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Immunoglobulins of the Chicken Antibody to Newcastle Disease Virus (Mukteswar and F Strain)*
Immunoglobulins of the Chicken Antibody to Newcastle Disease Virus (Mukteswar and F Strain)* MADAN L . K H A R E , 1 S. K U M A R 2 AND J. G R U N 3 13
Department of Animal Sciences (Pathobiology), Thompson Hall, Cook College, Rutgers University, New Brunswick, N.J. 08903, and 2Division of Bacteriology and Virology, Indian Veterinary Research Institute, Mukteswar (U.P.), India (Received for publication April 15, 1975)
POULTRY SCIENCE 55: 152-159, 1976
INTRODUCTION
I
N response to viral antigens, heterogeneity of immunoglobulins in various species is well documented (Cowon, 1973; Pike, 1967). In terms of antibodies, serological manifestation of an i m m u n e serum is a combined activity of the individual immunoglobulins participating in t h e serological test. All immunoglobulins may or may not be actively present in the immune sera at one time or all the time after infection or vaccination (Cowon, 1973). Individual immunoglobulins vary considerably in their degree of participation in a particular serological test. M o r e o v e r , sensitivity of a serological test
*Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University—The State University of New Jersey, Department of Animal Sciences, New Brunswick, New Jersey 08903. Presented in part at the 73rd Annual Meeting of the American Society for Microbiology, Miami, Florida, May 6-11, 1973. 1. In large part taken from M.V.Sc. (1971) thesis of the senior author submitted to Agra University, India.
system also varies in relation to the type of immunoglobulin present (Pike, 1967). Therefore, characterization of antibody r e s p o n s e s in terms of types of immunoglobulins, their induction times and their relative concentrations in t h e immune serum are n e c e s s a r y . Such studies might provide a more complete recognition of the infective or immune status of the host as compared with the d e m o n stration of antibody against a specific antigen or pathogen. It might also provide a better interpretation, thereby avoiding false and misleading serological analysis of immune sera. In the present communication we have reported the qualitative and quantitative induction of the chicken immunoglobulins in response to R 2 B (Mukteswar) and F strains of Newcastle disease virus (NDV). MATERIALS AND METHODS Experimental Birds. Forty-nine male and female White Leghorn chickens obtained from the Indian Veterinary R e s e a r c h Institute (Izatnagar, Uttar P r a d e s h , India) poultry farm ( P P L O free) were used in these studies. 152
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ABSTRACT The qualitative and quantitative development of chicken immunoglobulins in response to R 2 B (Mukteswar) and F strain of Newcastle disease virus was studied. One primary (R2B) and two secondary (R 2 B - R 2 B, F — R 2 B) vaccination trials were conducted. Sera was collected at weekly intervals and analyzed. There was an increase in total serum protein content in parallel to an increase in serum neutralizing (SN), hemagglutination inhibition (HI), and precipitating antibodies. The SN, HI and precipitating activities were detected both in IgM and IgG immunoglobulins when sera were treated with mercapto-ethanol. However, Sephadex G-200 fractionated sera showed only SN activity in the IgM fraction, whereas the IgG fraction showed both HI and SN activity. Serum IgM antibodies appeared during the first week following vaccination, then diminished, then rose again following secondary vaccination. Apparently, immunoglobulin induction and serological activities were not significantly influenced by the age of the chickens.
153
NEWCASTLE DISEASE
Vaccination. All chickens, with the exception of one-day-old chicks, were tested for preinoculation serum antibodies to NDV by the HI test. Chickens with even a doubtful HI titer were not included in the experiments. Experiment 1. Twenty-five chickens were distributed in five groups (A to E) according to age as detailed in Table 1. They were vaccinated subcutaneously with 1.0 ml. of R 2 B vaccine. Five more groups (C, to Cs) were kept as unvaccinated controls.
the R 2 B vaccine (primary vaccination), were divided into two groups. The first group (G) was revaccinated subcutaneously with 1.0 ml. of R 2 B vaccine (secondary vaccination), while the other group (I) was kept as uninoculated controls. All vaccinated and unvaccinated (contact infection) chickens from Experiments 1 and 2 were held in the same room in separate cages. Experiment 3. Twenty, one-day-old chicks were vaccinated intranasally with the " F " strain (primary vaccination). Twelve weeks post vaccination, six chickens (group H) were revaccinated subcutaneously with 1.0 ml. of R 2 B vaccine (secondary vaccination). Four chicks (group J) were kept as unvaccinated controls. The vaccinated birds from Experiment 3 were housed and cared for separately from the controls.
Experiment 2. Six chickens (20 to 24 weeks of age), vaccinated eight weeks earlier with
Collection of Sera. All chickens were bled before vaccination and at weekly intervals after vaccination. Collected serum samples were stored at -20° C. until used. No preservatives were added. When required, pooled serum samples were obtained by mix-
TABLE 1.—NDV vaccination schedule in chickens from Experiments 1, 2 and 3 Exp. I.
II. III.
Age of vaccination
Vaccine
Dose/i route
Groups
No. of chickens
Primary
Secondary
Primary
Secondary
A B C D E
3 8 3 6 5
adult 12-16 wks 10-12 wks. 8-10 wks. mixed
— — — — —
R2B R2B R2B R2B R2B
— — — — —
c,-c5*
t
G I** H J**
3 3 6 4
20-24 wks. 20-24 wks. 12 wks. 12 wks.
R2B R2B F F
R2B n.s. R2B n.s.
