- Email: [email protected]
Antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (H3N8)
Veterinary Microbiology, 19 (1989) 293-303 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
293
Antibody Isotype Responses in the Serum and Respiratory Tract to Primary and Secondary Infections with Equine Influenza Virus (H3N8) D. HANNANT, D.M. JESSETT, T.O'NEILL and J.A. MUMFORD
Department of Infectious Diseases, Animal Health Trust, Lanwades Park, Kennett, Nr. Newmarket, Suffolk CB8 7PN (Gt. Britain) (Accepted for publication 9 November 1988)
ABSTRACT Hannant, D., Jessett, D.M., O'Neill, T. and Mumford, J.A., 1989. Antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (H3N8). Vet. Microbiol., 19: 293-303. Serum antibody (IgGab, IgM and IgA) responses to primary and secondary infection with influenza A/equine/Newmarket/79 (H3N8) by nebulised aerosol were compared with local (nasopharyngeal and tracheal) antibody responses in ponies. Circulating IgGab antibody was of long duration after primary infection, whereas IgM responses were short-lived after both primary and secondary infections. The antigenic stimulation of secondary infection with equine influenza was sufficient to induce elevations of serum IgM and IgA in the presence of high levels of circulating IgG,b. These results support the potential of virus-specific IgM measurement for the detection of recent exposure to virus in horses which have high levels of circulating IgGa~, Unlike serum IgG~,,,, nasal and tracheal wash antibody of this isotype did not show long duration after primary infection, but local antibody memory was demonstrated by anamnestic responses on rechallenge. Nasopharyngeal IgA developed later than IgGal~and IgM, and was more durable.
INTRODUCTION
Protective immunity from rechallenge with equine influenza virus does not correlate absolutely with the level of circulating antibody. For example, although it has been shown that there is a close relationship between immunity and the level of circulating antibody to haemagglutinin in horses vaccinated with inactivated whole virus vaccines (Mumford et al., 1983, 1988a), effective protection from rechallenge in the absence of detectable circulating antibody has been demonstrated in ponies where the first exposure to virus antigens was by respiratory infection (Hannant et al., 1988). The duration of immunity after vaccination is rather short lived (Wood et al., 1983; Mumford et al., 1983 ), whereas partial clinical immunity (absence of coughing, reduction in pyrexic 0378-1135/89/$03.50
© 1989 Elsevier Science Publishers B.V.
294 responses) has been demonstrated in ponies rechallenged with equine influenza virus > 1 year after their first infection (Hannant et al., 1988). These studies emphasise that the duration of immunity to equine influenza may not be a function of the duration of serum antibody after infection or vaccination. Moreover, the kinetics of production and duration of circulating antibody in the latter study was shown to vary markedly depending upon the selection of serological tests. The short duration of circulating antibody to the haemagglutinin, as measured by single radial haemolysis (SRH), was a consistent feature. In contrast, antibody to whole virus proteins was of long duration and detected > 32 weeks after infection. The contribution of local (respiratory tract) antibody to protection from challenge with equine influenza is poorly understood, although studies have shown that nasopharyngeal antibody is stimulated (Rouse and Ditchfield, 1970; Kumanomido and Akiyama, 1975 ). The presence of a well developed mucosal immune system in horses has been described histologically (Mair et al., 1987a) and locally produced antibody has been detected in nasopharyngeal, tracheal and bronchial secretions of clinically normal horses (Mair et al., 1987b). Pahud and Mach ( 1972 ) showed that the predominant antibody in equine nasal secretions was secretory (dimeric) IgA and emphasised the homology between equine and human mucosal secretory immune systems. The importance of nasopharyngeal antibody in immunity to equine influenza may be inferred from human studies which showed that nasal IgA was associated with protection (Murphy et al., 1973; Johnson et al., 1985, 1986). The data on the kinetics of circulating antibody production (Hannant et al., 1988), and the possibility that local antibody responses may have contributed to protection from virus challenge, have highlighted the importance of studying antibody isotype responses to equine influenza infections. The purpose of the present study was to examine three serum antibody isotype responses (IgGab, IgM, IgA) to primary and secondary infections with equine influenza, and to compare these with local (nasopharyngeal and tracheal) antibody responses. A further objective was to assess the potential of antibody isotype in the differential diagnosis of influenza in animals primed by previous infection. MATERIALSAND METHODS
Challenge virus The equine influenza virus used for aerosol infection of Welsh mountain ponies, seronegative for H3N8 viruses, was the second egg passage of A/equine/ Newmarket/D55/79 (H3N8). Infection was by exposure to a standard nebulised aerosol of challenge virus as described previously (Mumford et al., 1988b).
