESULTS We have chosen 6 factors which we thought are likely to influence the final outcome of the plaque counts. These factors include (1) different cell culture media, (2) different buffers, (3) different numbers of cells seeded, (4) assay of cell-free and cell-associated HVT in the cell-associated types of vaccines, (5) different intervals of time between inoculation of cultures and plaque counts, and (6) demonstration of secondary plaque formation. All other factors which are necessary for growth and maintenance of cell cultures and not mentioned here remained as described previously (Kaleta and Siegmann, 1972). (1) Different Cell Culture Media. Table 1 shows the number of plaques counted after inoculation of cell-associated HVT into CEF cultures which were grown in different media. The largest difference in the number of plaques was found to be 0.54 log10 in experiment 1 and 0.47 log10 in experiment 2. The
TABLE 1.—Influence of different cell culture media for growth and maintenance of CEF cultures on the number of plaques formed following inoculation of a preparation of cell-associated HVT Culture medium Experiment 1 BME BME + TPB MEM MEM + TPB DMEM DMEM + TPB M 199 M 199 + TPB
4.70 4.57 4.44 4.35 4.83 4.47 4.78 4.29
Experiment 2 4.74 4.66 4.55 4.31 4.74 4.52 4.78 4.42
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
CEF Cultures. Ten day old embryos from eggs laid by our SPF White Leghorn flock were mechanically minced and dispersed by trypsin. The obtained cells were suspended in BME containing 5% fetal calf serum (FCS) and seeded at a rate of 4 million cells per 60 mm. plastic plate. The cultures were incubated in a moist 5% C 0 2 atmosphere at 37.0 to 37.5° C. An almost complete monolayer was formed after about 24 hours of incubation (Kottarides et al., 1969).
fication. Since the number of plaques per culture follows the Poisonian distribution (Lorenz, 1963; Stellmann and Bornarel, 1972) the estimate of the number of plaque forming units (PFU) per inoculum was based on methods described (Cavalli-Sforza, 1969; Kaleta and Siegmann, 1972).
2155
HERPESVIRUS VACCINES
(2) Different Buffers. Table 2 shows the results of titrations of cell-associated and cell-free preparations of HVT using BME supplemented with TPB and adjusted to pH 7.2 by either NaHC0 3 , HEPES- or TRISbuffer. The plaque formation of neither vaccine virus was significantiy influenced by the buffers used.
(3) Different Numbers of Cells Seeded. Figure 1 illustrates the influence of the number of cells seeded per plate on the number of plaques formed after inoculation of cell-associated and cell-free preparations of HVT into CEF cultures. The highest plaque TABLE 2.—Influence of different buffers used in counts were obtained in cultures seeded with BME+> on the number of plaques formed by three 1.5 million cells/ml. in case of cell-associated types of HVT vaccines vaccines. For the cell-free vaccine this value PFU/0.2 ml. vaccine (log,„) was 2.5 million cells per ml. Further, it can BME be seen that lower cellular densities reduce + Na BME BME Type of HVT HCQ3 + Hepes + Tris vaccine drastically the number of plaques, whereas frozen, the use of higher cellular densities results 4.14 cell-associated 4.14 4.13 in a slower decline of the number of plaques. lyophilized, 3.22 cell-free 3.25 3.26 (4) Assay of Cell-Free Virus in Cell-Associatlyophilized, ed Vaccines. Table 3 shows the results of cell-associated 3.63 3.62 3.62 assays of cell-associated and cell-free virus +) BME with Earle's salts, 10% TPB, antibiotics in 8 vaccines. For these titrations, cell-assoand FCS.