12-16 12-16 day day
wks. wks. old old
*Control groups (for respective infective groups) inoculated with normal saline only. "Controls: n.s. = normal saline; i.n. = intranasal. t C, = 1, C2 = 2, C3 = 2, C4 = 2, C5 = 3.
Primary 1 1 1 1 1
Secondary
— — — — —
ml./s.c. ml./s.c. ml./s.c. ml./s.c. ml./s.c.
1 ml./s.c. 1 ml./s.c. 0.2ml./i.n. 0.2 ml./i.n.
1 1 1 1
ml./s.c. ml./s.c. ml./s.c. ml./s.c.
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Vaccine. One-day-old chicks were vaccinated with the " F " strain of Newcastle disease virus (NDV) vaccine. For the primary and secondary vaccinations R 2 B (Mukteswar) strain of NDV with an ELD 5 0 titer greater than 10 8 /ml. was used. A chicken fibroblast cell culture adapted Mukteswar (R 2 B) strain of NDV was passed three times in chick embryos, collected, titrated and stored as seed virus at -20° C. Prior to vaccination, the seed virus was given another passage in chick embryos, titrated and used for primary and secondary vaccination (ELD 50 , 107ml.).
154
M. L. KHARE, S. KUMAR AND J. GRUN
ing equal quantities of individual serum samples. Serum Protein Estimation. The modified Biuret method (Weir, 1967) was used in determining total serum proteins. The test was read at 545 m^.. using Spectronic-20. Purified bovine serum (Armour) was used in preparing the standard curve.
3. Immunoelectrophoresis.
Immunoelec-
Assessment of Antibody Response. 1. Hemagglutination-Inhibition Test. The test was conducted according to the method described by Cunningham (1966). Dilutions of R 2 B virus having two or four hemagglutinating (HA) units were used as antigen, whereas test serum samples and their fractions were used as antisera. A constant volume (0.25 ml.) of test ingredients was employed in each test. The final HI titer of the serum sample was calculated by multiplying the observed serum titer by the HA units used. 2. Immunoprecipitin Test. A standard immunoprecipitin (IP) test was performed (Weir, 1967). Purified agar (1%) in phosphate buffer (pH 7.2) was utilized. Antigen and antiserum wells (two mm. in diameter) were five mm. from each other. A suspension of NDV (R 2 B) infected chorioallantoic membrane in allantoic fluid served as the antigen. Agar gels containing high (8%) and low (0.85%) concentrations of sodium chloride were utilized. Plates were incubated at room temperature (25 to 28° C.) for 48 to 72 hours and examined for the development of precipitin lines. 3. Serum Neutralization Test. The SN test was performed in a fibroblast cell culture system. Chick embryo fibroblast cell cultures
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Identification of Immunoglobulins. 1. Mercapto-Ethanol Treatment of Sera. For the detection of IgM and IgG immunoglobulins in test sera, serum samples were treated with 2-mercapto-ethanol (ME). IgM is ME sensitive, whereas IgG is resistant. A constant volume of individual serum sample was divided into two equal parts. One half was diluted with an equal volume of 0.2 M ME and incubated for one hour at 37° C. in a water bath. The other half, diluted with phosphate buffer saline alone and incubated at 37° C , was used as a control. After dialysis, ME treated and untreated serum samples were subjected to various serological tests and Immunoelectrophoresis. 2. Sephadex-Gel Chromatography. For the separation of IgM and IgG antibodies, test sera were fractionated on Sephadex G-200 (Upsala, Sweden) as described by Fischer (1969). A glass column (2.5 x 90 cm.) of 430 ml. capacity was used. Elution of the serum sample (2 ml.) was carried out at a flow rate of 2 ml./minute in 0.15 M normal saline solution. Fractions were collected and stored at 4° C. The optical density of fractions was measured at 280 mix. using an ultraviolet spectrophotometer (Beckman) and an elution curve was obtained utilizing the data. Immunoelectrophoresis and various serological tests were performed on the individual fractions. Also, fractions comprised of peaks in the elution curve were pooled, concentrated (by lyophilization) and subjected to similar analysis.
trophoresis (IE) was employed on microslides utilizing 1.5% agar gel in 0.025 M barbital buffer (pH 8.6). The same buffer was used in electrode vessels. Electrophoresis was carried out for 2.5 hours at a constant current of two milliamperes per slide. For the identification of immunoglobulins in the test sera, electrophoresed serum samples and their fractions were incubated at room temperature (28° C.) with rabbit anti-chicken serum and rabbit anti-chicken globulin. Resultant electropherograms were preserved and stained, as described by Weir (1967). Antisera against whole chicken serum and globulins (ammonium sulphate precipitated) were produced separately in rabbits (Weir, 1967).
155
NEWCASTLE DISEASE
ham, 1966). Cell culture adapted R 2 B strain of NDV in 0.1 ml. volumes containing 100 to 1000 TCID 50 (tissue culture infective dose) of virus was the antigen. The cell cultures were inoculated with either a mixture of virus and test serum, or virus alone (controls). Cultures were observed for 72 hours after infection for cytopathic effect (CPE) charac-
were prepared from 10 to 12 day-old embryos according to the standard method of Merchant et al. (1960). The growth media consisted of Hanks' balanced salt solution, supplemented with lactalbumin hydrolysate 0.5%, yeast extract 0.05% and 10% inactivated calf serum with penicillin. The test proper was conducted by the beta-procedure (Cunning-
TABLE 2.—Mean total serum protein profile of chickens from experiment one over a period of eight weeks Total protein (gm./lOO ml.) M.P.V.P. %Rise 27 5.80 13 3.70 17 5.25 8 5.00 34 5.97 9 4.42
P.V. 4.55 3.25 4.45 4.60 4.45 4.05
C2
c c,
% Fall
P.V. = Pre-vaccination. M.P.V.P. = Mean post-vaccination period. A-C = infected.
