Immunoglobulin class response to canine distemper virus in gnotobiotic dogs

Veterinary Immunology and Immunopathology, 5 (1983/1984) 209--215 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

~ B U L I N

209

CI2~S RESPONSE TO CANINE DISTEI~PER VIRUS IN 6~kK)TOBIOTIC DOGS

KAREN A. WII~rsHSI, MS; LAWRI~CE E. MATHES I, PhD; STEVI~ KRAKOWKA 1 , DVM, PhD; AND RICHARD G. OLSE~ 1'2, PhD IDepartment of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio.

2Reprint requests to Richard G. Olsen,

Goss Laboratory, 1925 Coffey Road, Col~nbus, Ohio 43210, USA 614-422-5661 (Accepted 7 June 1983) ABSTRACT Winters, K.A., Mathes, L.E., Krakowka, S. and Olsen, R.G., 1983. Immunoglobulin class response to canine distemper virus in gnotobiotic dogs. Vet. Inlaunol. ~ o p a t h o l . , 5: 209-215. Serial ser~n samples from 27 gnotobiotic dogs infected with R252-canine distemper virus (CDV) were tested for anti-viral IgG, IgM and IgA J/mxmoglobulins using an enzyme-linked immunosorbent assay (ELISA). The results were compared retrospectively to clinicopathological course of disease and to previously reported patterns of complement-fixing and virus neutralizing antibody titers determined in these same sera. Virus-specific IgA was never detected in the sera. High levels of IgG correlated with recovery from disease, whereas the antiviral IgM levels were equivalent in both persistently infected animals and those animals which recovered from disease. The inability to sustain a significant antiviral antibody response in either IgM or IgG classes was characteristic of dogs with fatal encephalitis. The data suggests that IgG is the most important Ig class for recovery from disease. 1-NTRODtL-~IGN

The importance of the antiviral htm~ral response during canine distemper virus (CDV) infection is well recognized (Appel 1969; Krakowka et al., 1975a). Gnotobiotic do~s infected with R252-CDV produce levels of antiviral

anti-

bodies, as measured by either cQmplement fixation (CF) or virus neutralization (VN), that vary inversely with the severity of clinical disease (Krakowka et al., 1975a).

Group

1 dogs developed acute

fatal encephalitis

following

infection with CDV and contain little or no detectable antibody activity in their ser~n. Group 2 animals survived the CDV infection, producing moderate antibody titers, necropsy.

but were

found to be chronically infected with CDV at

Group 3 did not exhibit clinical illness and did not have lesions

of active infection at necropsy.

These dogs produced the highest titers of

antibody.

0165-2427/83/$03.00

© 1983 Elsevier Science Publishers B.V.

210

Specific antibody classes present in the serum of dogs in these 3 groups have not been measured and,

therefore,

class in elimination and protection

the possible

role of each antibody

from CDV has not been determined.

The

objective of this study was to develop an enzyme-linked inmunosorbent assay (ELISA) to measure the IgG, IgM and IgA anti-CDV antibody and to apply the assay to sera from CDV-infected gnotobiotic dogs in each of these 3 groups in order to determine which specific ~ o g l o b u l i n

class is most significant for

recovery from disease.

MATERIALS AND METHODS Experimental Canine Sera Sera from 27 R252-CDV-infected gnotobiotic dogs were used.

The methods of

viral challenge, viral inoculum, sample collection, and clinical and pathological evaluation have been published al.,

1975a).

(McCullough et al.,

1974a;

Krakowka et

Serum samples were collected weekly and stored at -20C until

use.

Enzyme-Linked ~ o s o r b e n t An enzyme-linked

Assay (ELISA)

inrmlnosorbent assay was developed for detection of anti-

body to CDV by modifying (Pdce et al., green monkey 1975).

1981). kidney

the procedures used previously

The CDV antigen (Vero) cells

laboratory

as previously

described

(Confer

et al.,

Cell-associated virus from 75 mm 2 flasks was prepared by treating the

cell pellets with extract buffer 4.4,

in our

for basic coat was grown in African

0.5% NP40,

and

0.5%

(0.5M sodium chloride,

sodium deoxycholate)

cellular debris was re~noved by centrifugation.

for

0.02M Tris HCl, pH

1 to

2 hr,

22C.

A protease inhibitor,

The Apro-

tinin (10% v/v) , was added to the supernate and dilutions of this material was used as the CDV basic coat antigen. Linbro microtiter plates were coated with CDV antigen at a 1 :50 dilution in sodium carbonate buffer, pH 9.6.

After an overnight incubation at 4C, the

basic

plates

coat

(2000:I).

was

removed

and

the

were

washed

with

saline-tween-20

Duplicate test ser~n samples were diluted i/I0 and incubated in the

plates for 1 hr, 37C.

The plates were then washed and the anti-canine IgG,

IgA or IgM-horseradish peroxidase conjugates and incubated for an additional hr at 37C.

(Nakane et al., 1974) were added Finally,

the plates were again

washed and enzyme substrate was added and allowed to react for 1 hr.