PFU lO9
10
2,0
•• cell-ass. HVT x cell-ass. HVT •A cell-free HVT
10 0,50
0,75
uoo
1,25
150
2,00
2,50
aob~ million cells/ml
Fio. 1. Influence of the number of cells seeded on the number of plaques formed by cell-associated and cell-free preparations of HVT.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
highest number of plaques was found for DMEM in experiment 1 and for M 199 in experiment 2. All media supplemented with TPB gave rise to somewhat less plaques when compared with the same media used without TPB. However, the observed numerical differences are not significant. This fact is also documented by many authors who are successfully using various types of cell culture media (Ash and Barnhart, 1972; Calnek et al., 1972; Garrido et al, 1972; Kottarides et al, 1969; Kraft et al., 1973; Patrascu and Calnek, 1972).
2156
E. F. KALETA AND O. SIEGMANN
TABLE +> 3.—Assay of cell-associated and cell-free virus in cell-associated HVT vaccines using primary CEF cultures Number log I0 P F U / 0 . 2 ml. inoculum of Cell-associated Cell-free vaccine virus virus 1 2 3 4 5 6 7 8
3.77 3.95 4.20 4.99 5.18 5.45 5.50 5.06
1.15 1.61 1.74 2.13 2.32 2.57 2.56 2.48
,. .'
/
% 0.24 0.46 0.34 0.14 0.14 0.13 0.12 0.26
>FU 103 "
1 / /I
ll
/ I
/ /
1020
ciated vaccines were thawed and centrifuged for 5 min. at 500 g. The supernatant fluid was filtered through 300 nm. membrane filters. Filtrate and cellular sediment were titrated in the same batch of CEF cultures. It can be seen that the percentage of filtrable virus was in the range of 0.12 to 0.46% of that of the cellular inoculum. The relatively low proportion of the filtrable virus allows to neglect this virus fraction in the assay of cell-associated vaccines. (5) Time of Plaque Count and Formation of Secondary Plaques. The most important factor for the number of plaques was in our hands the length of the interval of time between inoculation of the cultures and the count of the plaques (Fig. 2). Plaques comprising only several rounded cells were first noted 3 days post inoculation (p. i.). In the following two days the number of plaques increased exponentially. Between the 5th and 6th day p. i. many secondary plaques occurred. On the 7th and 8th day p. i. the number of plaques increased at the same rate as between the 3rd and 5th day p. i. (6) Demonstration of Secondary Plaque Formation. The development of secondary plaques results in enhanced infectivity of the
3
/
A
5
6
.
exp1
»
°exp.2
7 8 days p i .
FIG. 2. Growth curve of HVT in CEF cultures.
cultures. However, the degree of secondary plaque formation is obviously dependent on the dose of HVT inoculated. This observation is illustrated in Figure 3. In this experiment one batch of CEF cultures was divided in two groups. The first group was inoculated with 640 PFU of HVT and the other group received 6400 PFU per plate, respectively. Cultures of both groups were assayed for infectivity at increasing intervals of time in newly prepared CEF cultures. Up to the 3rd day, cultures of both groups showed increasing infectivity. Between the 3rd and the 5th day, the infectivity remained at almost the same level. Thereafter, the lower infected cultures developed a definite second peak of increased infectivity. This peak coincides with secondary plaque formation as shown in Figure 2. However, little if any increase in infectivity was present in the highly inoculated cultures. With increasing age of the
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
I
-
+) Vaccines were centrifuged for 5 min. at 500 g. The supernatant was filtered through 300 nm. membrane filters. Filtrate and cellular sediment were titrated in the same batch of CEF cultures.
2157
HERPESVIRUS VACCINES
T T T T l
1
2
3
4
5
6
7
8
9
10 days p.i.