320
UJ I-
240
jZ
200 160
I
120 80 40
n i\
*
280
I. 1 .
A
7 n .i u
I:
If
— o——o-
~\
- + —*-
&L
5 6 7 WEEKS
320
TITER
_ z
1 2 640
280
D
560
240
480
200
400
r~~z //-— %
160 120
I r^
320 240
80
160
40
80
3
4
5
6
7
8
3
4
: Iff a w
:/
TOTAL lg
t^ • * - +-
4
5 6 7 WEEKS
M
lg G
*****
'&—>\ 5 6 7 WEEKS
5 6 WEEKS
+
A
IsrBssI
- * -
5 6 7 WEEKS
FIG. 1. ME sensitive (IgM) and ME resistant (IgG) HI titer of chicken serum from birds of various groups of experiment one: (A) Adult; (B) Age 12-16 weeks; (C) Age 10-12 weeks; (E) Mixed age, at the time of vaccination; (F) Control C, to C4.
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Group A C, B
M. L. KHARE, S. KUMAR AND J. GRUN
156
teristic of NDV. The highest dilution of serum showing no CPE was considered as the endpoint.
1200 1000
800
£
400
RESULTS
200
I
•• • .
/..?—*•**. V ^i*v
•—
/
1200
/ 800 |£
600 400
I
200 0
' I'll
...o
\ v
\\
H
TOTAL Iq M
if il *— /?/
V
:E3*3-|-Ei*L^§
Fio. 2. ME sensitive (IgM) and ME resistant (IgG) HI titer of chicken serum from experiment two: (G) Vaccinated; (I) Control; and experiment three: (H) Vaccinated; (I) Control.
FIG. 3. ME sensitive (IgM) and ME resistant (IgG) SN titer of vaccinated chicken: (K) Experiment two, adult; (L) Experiment three.
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In general, with the exception of mixed age group chickens, vaccinated birds showed an elevated level of total serum protein. The rise in serum protein content in chickens of the contact control group was significantly low. Results of protein analysis from groups A, B, C and their controls (Experiment 1) are presented in Table 2. All serum samples from primary and secondary vaccinated chickens contained HI, SN and precipitating antibodies against NDV. In the primary vaccinated chickens (Experiment 1), HI antibodies were detected in the first week and peaked during the second week (Figure 1). They were detectable throughout the observation period of eight weeks. There was no significant difference in the HI titer of the different age groups. Although SN antibodies were detectable in the first week, the highest SN titer was observed during the fourth week after vaccination (Figure 3K). A sharp rise in HI and SN titers was prominent in the serum samples of chickens following revaccination with R 2 B in both Experiments 2 and 3 (Figures 2G, 2H, 3K, and 3L). Sera from primary and secondary vaccination revealed two precipitation lines in the IP test. Precipitation lines of sera from Experiment 2 were sharper and more intense than the sera from Experiment 1. Precipitin titer in the respective sera was not determined. A low level of HI and SN antibodies titer was observed in chickens from the contact-infected group (Figures IF, 21). Detectable precipitating antibody was not present in these birds. Also, HI, SN and precipitating antibodies were not detected in controls from Experiment 3 (Figure 2J). Precipitating antibodies were also not detected in the IP test performed with 0.85% salt concentration,
• ••
600
157
NEWCASTLE DISEASE
FIG. 4. Sephadex G-200 profile of post vaccinal (seventh week) chicken serum from group A showing IgM and IgG peaks with their SN and HI titers.
body titers, also contained ME sensitive HI antibody. The number of observed precipitation lines in ME treated serum samples was reduced from two to one. Immunoelectrophoresis of ME treated and untreated sera from all three experiments exhibited no difference in the characteristics of different immunoglobulin bands. The result of ME treatment on SN activity of experimental sera was very similar to that obtained with HI activity. A comparative histogram is presented in Figure 3. DISCUSSION There was a definite rise in total serum protein in postvaccinal sera irrespective of age variations. This rise was also parallel with the development of immunoglobulins (antibodies) as evidenced by various serological tests. The appearance and increase in HI, SN and precipitating serum antibodies was detected both after primary and secondary vaccination. To determine which class of immunoglobulins were responsible for the manifestation of different serological tests, three different parameters were employed. Separation of IgM and IgG on Sephadex G-200 indicated that both IgM and IgG immunoglobulins contained serum neutralizing activities. Meyer and Dougherty (1972) reported similar findings in chicken serum against the avian leukosis complex (ALC) virus. However, participation of IgM and IgG in HI activity was different. HI activity was detected in the IgG fraction, whereas it was not detected in the IgM fraction of the sera. This was contradictory to the results obtained with ME treated serum. The presence of ME sensitive immunoglobulin (IgM) along with a subsequent reduction in HI titer was also observed in pooled sera. Considering the validity of the ME effect on the IgM, the lack of HI activity in the IgM fraction seems to be due to its low concentration. Possibly, the IgM
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irrespective of the source of the serum sample. A typical representative Sephadex G-200 profile of sera from group A (Experiment 1, primary R 2 B vaccination) is presented in Figure 4. Two major peaks, representing IgM and IgG, were obtained. The positioning of the peaks was further confirmed by Immunoelectrophoresis of individual and pooled fractions under the respective peaks (Figure 5). HI and SN titers in individual fractions were very low (two to eight). Although HI activity was not detected in the IgM fractions, high HI activity was present in the IgG fractions. SN titers were higher in the fractions of IgG range than the fractions of IgM range. The results of the ME treatment on the serological activities of serum samples is presented in Figures 1, 2 and 3. After ME treatment, a one to threefold reduction of HI titer was observed in the serum samples (Experiment 1). The sera of revaccinated birds (Figures 2G, 2H), having high HI anti-
158
M. L. KHARE, S. KUMAR AND J. GRUN
: T ••
^p-'
*.