295
Study design A pilot experiment was carried out in which four ponies were infected with influenza virus and rechallenged 8 weeks later, and serological assays were carried out at weekly intervals. The results (data not shown) confirmed earlier findings on the duration of circulating antibody of the IgGab isotype ( H a n n a n t et al., 1988). The experiment was repeated with three ponies also seronegative for H 3 N 8 viruses at the first infection. The sampling frequency was increased to daily intervals over the first week of primary infection and after rechallenge at 62 days later. The findings in both experiments were essentially the same and those of the second experiment are presented in detail.
Virological and clinical observations Regular sampling by nasopharyngeal swabs was carried out for the isolation of live virus as described by Mumford et al. (1983). Rectal temperatures were recorded at ~ 15.00 h daily in order to minimise diurnal variations and temperatures > 38.9 ° C were regarded as abnormal. Clinical records of nasal/ocular discharges, coughing, etc., were made for each animal throughout the observation period.
Nasal and tracheal wash samples Nasal washes were collected by flushing the nasopharynx with ~ 100 ml sterile phosphate-buffered saline, p H 7.2, using a flexible polythene tubing attached to a large syringe. Tracheal washes were collected from conscious ponies with the aid of a bronchoendoscope as described (Burrell et al., 1986). Samples were stored on ice and frozen at - 2 0 ° C after the addition of 0.02% NAN3. Before antibody measurements were carried out, samples were concentrated ~ 10-fold by dialysis against Aquacide (Calbiochem).
Antibody measurements All samples were measured by a radioisotopic antiglobulin binding assay (RABA) as described by H a n n a n t et al. ( 1988 ). Nasal and tracheal wash samples were also assayed for equine serum albumin (ESA) content by an inhibition radioimmunoassay using reagents prepared in this laboratory. Antibody measurements were expressed in terms of micrograms ESA in each sample. Antibody isotypes were identified using monospecific polyclonal antiglobulin reagents prepared in this laboratory. These were necessary to amplify the radioisotopic signal derived from binding of radiolabelled Staphylococcus aureus protein A (Protein A) or Protein G, prepared from Group G streptococci. Virus-specific equine IgGab was detected with a rabbit antiserum as revealed
296
by the subsequent uptake of ~2SI-labelled Protein A. The antiglobulin reagent was used in this case because preliminary tests confirmed the findings of Goudswaard et al. (1978) that Protein A had low or no affinity for equine IgG. Similarly, sheep antiglobulin reagents to equine IgA and IgM have been shown to have poor reactivity with Protein A (Surolia et al., 1982). Therefore, the binding of these reagents was detected using Protein G (Akerstrom et al., 1985 ). ~2SI-labelled Protein A was prepared as described ( H a n n a n t et al., 1985) and ~2SI-labelled Protein G was supplied by Amersham International plc.
Data analysis Antibody activity was recorded in terms of counts m i n - 1 ~__S.D. of triplicate samples and results were subjected to analysis of variance. Comparisons of antibody levels between sequential sera of individuals and between groups was made by Student's t-test. RESULTS
Serum antibody responses Titration of pre-infection and post-infection (Day 14) sera from the same individual showed that there was a considerable non-specific background activity at low dilutions (Fig. 1). In order to discriminate between control and antibody-positive sera, all samples were analysed at a dilution of 10 -2. Evidence for the long duration of the virus-specific IgGab isotype after primary infection is given in Fig. 2A, which shows that the initial rise in antibody was maintained until second challenge 62 days later. There was no evidence of a significant anamnestic increase in antibody after rechallenge, but increases in levels were detected at 8 days. The high levels of circulating IgGa~, were 20o x15. V 10-
O'
i
I
In
20
,
l
i
,
,
,
40 80 150 320 640 1280 RECIPROCAL DILIIT[ON OF SERUM
i
2560
Fig. 1. Titration of pre-infection and post-infection serum by RABA.
297 12lO-
A
x
8
4-
w
~ O--
!
I
I
I
I
I
I
O 4 8 12 FIRST INFECTION
!
I
I
16
0
[
I
I
!
I
4
-T-
I
8
42
I SECOND INFECTION
46 DAYS
12 +, 10 8-
x
7
z
6--
48 2-
0-
I I 0
I 4
FIRST
!
I I ! I I 8 12 16
INFECTION
i
I I I I I I T 0 q 8 42 i SECOND INFECTION
46 DAYS •
12
+~ 10
o × 7 £
¢
8 6
T
4
8
T7£
O-
I
;
' I ' ~ '1'2 ' 1~ FIRST INFECTION i
46 DAYS
!