FIG. 3. Assay of infectivity of CEF cultures inoculated with the FC 126 strain of HVT.
cultures the cells deteriorate and loose rapidly infectivity as reflected by the dramatic drop of titers in both groups. DISCUSSION HVT produces pock-like lesions on the chorio-allantoic membrane (CAM) following inoculation of the yolk sac or the dropped CAM of chicken embryos (Biggs and Milne, 1971; Biilow, 1969). Since the use of embryonated eggs for quantitative studies of HVT is not reliable enough and, therefore, not recommended by the Workshop Conference of the Leukosis Committee of the American Association of Avian Pathologists (1970), we concentrated our efforts on studies with primary chicken embryo fibroblast (CEF) cultures. Theoretically, numerous factors may influence the degree of adsorption, penetration, and multiplication of the virus in cell cultures. Some of these ones may stimulate and others may inhibit viral growth and plaque forma-
tion. Culture conditions, mode of inoculation of cultures and procedures of plaque counts vary in different laboratories (Adldinger and Calnek, 1971; Ash and Barnhart, 1972; Biilow and Lorenz, 1973; Calnek et al., 1972; Kaleta and Siegmann, 1972; Kraft era/., 1973; Patrascu and Calnek, 1972) and make meaningful comparisons of quantitative results sometimes difficult. However, the data presented here clearly indicate that the type of cell culture media and buffers are of minor if any importance. The cellular density of the monolayers is especially important for attachment of cellfree HVT and less for cell-associated virus. The dynamics of plaque formation require that plaques should not be counted before all primary plaques are clearly visible (5th day p. i.). In case that the plaques are to be counted somewhat later the still smaller secondary plaques should not be included in the final assessment of the viral content of the vaccine.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
T
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E. F. KALETA AND O. SIEGMANN
REFERENCES American Association of Avian Pathologists' Workshop, 1970. Methods in Marek's disease research. Avian Dis. 14: 820-828. Adldinger, H. K., and B. W. Calnek, 1971. An improved in vitro assay for cell-free Marek's disease virus. Arch. ges. Virusforsch. 34: 391-395. Ash, R. J., and R. E. Barnhart, 1972. Factors affecting the propagation and assay of a herpesvirus of turkeys. Avian Dis. 16: 285-290. Biggs, P. M., and B. S. Milne, 1971. Use of the embryonating egg in studies on Marek's disease. Am. J. Vet. Res. 32: 1795-1809. Biilow, von V., 1969. Marek'sche Huhnerlahmung: Reaktionen im experimentell infizierten embryonierten Ei. Zbl. Vet. Med. B 16: 97-114. Biilow, von V., and R. J. Lorenz, 1973. Plaque assay of turkey herpesvirus and attenuated Marek's disease virus. Zbl. Vet. Med. B 20: 161-165. Burmester, B. R., H. G. Purchase, R. L. Witter and W. Okazaki, 1970. Developing and testing of a Marek's disease vaccine. Proc. 19th Western Poultry Disease Conf. and 4th Poultry Health Symposium, 1970, Davis, Calif.: pp. 90-93. Calnek, B. W., C. Garrido, W. Okazaki and I. V. Patrascu, 1972. In vitro methods for assay of turkey herpesvirus. Avian Dis. 16: 52-56. Calnek, B. W., and M. W. Smith, 1972. Vaccination against Marek's disease with cell-free turkey
herpesvirus: Interference by maternal antibody. Avian Dis. 16: 954-957. Cavalli-Sforza, L., 1969. Biometrie: Grundzuge biologisch-medizinischer Statistik. G. Fischer Verlag, Stuttgart, 2. Aufl. S: 191-204. Eidson, C. S., S. H. Kleven and D. P. Anderson, 1972. Vaccination against Marek's disease. I.A.R.C. Scientific Publications No. 2: 147-152. Garrido, C , W. Okazaki, H. G. Purchase and B. R. Burmester, 1972. Storage of a cell-associated herpesvirus of turkeys (HVT Strain FC126) vaccine. Avian Dis. 16: 45-51. Kaleta, E. F., und O. Siegmann, 1972. Titration des Putenherpesvirus in Vaccinen zum Schutze gegen die Mareksche Geflugellahme. Dtsch. tierarztl. Wschr. 79: 428-430. Kottarides, S. D., R. E. Luginbuhl and T. N. Frederickson, 1969. Infectivity of Marek's disease agent after different treatments used in vaccine production. Am. J. Vet. Res. 30: 1851-1855. Kraft, V., P. Ingwersen und G. Monreal, 1973. Zur Methodik der Titration von lyophilisierten und zellassoziierten Marek-Vakzinen. Berl. Munch, tierarztl. Wschr. 86: 283-285. Lorenz, R. J., 1963. Zur Statistik des Plaque-Testes. Arch. ges. Virusforsch. 12: 108-137. Okazaki, W., H. G. Purchase and B. R. Burmester, 1971. The temporal relationship between vaccination with the herpesvirus of turkeys and challenge with virulent Marek's disease virus. Avian Dis. 15: 752761. Patrascu, I. V., and B. W. Calnek, 1972. In vitro assay of cell-free turkey herpesvirus. Avian Dis. 16: 397-413. Purchase, H. G., W. Okazaki and B. R. Burmester, 1971. Field trials with the herpesvirus of turkeys (HVT) strain FC 126 as a vaccine against Marek's disease. Poultry Sci. 50: 775-783. Stellmann, C , and P. Bornarel, 1972. Titration of viruses in cell cultures. Pharmacology or single particle hypothesis. Arch. ges. Virusforsch. 36: 205-217. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus anti-genetically related to Marek's disease virus. Am. J. Vet. Res. 31: 525-538.