.
=#
'
I9 G
^f,
MM ^
lr
FIG. 5A. Immiinoelectropherograms of Sephadex chromatographed individual and pooled fractions of chicken serum showing (arrows) IgM and IgG immunoglobulins (photographic). FIG. 5B. Drawings of the Fig. 5A immunoelectropherograms. RG = Rabbit anti-chicken globulin serum RS = Rabbit anti-chicken whole serum 56, 53 and 42 = Sephadex chromatographed serum fractions S = Unfractionated chicken serum.
concentration was not high enough for the detection of HI activity. Apparently, the optimal concentration of IgM needed to reveal SN activity is lower than that needed to reveal HI activity. Mercapto-ethanol treatment of sera indicated the predominance of ME sensitive (IgM) HI antibodies in the first week postvaccination sera. It was also observed that ME sensitive antibodies persisted in the serum of all age groups up to the eighth week of observation. However, their titer gradually declined with time. Our findings are in agreement with the general consensus of opinion (Pike, 1967), although they disagree with the
findings of Hashiguchi (1969). Utilizing an NDV living vaccine (B, strain) in chickens, Hashiguchi observed that antibody sensitive to ME appeared in serum during the first and second weeks after vaccination and was replaced with resistant antibody in the following weeks. In the contact-infected control group, we found that antibodies were ME sensitive. This indicated that IgM is induced and persists with no detectable IgG antibodies coming into the circulation in chickens which contract the infection naturally. Incidentally, this also shows the difference in antibody response when chickens are artificially infected as against natural infections. In sec-
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l 9
159
NEWCASTLE DISEASE
Reduction in one precipitin line after ME treatment might in part suggest the presence of both IgM and IgG precipitating antibodies. Our results indicated that the general pattern of SN antibodies was similar to HI antibodies and that IgM and IgG immunoglobulins were involved in virus neutralization. Similar findings have been reported for foot-and-mouth virus in guinea pigs (Cowon and Traurtman, 1965), polio virus (Svehag and Mandel, 1965), influenza A virus in mice (Berlin, 1963), and for several arboviruses in guinea pigs (Bellanti et al., 1965). It is apparent from our findings that IgM antibodies appear in chicken sera during the primary response, and also during the secondary response. Our findings suggest that, in the chicken system, the sequential appearance of immunoglobulins in response to viral antigen differs from that described for mammalian systems (Pike, 1967). ACKNOWLEDGMENT The authors are thankful to Dr. M. P. Bansal, I.V.R.I., Izatnagar, India, for his help
in serum neutralization tests in tissue culture during the course of study. The authors are also grateful to Dr. Eugene V. Adams, Department of Animal Sciences, Cook College, Rutgers University for his aid in the preparation of this manuscript. REFERENCES Bellanti, J. A., S. E. Russ, G. E. Holmes and E. L. Buescher, 1965. The nature of antibodies following experimental arbovirus infection in guinea pigs. Immunology, 94: 1-11. Berlin, B. A., 1963. The nature of antibodies in mice infected with influenza virus vaccine. Proc. Soc. Expt. Biol. Med. 113: 1013-1016. Cowon, K. M., 1973. Antibody response to viral antigens. Ad. Imm. 17: 195-215. Cowon, K. M., and R. Traurtman, 1965. Antibodies produced by guinea pigs infected with foot-andmouth disease virus. J. Immunol. 94: 858-867. Cunningham, C. H., 1966. A Laboratory Guide in Virology. Sixth ed. Burgess Publishing Company, Minneapolis, Minnesota. Fischer, L., 1969. An Introduction to Gel Chromatography. North Holland Publications, Netherlands. Hamper, B., I. M. Martos, M. Chakrabarty and M. A. K. Burroughs, 1970. Late 19S rabbit antibody neutralization test for differentiating herpes simplex virus types 1 and 2. J. Immunol. 104: 593-598. Hashiguchi, Y., 1969. Relationship between antibody and immunity in chickens vaccinated with Newcastle disease living vaccine (B, strain). Virus, 19(4): 143-154. Merchant, D. J., R. H. Kahn and W. H. Murphy, Jr., 1960. In: Handbook of Cell and Organ Culture. Burgess Publishing Company, Minneapolis, Minnesota. Meyer, P., and R. M. Dougherty, 1972. Analysis of immunoglobulins in chicken antibody to avian leukosis virus. Immunol. 23: 1-4. Pike, R. M., 1967. Antibody heterogeneity and serological reactions. Bact. Rev. 31(1): 157-174. Svehag, S. E., and B. Mandel, 1964. The formation and properties of poliovirus-neutralizing antibody. I. 19S and 7S antibody formation: difference in. J. Exp. Med. 119: 1-9. Szenberg, A., P. Lind and K. Clarke, 1965. IgG and IgM antibodies in fowl serum. Aust. J. Exp. Biol. Med. Sc. 43: 451-454. Weir, D. M., 1967. Handbook of Experimental Immunology. Edt. Blackwell Scientific Publications, Oxford, U.K.