I
I
I
i
=r"
0 4 8 42 I SECOND INFECTION
Fig. 2. Serum antibody responses to equine influenza virus after primary and secondary infections. Results are mean 4- S.D. for three ponies. A -- IgGab; B = IgM; C -- IgA.
maintained at least until 32 days after rechallenge when sampling was stopped. Virus-specific IgM development is summarised in Fig. 2B. (The background, Day 0, counts are higher than for other isotypes in this assay which is a feature
298 of the antiglobulin reagents.) The data show that there was a rise in serum IgM on primary infection which had declined by ~ 50% at the time of rechallenge. There was a further stimulation of IgM after rechallenge and this was also of relatively short duration, showing a marked decline by 32 days after secondary infection. This contrasted markedly with the kinetics of serum IgA production (Fig. 2C), which increased much more slowly after primary infection. At the time of rechallenge, serum IgA levels had declined by ~ 20%. A strong and more rapid response was seen on rechallenge and the high levels were maintained at least for a further 32 days. Antibody responses in the respiratory tract In order to correct for variable sample dilution and serum transudation, antibody measurements in nasal and tracheal washes were expressed in terms of ESA content. The kinetics of virus-specific IgGab production in the nasopharynx after primary infection are shown in Fig. 3A and appeared to be similar to that of serum IgGab in primary infections (Fig. 2A). However, there was a marked decline in nasal wash antibody by the time of secondary infection. A typical anamnestic antibody response was observed on rechallenge, illustrated by a significant antibody rise at 4 days after secondary infection. In addition to the nasal wash samples, tracheal washes were available for virus-specific IgGab estimations and these also confirmed the secondary response on rechallenge (Fig. 3A). Therefore, virus-specific IgGab in the nasopharynx and trachea did not show the features of long duration after primary infection, as seen with serum antibody of this isotype. Nasopharyngeal IgM responses to primary infection with influenza virus were rather transient and had declined by ~ 50% by the time of secondary infection (Fig. 3B). In contrast to the serum IgM response, secondary infection did not induce a marked re-stimulation of virus-specific IgM in the nasopharynx. However, the level of nasal IgM following secondary infection seemed to be more durable and had not declined by 42 days. Nasal wash IgA responses to primary infection were in accordance with those of serum IgA, but virus rechallenge failed to stimulate a strong or rapid secondary response (Fig. 3C). The levels of nasopharyngeal IgA achieved at 14 days after rechallenge appeared to be maintained until the end of the sampling period 28 days later. Clinical responses Primary infection with equine influenza induced a severe, self-limiting respiratory disease characterised by pyrexia, dyspnoea and cough (Fig. 4). Infectious virus was recovered from nasopharyngeal swabs from 2 to 6 days after infection and all the animals coughed for > 8 days. In contrast, after secondary
299
~I0"
%8 #
.m 0
I
0
I
I
4 FIRST
I
I
I
I
-r-
I
0'
' 4' ' 8' ' 12' ' i SECOND INFECTION
I'6
8 12 INFECTION
I
42
46 DAYS
~1o +1
8 x
T
6
2-L S
2 I
I
0
I
I
4
|
I
8
FIRST
I
I
12
I
|
1~
0
|
INFECTION
I
I
I
4
I
l
8
SECOND
t
12
42
INFECTION
46 DAYS
~1o +1
o
8
T
6
z
¢
4
2 w
0
s
~)'
L~'
FIRST
8I
i
1'2
INFECTION
!
16
!
0 46 DAYS
|
i
1
1
1
I
i
4 8 12 SECOND INFECTION
"1""
42
Fig. 3. Nasopharyngeal antibody responses to equine influenza virus after primary and secondary infections. Symbols are as for Fig. 2. Secondary IgGab responses in the trachea are shown by the broken line in A. * = significant rise in antibody at Day 4 after secondary infection (P < 0.01 ).
infection, there were no clinical signs of influenza and no virus was isolated from the nasopharynx. These data indicate that clinical protection from rechallenge was stimulated by primary infection 62 days previously.
300 +
+
+
+
+
+
+
+
+
+
+
I 7
• g
I 9
C,
+
© 40-
39"
38-
Ld 37"
I 0
I 1
I 2
! 3
FIRST
! 4
I 5
I 6
I 0
INFECTION
I 1
I ?