APRIL 21-24, 1975. THIRD INTERNATIONAL SYMPOSIUM ON LIVESTOCK WASTE, UNIVERSITY OF ILLINOIS, URBANA-CHAMPAIGN, ILLINOIS.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
The intended prevention of secondary plaque formation by the application of an agar overlay 24 hours after inoculation (Billow and Lorenz, 1973; Kraft et al., 1973) cannot be recommended generally for several reasons. The use of the overlay technique implies the introduction of a biological product which is difficult to standardize into the assay system. A detailed study on agar overlay techniques is presently under way which will prove that source and type of agar, time of application and concentration of agar in the overlay markedly influences the number of plaques.
Institute of Poultry Diseases, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-3000 Hannover, Federal Republic of Germany (Received for publication March 11, 1974)
POULTRY SCIENCE 53: 2153-2158, 1974
INTRODUCTION HE degree of protection provided by turkey herpesvirus (HTV) against the development of clinically overt Marek's disease (MD) in chickens depends among others on the dose of virus inoculated right after hatch (Calnek and Smith, 1972; Eidson et al., 1972, Okazaki et al., 1971; Purchase et al., 1971). Therefore, legal authorities in our country require a minimum threshold level of infectious virus in vaccines. Consequently, manufacturers and independent research laboratories need for the control of the viral content in cell-associated and lyophilized vaccines a valid in vitro system for quantitative assays of HVT (Burmester et al, 1970). As far as the mode of preservation of viral infectivity is concerned, three types of HVT vaccines are presently available in the Federal Republic of Germany. These are (i) frozen cell-associated vaccine, (ii) lyophilized vaccine prepared from sonically disrupted infectious cells, and (iii) lyophilized vaccine
T
1. Partially supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. 2. Based on a paper presented at the Symposium of the International Association for Biological Standardisation, Lyon, France, 28-30 August, 1973.
prepared from whole intact infectious cells. It was the aim of this study to investigate the influence of some factors on the growth of chicken embryo fibroblasts and on the plaque formation of HVT in order to establish an in vitro assay system which is equally sensitive and reproducible in detecting infectious HVT in all types of commercially available vaccines. MATERIALS AND METHODS If not otherwise stated, the following materials and basic methods were used for production and maintenance of cell cultures and for plaque counts: Media. Eagle's basal medium (BME) containing Earle's salts was used in most of the experiments. In addition, minimum essential medium (MEM), Dulbecco's modification of MEM (DMEM) and medium 199 (M 199) were employed. These media were supplemented with 10% tryptose phosphate broth (TPB) in some cases. All media were obtained from Flow Laboratories, Bonn. Buffers. Sodium bicarbonate (NaHCO,) was generally used as buffer. In addition, N-2-hydroxyethylpiperazin-N'-ethanesulfon-
2153
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
ABSTRACT The type of the cell culture medium and the type of the buffers used in this study do not seem to be very important factors for quantitative assays of herpesvirus of turkeys (HVT) in vaccines against Marek's disease of chickens. A dense monolayer should be used in order to gain high plaque counts induced by infectious HVT. This is especially important for assays of cell-free virus. The low proportion of cell-free virus in cell-associated vaccines allows to neglect this viral fraction. The most important factor was in our hands the interval of time between inoculation of the cultures and the plaque count. Our data indicate that plaques should be counted on the 5th day post inoculation.