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ondary vaccination trials (Experiments 2 and 3) both in R 2 B and F - R 2 B series ME sensitive (IgM) antibodies were detected in greater amounts in comparison to ME resistant (IgG) antibodies. Evidently, a high IgM titer is accompanied by a high HI titer. Indirectly, this indicates that IgM is mostly responsible for the HI activity of the serum. Persistence of serum IgM antibodies in the secondary response has also been reported in chickens with influenza A infection (Szenberg, 1965). It seems possible that the induction of a high IgM response due to a secondary antigenic stimulus is a property of the mesogenic R 2 B strain of NDV. This property might be useful in differentiating NDV strains. Hamper et al. (1970), used this difference in IgM response for the differentiation of herpes simplex virus types.
Department of Animal Sciences (Pathobiology), Thompson Hall, Cook College, Rutgers University, New Brunswick, N.J. 08903, and 2Division of Bacteriology and Virology, Indian Veterinary Research Institute, Mukteswar (U.P.), India (Received for publication April 15, 1975)
POULTRY SCIENCE 55: 152-159, 1976
INTRODUCTION
I
N response to viral antigens, heterogeneity of immunoglobulins in various species is well documented (Cowon, 1973; Pike, 1967). In terms of antibodies, serological manifestation of an i m m u n e serum is a combined activity of the individual immunoglobulins participating in t h e serological test. All immunoglobulins may or may not be actively present in the immune sera at one time or all the time after infection or vaccination (Cowon, 1973). Individual immunoglobulins vary considerably in their degree of participation in a particular serological test. M o r e o v e r , sensitivity of a serological test
*Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University—The State University of New Jersey, Department of Animal Sciences, New Brunswick, New Jersey 08903. Presented in part at the 73rd Annual Meeting of the American Society for Microbiology, Miami, Florida, May 6-11, 1973. 1. In large part taken from M.V.Sc. (1971) thesis of the senior author submitted to Agra University, India.
system also varies in relation to the type of immunoglobulin present (Pike, 1967). Therefore, characterization of antibody r e s p o n s e s in terms of types of immunoglobulins, their induction times and their relative concentrations in t h e immune serum are n e c e s s a r y . Such studies might provide a more complete recognition of the infective or immune status of the host as compared with the d e m o n stration of antibody against a specific antigen or pathogen. It might also provide a better interpretation, thereby avoiding false and misleading serological analysis of immune sera. In the present communication we have reported the qualitative and quantitative induction of the chicken immunoglobulins in response to R 2 B (Mukteswar) and F strains of Newcastle disease virus (NDV). MATERIALS AND METHODS Experimental Birds. Forty-nine male and female White Leghorn chickens obtained from the Indian Veterinary R e s e a r c h Institute (Izatnagar, Uttar P r a d e s h , India) poultry farm ( P P L O free) were used in these studies. 152
Downloaded from http://ps.oxfordjournals.org/ at Osaka Daigaku Ningen on May 7, 2015
ABSTRACT The qualitative and quantitative development of chicken immunoglobulins in response to R 2 B (Mukteswar) and F strain of Newcastle disease virus was studied. One primary (R2B) and two secondary (R 2 B - R 2 B, F — R 2 B) vaccination trials were conducted. Sera was collected at weekly intervals and analyzed. There was an increase in total serum protein content in parallel to an increase in serum neutralizing (SN), hemagglutination inhibition (HI), and precipitating antibodies. The SN, HI and precipitating activities were detected both in IgM and IgG immunoglobulins when sera were treated with mercapto-ethanol. However, Sephadex G-200 fractionated sera showed only SN activity in the IgM fraction, whereas the IgG fraction showed both HI and SN activity. Serum IgM antibodies appeared during the first week following vaccination, then diminished, then rose again following secondary vaccination. Apparently, immunoglobulin induction and serological activities were not significantly influenced by the age of the chickens.
153
NEWCASTLE DISEASE
Vaccination. All chickens, with the exception of one-day-old chicks, were tested for preinoculation serum antibodies to NDV by the HI test. Chickens with even a doubtful HI titer were not included in the experiments. Experiment 1. Twenty-five chickens were distributed in five groups (A to E) according to age as detailed in Table 1. They were vaccinated subcutaneously with 1.0 ml. of R 2 B vaccine. Five more groups (C, to Cs) were kept as unvaccinated controls.
the R 2 B vaccine (primary vaccination), were divided into two groups. The first group (G) was revaccinated subcutaneously with 1.0 ml. of R 2 B vaccine (secondary vaccination), while the other group (I) was kept as uninoculated controls. All vaccinated and unvaccinated (contact infection) chickens from Experiments 1 and 2 were held in the same room in separate cages. Experiment 3. Twenty, one-day-old chicks were vaccinated intranasally with the " F " strain (primary vaccination). Twelve weeks post vaccination, six chickens (group H) were revaccinated subcutaneously with 1.0 ml. of R 2 B vaccine (secondary vaccination). Four chicks (group J) were kept as unvaccinated controls. The vaccinated birds from Experiment 3 were housed and cared for separately from the controls.
Experiment 2. Six chickens (20 to 24 weeks of age), vaccinated eight weeks earlier with
Collection of Sera. All chickens were bled before vaccination and at weekly intervals after vaccination. Collected serum samples were stored at -20° C. until used. No preservatives were added. When required, pooled serum samples were obtained by mix-
TABLE 1.—NDV vaccination schedule in chickens from Experiments 1, 2 and 3 Exp. I.