The

substrate consisted of 9 parts 0.08% 5-amino salicylic acid at pH 6, and 1 part 0.6% H202.

One percent NaN 3 was added to each well to stop the reaction.

Positive and negative controls were included in each test plate.

211

Standard curves for absorbency vs. ng bound for each conjugate

(of known

protein concentration) were constructed using 4 replicates/dilution and 20 ~I conjugate dilutions per well, for each of the anti-canine Ig conjugates as described (Rice et al., 1982).

From this, optical density (absorbency) values

for each sample were determined and recorded as mean values of Ig bound per I0 ~i serum. RESULTS Mean anti-CDV antibody levels for dogs of group 1 (n=9) are shown in figure i.

These dogs developed acute fatal encephalitis following infection with CDV

1.0

E 0.9

co

0.8

>-J c:~ b..I rO >., 0 . 7 I

...o

A

~ IgG

C

O

IgM

•~ ..o 0 . 6 rCD <[-,-

o 0.5 O

~-

~ E3 0 . 4 fl,)

- - "o 0 . 3 o ~

.,__ 0 . 2

r--

o4 m

r-

0.1

0 -7

0

7

14

21

28

:55 4 2

49

I 56

Doys Post Infection Figure i. Anti-canine d i s ~ r virus ser~n antibody in 9 dogs fatally infected with R252-CDV. IgG (0.3 ng/10 ~i ser~n) and IgM (0.09 ng/10 ~i ser~n) were first detected on post-infection day (PID) 7 and gradually decreased to background by PID 42.

212

and survived no longer than 49 days after infection.

Trace amounts of IgG

(0.3 ng/10 pl) and IgM (0.09 ng/10 pl) were detected on PID 7 and gradually decreased to undetectable levels thereafter. Group 2 dogs (n=9) survived the acute phase of infection but were found to have persistent infection and chronic encephalitis at necropsy.

These dogs

produced beth anti-CDV IgG and IgM antibody throughout the observation period (Figure 2).

However, mean values remained at approximately 0.5 ng/10 ~i for

1.0

E

0.9 c~ co O. 8 >.__1 clw 0.7 I

a

IgG

o

IgM

..Q

•~ . ~ c-

o

~

0.6


w-- a~ O ~Q) -

~r7

0.4

y,°

o.3

~

0.2

(D

E

~

r-

0.1 0

-7

0

7

14

21

28

35 42 49

56

63 70 77

84

Doys Post Infection

Figure 2. Anti-canine distenlper virus seru~ antibody in 9 dogs persistently infected with R252-C~V. IgG and IgM were detected by PID 7 and continued to be produced throughout the study at mean values of approximately 0.5 ng IgG/10 ~i serum and 0.2 ng IgM/10 ~i serum.

213

IgG and 0.2 ng/10 ~i for IgM.

The level of IgG on PID 7 was equivalent to

that measured on PID 7 for group I, but by PID 14, group 2 IgG had increased to 0.43 ng/10 ~I, wherein group 1 values had dropped to 0.09 ng/10 pl. Dogs of group 3 (n=9) did not exhibit clinical illness and had no lesions of active infection at necropsy.

Anti-CDV IgG (0.47 ng/10 pl) and IgM (0.21

ng/10 ~I) were detected by PID 7 (Figure 3). values on PID 21 of 0.96 ng/10 pl.

IgG values rose rapidly to peak

From PID 28 to 56, levels re~ained fairly

constant (0.80 ng/10 pl) and then gradually declined to 0.21 ng/10 pl by PID 84.

IgM

also

1.0

E

0.9 oOcf) >._-J 0.8 all 0.7

peaked

on PID 21 (0.31 ng/10 pl)

and

gradually

decreased

B

~

IgG

B

m

I

"-= --o 0 . 6 <~-,O

o 0.5 +-

~r"~ 0.4

m

(D

--o

y,°

0.3

~

0.2

..Q

r-

o~ ~

c-

0.1

0

-7

0

i

J

l

I

7

14

21

28

[

[

55 42

i

[

49

56

[

l

[

65 70 7 7

~

84

Doys Post Infection

Figure 3. Anti-canine distemper virus serum antibody in 9 convalescent dogs infected with R252-CDV. IgG and IgM were detected by PID 7 with IgG values rising to 0.96 ng/10 pl sert~ by PID 21, and then gradually dropping to 0.21 ng/10 pl by PID 84.

214 to 0.07 nq/lO ul by PID 84. Thirty-five serum samples from dogs in groups 1, 2 and 3 were tested in ELISA for anti-CDV IqA. Virus-specific IqA was not detected in any of the samples tested.

DISCUSSION Previous study has shm

that sraananti-CDV titers as rnaasured. by either

CF or VN varied inversely with the severity of the disease (Krakmka gal., 1975a). The results obtained with ELISA confirm these findings, and indicate that the antibody class nrxt prcxainentduring recovery from infection is I$. Others (Bernards

&.,

1982), using the ELISA technique, have -hasized

the

importance of virus-specific IqG in the evolution of anti-virus inrnunity following vaccination with a modified-live vaccination product. Dogs persistently infected with CDV produced 1~

levels of IqG, while those dogs which

recovered fram infection producted high levels of IqG.