2154
E. F. KALETA AND O. SIEGMANN
ic acid (HEPES) and tris-(hydroxymethyl)amino-methane (TRIS) were employed.
HVT Vaccines. All types of vaccines were obtained either directly from the manufacturers or sent to us by veterinarians for the control of the viral content. In addition to these vaccines, own preparations of HVT (strain FC 126) were employed in some cases (21). Titration. Tenfold dilutions in BME were prepared from both types of cell-associated vaccines and were inoculated into the growth medium using a volume of 0.2 ml. per plate. The growth medium was removed 24 hours later and replaced by maintenance medium. The latter had the same composition as the growth medium, except the fetal calf serum (FCS) content was reduced from 5 to 2%. Maintenance medium was changed every other day. Tenfold dilutions of lyophilized cell-free vaccine virus were inoculated onto drained cultures to facilitate viral adsorption (45 min.) to the cells. A volume of 0.05 ml. per plate was used (Adldinger and Calnek, 1971; Biilow and Lorenz, 1973; Patrascu and Calnek, 1972). Thereafter 4 ml. of maintenance medium was added and changed every other day. At least two plates per dilution were used for all titrations. Plaques were counted 5 days after inoculation of the cultures by the aid of an inverted microscope at a 60-fold magni-
RESULTS We have chosen 6 factors which we thought are likely to influence the final outcome of the plaque counts. These factors include (1) different cell culture media, (2) different buffers, (3) different numbers of cells seeded, (4) assay of cell-free and cell-associated HVT in the cell-associated types of vaccines, (5) different intervals of time between inoculation of cultures and plaque counts, and (6) demonstration of secondary plaque formation. All other factors which are necessary for growth and maintenance of cell cultures and not mentioned here remained as described previously (Kaleta and Siegmann, 1972). (1) Different Cell Culture Media. Table 1 shows the number of plaques counted after inoculation of cell-associated HVT into CEF cultures which were grown in different media. The largest difference in the number of plaques was found to be 0.54 log10 in experiment 1 and 0.47 log10 in experiment 2. The
TABLE 1.—Influence of different cell culture media for growth and maintenance of CEF cultures on the number of plaques formed following inoculation of a preparation of cell-associated HVT Culture medium Experiment 1 BME BME + TPB MEM MEM + TPB DMEM DMEM + TPB M 199 M 199 + TPB
4.70 4.57 4.44 4.35 4.83 4.47 4.78 4.29
Experiment 2 4.74 4.66 4.55 4.31 4.74 4.52 4.78 4.42
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
CEF Cultures. Ten day old embryos from eggs laid by our SPF White Leghorn flock were mechanically minced and dispersed by trypsin. The obtained cells were suspended in BME containing 5% fetal calf serum (FCS) and seeded at a rate of 4 million cells per 60 mm. plastic plate. The cultures were incubated in a moist 5% C 0 2 atmosphere at 37.0 to 37.5° C. An almost complete monolayer was formed after about 24 hours of incubation (Kottarides et al., 1969).
fication. Since the number of plaques per culture follows the Poisonian distribution (Lorenz, 1963; Stellmann and Bornarel, 1972) the estimate of the number of plaque forming units (PFU) per inoculum was based on methods described (Cavalli-Sforza, 1969; Kaleta and Siegmann, 1972).