II. III.
Age of vaccination
Vaccine
Dose/i route
Groups
No. of chickens
Primary
Secondary
Primary
Secondary
A B C D E
3 8 3 6 5
adult 12-16 wks 10-12 wks. 8-10 wks. mixed
— — — — —
R2B R2B R2B R2B R2B
— — — — —
c,-c5*
t
G I** H J**
3 3 6 4
20-24 wks. 20-24 wks. 12 wks. 12 wks.
R2B R2B F F
R2B n.s. R2B n.s.
12-16 12-16 day day
wks. wks. old old
*Control groups (for respective infective groups) inoculated with normal saline only. "Controls: n.s. = normal saline; i.n. = intranasal. t C, = 1, C2 = 2, C3 = 2, C4 = 2, C5 = 3.
Primary 1 1 1 1 1
Secondary
— — — — —
ml./s.c. ml./s.c. ml./s.c. ml./s.c. ml./s.c.
1 ml./s.c. 1 ml./s.c. 0.2ml./i.n. 0.2 ml./i.n.
1 1 1 1
ml./s.c. ml./s.c. ml./s.c. ml./s.c.
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Vaccine. One-day-old chicks were vaccinated with the " F " strain of Newcastle disease virus (NDV) vaccine. For the primary and secondary vaccinations R 2 B (Mukteswar) strain of NDV with an ELD 5 0 titer greater than 10 8 /ml. was used. A chicken fibroblast cell culture adapted Mukteswar (R 2 B) strain of NDV was passed three times in chick embryos, collected, titrated and stored as seed virus at -20° C. Prior to vaccination, the seed virus was given another passage in chick embryos, titrated and used for primary and secondary vaccination (ELD 50 , 107ml.).
154
M. L. KHARE, S. KUMAR AND J. GRUN
ing equal quantities of individual serum samples. Serum Protein Estimation. The modified Biuret method (Weir, 1967) was used in determining total serum proteins. The test was read at 545 m^.. using Spectronic-20. Purified bovine serum (Armour) was used in preparing the standard curve.
3. Immunoelectrophoresis.
Immunoelec-
Assessment of Antibody Response. 1. Hemagglutination-Inhibition Test. The test was conducted according to the method described by Cunningham (1966). Dilutions of R 2 B virus having two or four hemagglutinating (HA) units were used as antigen, whereas test serum samples and their fractions were used as antisera. A constant volume (0.25 ml.) of test ingredients was employed in each test. The final HI titer of the serum sample was calculated by multiplying the observed serum titer by the HA units used. 2. Immunoprecipitin Test. A standard immunoprecipitin (IP) test was performed (Weir, 1967). Purified agar (1%) in phosphate buffer (pH 7.2) was utilized. Antigen and antiserum wells (two mm. in diameter) were five mm. from each other. A suspension of NDV (R 2 B) infected chorioallantoic membrane in allantoic fluid served as the antigen. Agar gels containing high (8%) and low (0.85%) concentrations of sodium chloride were utilized. Plates were incubated at room temperature (25 to 28° C.) for 48 to 72 hours and examined for the development of precipitin lines. 3. Serum Neutralization Test. The SN test was performed in a fibroblast cell culture system. Chick embryo fibroblast cell cultures
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Identification of Immunoglobulins. 1. Mercapto-Ethanol Treatment of Sera. For the detection of IgM and IgG immunoglobulins in test sera, serum samples were treated with 2-mercapto-ethanol (ME). IgM is ME sensitive, whereas IgG is resistant. A constant volume of individual serum sample was divided into two equal parts. One half was diluted with an equal volume of 0.2 M ME and incubated for one hour at 37° C. in a water bath. The other half, diluted with phosphate buffer saline alone and incubated at 37° C , was used as a control. After dialysis, ME treated and untreated serum samples were subjected to various serological tests and Immunoelectrophoresis. 2. Sephadex-Gel Chromatography. For the separation of IgM and IgG antibodies, test sera were fractionated on Sephadex G-200 (Upsala, Sweden) as described by Fischer (1969). A glass column (2.5 x 90 cm.) of 430 ml. capacity was used. Elution of the serum sample (2 ml.) was carried out at a flow rate of 2 ml./minute in 0.15 M normal saline solution. Fractions were collected and stored at 4° C. The optical density of fractions was measured at 280 mix. using an ultraviolet spectrophotometer (Beckman) and an elution curve was obtained utilizing the data. Immunoelectrophoresis and various serological tests were performed on the individual fractions. Also, fractions comprised of peaks in the elution curve were pooled, concentrated (by lyophilization) and subjected to similar analysis.
trophoresis (IE) was employed on microslides utilizing 1.5% agar gel in 0.025 M barbital buffer (pH 8.6). The same buffer was used in electrode vessels. Electrophoresis was carried out for 2.5 hours at a constant current of two milliamperes per slide. For the identification of immunoglobulins in the test sera, electrophoresed serum samples and their fractions were incubated at room temperature (28° C.) with rabbit anti-chicken serum and rabbit anti-chicken globulin. Resultant electropherograms were preserved and stained, as described by Weir (1967). Antisera against whole chicken serum and globulins (ammonium sulphate precipitated) were produced separately in rabbits (Weir, 1967).