Similar amDunts of

IqM were found in both the recovered and persistently infected dogs. inability to mount an adequate hmral

The

response, as observed in fatally

infected dogs, resulted in acute encephalitis and, ultimately, death. The ELISA method used in this study relies upon the ability of the antiglobulin conjugates to detect virus Iq class-specific responses in unfractionated dilute serum samples. Accordingly, campetition for bound antigen by each Iq class could have resulted in the underestimationof the actual aunt of each individual Iq class bound. For this reason, the ratios of detectable In either

IqG vs IqM may be a aore realistic way to express these data.

case, however, our findings are clear; prompt developsent of high levels of CDV-specific IqG correlates with eventual recovery from infection. A critical feature in the pathcqenesis of infection is CDV-associated 1yMphoid depletion and necrosis (McCullouqhet al., 1974a), lynphopenia and accospanyinq Wosuppression 1982).

(Krak~ka SC.,

197513,1977, 1980; Krakmka,

The duration and deqree of lyn-phoidtissue damage also appears to

vary with the severity of the disease. The relative failure to produce high levels of IgG in persistently infected.dogs suggests that the inxnunosuppression associated with CDV may act primarily on the T-lymphocyte population. The production of IqG antibody is knm

to be a T-cell-dependent event,

perhaps involving the IqM to IqG switch (Sellanti, 1978). possible that some T-lphccyte

It is thus

functions in persistently infected dogs are

compromised by infection, thereby resulting in depressed levels of CDVspecific IqG with near normal response in the IqM class. A rapid and mre extensive destruction of the imrolne system would prevent the animal from responding in an appropriate manner with either IqN or IqG and thus result in

215

a fatal infection.

Further study is needed to determine if this apparent

failure of immunoglobulin class switch in persistently infected dogs is due to a generalized immunosuppressive state induced by CDV or to a more specific inhibition of helper T-cell function in these animals. ACKNCWLEDG~ This research was supported by The State of Ohio Canine Research Funds and grant no.

2ROI NS 14821-13, National Institutes of Health,

Public Health

Service. REFERENCES Appel, M.J.G., 1969. Pathogenesis of canine distemper. Am. J. Vet. Res. 30, 1167-1182. Bernard, S.L., Shen, D.T., and Gorham, J.R. 1982. Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens. Am. J. Vet. Res. 43, 22662269. Belanti, J.L. 1978. Immunology. Philadelphia, PA, W.B. Saunders Co., 180-185. Confer, A.W., Kahn, D.E., Koestner, A., and Krakowka, S. 1975. Biological properties of a canine distemper virus isolate associated with dermyelinating encephalomyelitis. Infect. Immun. Ii, 835-844. Krakowka, S., Olsen, R., Confer, A., Koestner, A., and McCullough, B. 1975a. Serologic response to canine distemper viral antigens in gnotobiotic dogs infected with canine distemper virus. J. Infect. Dis. 132, 384-392. Krakowka, S., Cockerell, G., and Koestner, A. 1975b. Effects of canine dister~0er virus on ly~ohoid function in vitro and in vivo. Infect. Immun. ii, 1069-1075. Krakowka, S. and Koestner, A. 1977. Comparison of canine dister~r virus strains in gnotobiotic dogs: Effects on lymphoid tissues. Am. J. Vet. Res. 38, 1919-1922. Krakowka, S., Higgins, R.J., and Koestner, A. 1980. Canine diste~oer virus: Review of structural and functional modulation in lymphoid tissues. Am. J. Vet. Res. 41, 284-292. Krakowka, S. 1982. Mechanisms of in vitro immunosuppression in canine distemper virus infection. J. Clin. Lab. Ir~m/nol. 8, 187-196. McCullough, B., Krakowka, S., and Koestner, A. 1974a. Experimental canine disten~er virus-induced lymphoid depletion. Am. J. Path. 74, 155-166. McCA/llough, B., Krakowka, S., Koestner, A., and Shattuck, J.A. 1974b. Denlyelinating activity of canine dister~0er virus isolates in gnotobiotic dogs. J. Infect. Dis. 130, 343-350. Nakane, P.K. and Kawaoi, A.I. 1974. Peroxidase-labeled antibody - a new method of conjugation. J. Histochem. Cytochem. 22, 1084-1091. Rice, J.B. and Olsen, R.G. 1981. Feline oncornavirus-associated cell membrane antigen and feline leukemia virus group-specific antigen expression in bone marrow and sert~n. J. Natl. Cancer Inst. 66, 737-743. Rice, J.B., Winters, K.G., Krakowka, S., and Olsen, R.G. 1982. Con~oarison of systemic and local immunity in dogs with canine parvovirus enteritis. Infect. Inm~m. 38, 1003-1009.