2155
HERPESVIRUS VACCINES
(2) Different Buffers. Table 2 shows the results of titrations of cell-associated and cell-free preparations of HVT using BME supplemented with TPB and adjusted to pH 7.2 by either NaHC0 3 , HEPES- or TRISbuffer. The plaque formation of neither vaccine virus was significantiy influenced by the buffers used.
(3) Different Numbers of Cells Seeded. Figure 1 illustrates the influence of the number of cells seeded per plate on the number of plaques formed after inoculation of cell-associated and cell-free preparations of HVT into CEF cultures. The highest plaque TABLE 2.—Influence of different buffers used in counts were obtained in cultures seeded with BME+> on the number of plaques formed by three 1.5 million cells/ml. in case of cell-associated types of HVT vaccines vaccines. For the cell-free vaccine this value PFU/0.2 ml. vaccine (log,„) was 2.5 million cells per ml. Further, it can BME be seen that lower cellular densities reduce + Na BME BME Type of HVT HCQ3 + Hepes + Tris vaccine drastically the number of plaques, whereas frozen, the use of higher cellular densities results 4.14 cell-associated 4.14 4.13 in a slower decline of the number of plaques. lyophilized, 3.22 cell-free 3.25 3.26 (4) Assay of Cell-Free Virus in Cell-Associatlyophilized, ed Vaccines. Table 3 shows the results of cell-associated 3.63 3.62 3.62 assays of cell-associated and cell-free virus +) BME with Earle's salts, 10% TPB, antibiotics in 8 vaccines. For these titrations, cell-assoand FCS.
PFU lO9
10
2,0
•• cell-ass. HVT x cell-ass. HVT •A cell-free HVT
10 0,50
0,75
uoo
1,25
150
2,00
2,50
aob~ million cells/ml
Fio. 1. Influence of the number of cells seeded on the number of plaques formed by cell-associated and cell-free preparations of HVT.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
highest number of plaques was found for DMEM in experiment 1 and for M 199 in experiment 2. All media supplemented with TPB gave rise to somewhat less plaques when compared with the same media used without TPB. However, the observed numerical differences are not significant. This fact is also documented by many authors who are successfully using various types of cell culture media (Ash and Barnhart, 1972; Calnek et al., 1972; Garrido et al, 1972; Kottarides et al, 1969; Kraft et al., 1973; Patrascu and Calnek, 1972).
2156
E. F. KALETA AND O. SIEGMANN
TABLE +> 3.—Assay of cell-associated and cell-free virus in cell-associated HVT vaccines using primary CEF cultures Number log I0 P F U / 0 . 2 ml. inoculum of Cell-associated Cell-free vaccine virus virus 1 2 3 4 5 6 7 8
3.77 3.95 4.20 4.99 5.18 5.45 5.50 5.06
1.15 1.61 1.74 2.13 2.32 2.57 2.56 2.48
,. .'
/
% 0.24 0.46 0.34 0.14 0.14 0.13 0.12 0.26
>FU 103 "
1 / /I
ll
/ I
/ /
1020
ciated vaccines were thawed and centrifuged for 5 min. at 500 g. The supernatant fluid was filtered through 300 nm. membrane filters. Filtrate and cellular sediment were titrated in the same batch of CEF cultures. It can be seen that the percentage of filtrable virus was in the range of 0.12 to 0.46% of that of the cellular inoculum. The relatively low proportion of the filtrable virus allows to neglect this virus fraction in the assay of cell-associated vaccines. (5) Time of Plaque Count and Formation of Secondary Plaques. The most important factor for the number of plaques was in our hands the length of the interval of time between inoculation of the cultures and the count of the plaques (Fig. 2). Plaques comprising only several rounded cells were first noted 3 days post inoculation (p. i.). In the following two days the number of plaques increased exponentially. Between the 5th and 6th day p. i. many secondary plaques occurred. On the 7th and 8th day p. i. the number of plaques increased at the same rate as between the 3rd and 5th day p. i. (6) Demonstration of Secondary Plaque Formation. The development of secondary plaques results in enhanced infectivity of the
3
/
A
5
6
.
exp1
»
°exp.2
7 8 days p i .