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NEWCASTLE DISEASE
ham, 1966). Cell culture adapted R 2 B strain of NDV in 0.1 ml. volumes containing 100 to 1000 TCID 50 (tissue culture infective dose) of virus was the antigen. The cell cultures were inoculated with either a mixture of virus and test serum, or virus alone (controls). Cultures were observed for 72 hours after infection for cytopathic effect (CPE) charac-
were prepared from 10 to 12 day-old embryos according to the standard method of Merchant et al. (1960). The growth media consisted of Hanks' balanced salt solution, supplemented with lactalbumin hydrolysate 0.5%, yeast extract 0.05% and 10% inactivated calf serum with penicillin. The test proper was conducted by the beta-procedure (Cunning-
TABLE 2.—Mean total serum protein profile of chickens from experiment one over a period of eight weeks Total protein (gm./lOO ml.) M.P.V.P. %Rise 27 5.80 13 3.70 17 5.25 8 5.00 34 5.97 9 4.42
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FIG. 1. ME sensitive (IgM) and ME resistant (IgG) HI titer of chicken serum from birds of various groups of experiment one: (A) Adult; (B) Age 12-16 weeks; (C) Age 10-12 weeks; (E) Mixed age, at the time of vaccination; (F) Control C, to C4.
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Group A C, B
M. L. KHARE, S. KUMAR AND J. GRUN
156
teristic of NDV. The highest dilution of serum showing no CPE was considered as the endpoint.
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FIG. 3. ME sensitive (IgM) and ME resistant (IgG) SN titer of vaccinated chicken: (K) Experiment two, adult; (L) Experiment three.
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In general, with the exception of mixed age group chickens, vaccinated birds showed an elevated level of total serum protein. The rise in serum protein content in chickens of the contact control group was significantly low. Results of protein analysis from groups A, B, C and their controls (Experiment 1) are presented in Table 2. All serum samples from primary and secondary vaccinated chickens contained HI, SN and precipitating antibodies against NDV. In the primary vaccinated chickens (Experiment 1), HI antibodies were detected in the first week and peaked during the second week (Figure 1). They were detectable throughout the observation period of eight weeks. There was no significant difference in the HI titer of the different age groups. Although SN antibodies were detectable in the first week, the highest SN titer was observed during the fourth week after vaccination (Figure 3K). A sharp rise in HI and SN titers was prominent in the serum samples of chickens following revaccination with R 2 B in both Experiments 2 and 3 (Figures 2G, 2H, 3K, and 3L). Sera from primary and secondary vaccination revealed two precipitation lines in the IP test. Precipitation lines of sera from Experiment 2 were sharper and more intense than the sera from Experiment 1. Precipitin titer in the respective sera was not determined. A low level of HI and SN antibodies titer was observed in chickens from the contact-infected group (Figures IF, 21). Detectable precipitating antibody was not present in these birds. Also, HI, SN and precipitating antibodies were not detected in controls from Experiment 3 (Figure 2J). Precipitating antibodies were also not detected in the IP test performed with 0.85% salt concentration,
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157
NEWCASTLE DISEASE
FIG. 4. Sephadex G-200 profile of post vaccinal (seventh week) chicken serum from group A showing IgM and IgG peaks with their SN and HI titers.
body titers, also contained ME sensitive HI antibody. The number of observed precipitation lines in ME treated serum samples was reduced from two to one. Immunoelectrophoresis of ME treated and untreated sera from all three experiments exhibited no difference in the characteristics of different immunoglobulin bands. The result of ME treatment on SN activity of experimental sera was very similar to that obtained with HI activity. A comparative histogram is presented in Figure 3. DISCUSSION There was a definite rise in total serum protein in postvaccinal sera irrespective of age variations. This rise was also parallel with the development of immunoglobulins (antibodies) as evidenced by various serological tests. The appearance and increase in HI, SN and precipitating serum antibodies was detected both after primary and secondary vaccination. To determine which class of immunoglobulins were responsible for the manifestation of different serological tests, three different parameters were employed. Separation of IgM and IgG on Sephadex G-200 indicated that both IgM and IgG immunoglobulins contained serum neutralizing activities. Meyer and Dougherty (1972) reported similar findings in chicken serum against the avian leukosis complex (ALC) virus. However, participation of IgM and IgG in HI activity was different. HI activity was detected in the IgG fraction, whereas it was not detected in the IgM fraction of the sera. This was contradictory to the results obtained with ME treated serum. The presence of ME sensitive immunoglobulin (IgM) along with a subsequent reduction in HI titer was also observed in pooled sera. Considering the validity of the ME effect on the IgM, the lack of HI activity in the IgM fraction seems to be due to its low concentration. Possibly, the IgM
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irrespective of the source of the serum sample. A typical representative Sephadex G-200 profile of sera from group A (Experiment 1, primary R 2 B vaccination) is presented in Figure 4. Two major peaks, representing IgM and IgG, were obtained. The positioning of the peaks was further confirmed by Immunoelectrophoresis of individual and pooled fractions under the respective peaks (Figure 5). HI and SN titers in individual fractions were very low (two to eight). Although HI activity was not detected in the IgM fractions, high HI activity was present in the IgG fractions. SN titers were higher in the fractions of IgG range than the fractions of IgM range. The results of the ME treatment on the serological activities of serum samples is presented in Figures 1, 2 and 3. After ME treatment, a one to threefold reduction of HI titer was observed in the serum samples (Experiment 1). The sera of revaccinated birds (Figures 2G, 2H), having high HI anti-
158
M. L. KHARE, S. KUMAR AND J. GRUN
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FIG. 5A. Immiinoelectropherograms of Sephadex chromatographed individual and pooled fractions of chicken serum showing (arrows) IgM and IgG immunoglobulins (photographic). FIG. 5B. Drawings of the Fig. 5A immunoelectropherograms. RG = Rabbit anti-chicken globulin serum RS = Rabbit anti-chicken whole serum 56, 53 and 42 = Sephadex chromatographed serum fractions S = Unfractionated chicken serum.
concentration was not high enough for the detection of HI activity. Apparently, the optimal concentration of IgM needed to reveal SN activity is lower than that needed to reveal HI activity. Mercapto-ethanol treatment of sera indicated the predominance of ME sensitive (IgM) HI antibodies in the first week postvaccination sera. It was also observed that ME sensitive antibodies persisted in the serum of all age groups up to the eighth week of observation. However, their titer gradually declined with time. Our findings are in agreement with the general consensus of opinion (Pike, 1967), although they disagree with the
findings of Hashiguchi (1969). Utilizing an NDV living vaccine (B, strain) in chickens, Hashiguchi observed that antibody sensitive to ME appeared in serum during the first and second weeks after vaccination and was replaced with resistant antibody in the following weeks. In the contact-infected control group, we found that antibodies were ME sensitive. This indicated that IgM is induced and persists with no detectable IgG antibodies coming into the circulation in chickens which contract the infection naturally. Incidentally, this also shows the difference in antibody response when chickens are artificially infected as against natural infections. In sec-
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l 9
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NEWCASTLE DISEASE
Reduction in one precipitin line after ME treatment might in part suggest the presence of both IgM and IgG precipitating antibodies. Our results indicated that the general pattern of SN antibodies was similar to HI antibodies and that IgM and IgG immunoglobulins were involved in virus neutralization. Similar findings have been reported for foot-and-mouth virus in guinea pigs (Cowon and Traurtman, 1965), polio virus (Svehag and Mandel, 1965), influenza A virus in mice (Berlin, 1963), and for several arboviruses in guinea pigs (Bellanti et al., 1965). It is apparent from our findings that IgM antibodies appear in chicken sera during the primary response, and also during the secondary response. Our findings suggest that, in the chicken system, the sequential appearance of immunoglobulins in response to viral antigen differs from that described for mammalian systems (Pike, 1967). ACKNOWLEDGMENT The authors are thankful to Dr. M. P. Bansal, I.V.R.I., Izatnagar, India, for his help
in serum neutralization tests in tissue culture during the course of study. The authors are also grateful to Dr. Eugene V. Adams, Department of Animal Sciences, Cook College, Rutgers University for his aid in the preparation of this manuscript. REFERENCES Bellanti, J. A., S. E. Russ, G. E. Holmes and E. L. Buescher, 1965. The nature of antibodies following experimental arbovirus infection in guinea pigs. Immunology, 94: 1-11. Berlin, B. A., 1963. The nature of antibodies in mice infected with influenza virus vaccine. Proc. Soc. Expt. Biol. Med. 113: 1013-1016. Cowon, K. M., 1973. Antibody response to viral antigens. Ad. Imm. 17: 195-215. Cowon, K. M., and R. Traurtman, 1965. Antibodies produced by guinea pigs infected with foot-andmouth disease virus. J. Immunol. 94: 858-867. Cunningham, C. H., 1966. A Laboratory Guide in Virology. Sixth ed. Burgess Publishing Company, Minneapolis, Minnesota. Fischer, L., 1969. An Introduction to Gel Chromatography. North Holland Publications, Netherlands. Hamper, B., I. M. Martos, M. Chakrabarty and M. A. K. Burroughs, 1970. Late 19S rabbit antibody neutralization test for differentiating herpes simplex virus types 1 and 2. J. Immunol. 104: 593-598. Hashiguchi, Y., 1969. Relationship between antibody and immunity in chickens vaccinated with Newcastle disease living vaccine (B, strain). Virus, 19(4): 143-154. Merchant, D. J., R. H. Kahn and W. H. Murphy, Jr., 1960. In: Handbook of Cell and Organ Culture. Burgess Publishing Company, Minneapolis, Minnesota. Meyer, P., and R. M. Dougherty, 1972. Analysis of immunoglobulins in chicken antibody to avian leukosis virus. Immunol. 23: 1-4. Pike, R. M., 1967. Antibody heterogeneity and serological reactions. Bact. Rev. 31(1): 157-174. Svehag, S. E., and B. Mandel, 1964. The formation and properties of poliovirus-neutralizing antibody. I. 19S and 7S antibody formation: difference in. J. Exp. Med. 119: 1-9. Szenberg, A., P. Lind and K. Clarke, 1965. IgG and IgM antibodies in fowl serum. Aust. J. Exp. Biol. Med. Sc. 43: 451-454. Weir, D. M., 1967. Handbook of Experimental Immunology. Edt. Blackwell Scientific Publications, Oxford, U.K.
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ondary vaccination trials (Experiments 2 and 3) both in R 2 B and F - R 2 B series ME sensitive (IgM) antibodies were detected in greater amounts in comparison to ME resistant (IgG) antibodies. Evidently, a high IgM titer is accompanied by a high HI titer. Indirectly, this indicates that IgM is mostly responsible for the HI activity of the serum. Persistence of serum IgM antibodies in the secondary response has also been reported in chickens with influenza A infection (Szenberg, 1965). It seems possible that the induction of a high IgM response due to a secondary antigenic stimulus is a property of the mesogenic R 2 B strain of NDV. This property might be useful in differentiating NDV strains. Hamper et al. (1970), used this difference in IgM response for the differentiation of herpes simplex virus types.