FIG. 2. Growth curve of HVT in CEF cultures.
cultures. However, the degree of secondary plaque formation is obviously dependent on the dose of HVT inoculated. This observation is illustrated in Figure 3. In this experiment one batch of CEF cultures was divided in two groups. The first group was inoculated with 640 PFU of HVT and the other group received 6400 PFU per plate, respectively. Cultures of both groups were assayed for infectivity at increasing intervals of time in newly prepared CEF cultures. Up to the 3rd day, cultures of both groups showed increasing infectivity. Between the 3rd and the 5th day, the infectivity remained at almost the same level. Thereafter, the lower infected cultures developed a definite second peak of increased infectivity. This peak coincides with secondary plaque formation as shown in Figure 2. However, little if any increase in infectivity was present in the highly inoculated cultures. With increasing age of the
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
I
-
+) Vaccines were centrifuged for 5 min. at 500 g. The supernatant was filtered through 300 nm. membrane filters. Filtrate and cellular sediment were titrated in the same batch of CEF cultures.
2157
HERPESVIRUS VACCINES
T T T T l
1
2
3
4
5
6
7
8
9
10 days p.i.
FIG. 3. Assay of infectivity of CEF cultures inoculated with the FC 126 strain of HVT.
cultures the cells deteriorate and loose rapidly infectivity as reflected by the dramatic drop of titers in both groups. DISCUSSION HVT produces pock-like lesions on the chorio-allantoic membrane (CAM) following inoculation of the yolk sac or the dropped CAM of chicken embryos (Biggs and Milne, 1971; Biilow, 1969). Since the use of embryonated eggs for quantitative studies of HVT is not reliable enough and, therefore, not recommended by the Workshop Conference of the Leukosis Committee of the American Association of Avian Pathologists (1970), we concentrated our efforts on studies with primary chicken embryo fibroblast (CEF) cultures. Theoretically, numerous factors may influence the degree of adsorption, penetration, and multiplication of the virus in cell cultures. Some of these ones may stimulate and others may inhibit viral growth and plaque forma-
tion. Culture conditions, mode of inoculation of cultures and procedures of plaque counts vary in different laboratories (Adldinger and Calnek, 1971; Ash and Barnhart, 1972; Biilow and Lorenz, 1973; Calnek et al., 1972; Kaleta and Siegmann, 1972; Kraft era/., 1973; Patrascu and Calnek, 1972) and make meaningful comparisons of quantitative results sometimes difficult. However, the data presented here clearly indicate that the type of cell culture media and buffers are of minor if any importance. The cellular density of the monolayers is especially important for attachment of cellfree HVT and less for cell-associated virus. The dynamics of plaque formation require that plaques should not be counted before all primary plaques are clearly visible (5th day p. i.). In case that the plaques are to be counted somewhat later the still smaller secondary plaques should not be included in the final assessment of the viral content of the vaccine.
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REFERENCES American Association of Avian Pathologists' Workshop, 1970. Methods in Marek's disease research. Avian Dis. 14: 820-828. Adldinger, H. K., and B. W. Calnek, 1971. An improved in vitro assay for cell-free Marek's disease virus. Arch. ges. Virusforsch. 34: 391-395. Ash, R. J., and R. E. Barnhart, 1972. Factors affecting the propagation and assay of a herpesvirus of turkeys. Avian Dis. 16: 285-290. Biggs, P. M., and B. S. Milne, 1971. Use of the embryonating egg in studies on Marek's disease. Am. J. Vet. Res. 32: 1795-1809. Biilow, von V., 1969. Marek'sche Huhnerlahmung: Reaktionen im experimentell infizierten embryonierten Ei. Zbl. Vet. Med. B 16: 97-114. Biilow, von V., and R. J. Lorenz, 1973. Plaque assay of turkey herpesvirus and attenuated Marek's disease virus. Zbl. Vet. Med. B 20: 161-165. Burmester, B. R., H. G. Purchase, R. L. Witter and W. Okazaki, 1970. Developing and testing of a Marek's disease vaccine. Proc. 19th Western Poultry Disease Conf. and 4th Poultry Health Symposium, 1970, Davis, Calif.: pp. 90-93. Calnek, B. W., C. Garrido, W. Okazaki and I. V. Patrascu, 1972. In vitro methods for assay of turkey herpesvirus. Avian Dis. 16: 52-56. Calnek, B. W., and M. W. Smith, 1972. Vaccination against Marek's disease with cell-free turkey
herpesvirus: Interference by maternal antibody. Avian Dis. 16: 954-957. Cavalli-Sforza, L., 1969. Biometrie: Grundzuge biologisch-medizinischer Statistik. G. Fischer Verlag, Stuttgart, 2. Aufl. S: 191-204. Eidson, C. S., S. H. Kleven and D. P. Anderson, 1972. Vaccination against Marek's disease. I.A.R.C. Scientific Publications No. 2: 147-152. Garrido, C , W. Okazaki, H. G. Purchase and B. R. Burmester, 1972. Storage of a cell-associated herpesvirus of turkeys (HVT Strain FC126) vaccine. Avian Dis. 16: 45-51. Kaleta, E. F., und O. Siegmann, 1972. Titration des Putenherpesvirus in Vaccinen zum Schutze gegen die Mareksche Geflugellahme. Dtsch. tierarztl. Wschr. 79: 428-430. Kottarides, S. D., R. E. Luginbuhl and T. N. Frederickson, 1969. Infectivity of Marek's disease agent after different treatments used in vaccine production. Am. J. Vet. Res. 30: 1851-1855. Kraft, V., P. Ingwersen und G. Monreal, 1973. Zur Methodik der Titration von lyophilisierten und zellassoziierten Marek-Vakzinen. Berl. Munch, tierarztl. Wschr. 86: 283-285. Lorenz, R. J., 1963. Zur Statistik des Plaque-Testes. Arch. ges. Virusforsch. 12: 108-137. Okazaki, W., H. G. Purchase and B. R. Burmester, 1971. The temporal relationship between vaccination with the herpesvirus of turkeys and challenge with virulent Marek's disease virus. Avian Dis. 15: 752761. Patrascu, I. V., and B. W. Calnek, 1972. In vitro assay of cell-free turkey herpesvirus. Avian Dis. 16: 397-413. Purchase, H. G., W. Okazaki and B. R. Burmester, 1971. Field trials with the herpesvirus of turkeys (HVT) strain FC 126 as a vaccine against Marek's disease. Poultry Sci. 50: 775-783. Stellmann, C , and P. Bornarel, 1972. Titration of viruses in cell cultures. Pharmacology or single particle hypothesis. Arch. ges. Virusforsch. 36: 205-217. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus anti-genetically related to Marek's disease virus. Am. J. Vet. Res. 31: 525-538.
APRIL 21-24, 1975. THIRD INTERNATIONAL SYMPOSIUM ON LIVESTOCK WASTE, UNIVERSITY OF ILLINOIS, URBANA-CHAMPAIGN, ILLINOIS.
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The intended prevention of secondary plaque formation by the application of an agar overlay 24 hours after inoculation (Billow and Lorenz, 1973; Kraft et al., 1973) cannot be recommended generally for several reasons. The use of the overlay technique implies the introduction of a biological product which is difficult to standardize into the assay system. A detailed study on agar overlay techniques is presently under way which will prove that source and type of agar, time of application and concentration of agar in the overlay markedly influences the number of plaques.