! 3
SECOND
i
46 DAYS
I 4
I 5
f (,
I 7
I 8
I 9
INFECTION
I
Fig. 4. Mean rectal temperatures, virus isolation and coughing responses in ponies after primary and secondary infections with equine influenza virus. V.I., virus isolation from nasal swabs: + =positive; - =negative. C, coughing recorded during sampling: + =positive; - =negative.
DISCUSSION
The RABA proved to be a very sensitive method for measuring antibody to equine influenza virus in horses. Rearden et al. (1982) used a similar assay for equine serum antibody to Escherichia coli, but employed a fixed dilution of 10 -1 test serum. In the present study, there was considerable non-specific binding of pre-infection horse serum at low dilution to the antigen-coated plates (Fig. 1). It was necessary to use a dilution of 10 -2 before it was possible to discriminate between pre-infection and convalescent samples or to detect increases in antibody levels in sequential samples. The present study has confirmed earlier findings on the long duration of circulating IgGab antibody after infection with equine influenza virus (Hannant et al., 1988). The serum IgM responses were short-lived after both primary and secondary infections (Fig. 2B ), and showed features of duration consistent with those observed using the S R H technique ( H a n n a n t et al., 1988). It is likely that equine IgM would function in a test based on complementmediated lysis (McGuire et al., 1973 ) such as SRH, in addition to IgGal, However, it is not known if the transient IgM response during primary and secondary infections showed similar specificity to the antibodies defined by SRH. In contrast to IgGab and IgM, serum IgA development was rather slow after primary infection. There was a more rapid rise in serum IgA on rechallenge and the high levels of antibody which resulted showed features of long duration. Therefore, the antigenic stimulation of a secondary infection of equine influenza was sufficient to induce elevations of serum IgM and IgA under the cover of high levels of circulating IgGab. The high levels of IgM stimulated by primary infection had declined by > 50% after ~ 50 days (Fig. 2B), which suggests that
301
the measurement of virus-specific IgM may be useful in the detection of recent exposure to virus in horses which have high levels of circulating IgGab (e.g., heavily vaccinated animals ). In order for a secondary stimulation of antibody to occur, it must be assumed that some virus infection/replication took place in previously infected ponies. Occasional low levels of virus recovery have been described in experiments similar to those described here (Hannant et al., 1988). Moreover, it has been shown that seroconversions did not occur in ponies exposed to inactivated virus of the same antigenic mass used for nebulised aerosol infection with live virus (D. Hannant and J.A. Mumford, unpublished results, 1988). Unlike serum IgGab, nasal and tracheal wash antibody of this isotype did not show long duration after primary infection (Fig. 3A). On rechallenge, local antibody memory was demonstrated by a rapid production of antibody at Day 4. This response was consistent with the anamnestic serum antibody responses described in a previous report (Hannant et al., 1988). The decline in local IgGab and its anamnestic stimulation in the presence of high levels of circulating antibody contrasts with the suggestion that nasal wash IgG was a transudate from serum in human influenza infections (Wagner et al., 1987). Some evidence in support of local antibody production in the nasopharynx and trachea after equine influenza infection was obtained by applying the formulae described by Stockley and Burnett (1980). The proportion of locally produced antibody for randomly selected samples (data not shown) varied from 15 to 60%. However, estimations of the degree of local antibody production in the presence of an inflammatory response after influenza infection is liable to error unless it is possible to measure components of the antibody molecules that are unique to the secretory immune system. Therefore, studies are in progress to measure secretory IgA in the horse based on two-site assays specific for the secretory component and alpha chains of immunoglobulin. These components have been described for equine secretory IgA (Pahud and Mach, 1972 ). Butler et al. (1967) studied the mechanism of appearance of antibodies in the nasal secretions of man and suggested that IgA was derived in part from circulating monomeric IgA as well as being synthesised de novo as dimeric secretory IgA in the tissues of the upper respiratory tract. Rhinovirus-specific secretory IgA was also shown to develop in the nasal secretions after the acute phase of respiratory disease (Butler et al., 1970). Increased levels of secretory IgA were also a feature of chronic obstructive pulmonary disease in humans where there was no defined aetiological agent (Burnett et al., 1987). These studies and those carried out in horses (Galan and Timoney, 1985; Mair et al., 1987a, 1987b ) highlight the potential importance of locally produced antibody in the protection and recovery from viral and bacterial infections of the respiratory tract. The present study suggested that nasopharyngeal IgA developed later than
302 IgGab a n d I g M , a n d w a s also m o r e d u r a b l e in t h a t it h a d n o t declined to p r e c h a l l e n g e levels 62 d a y s a f t e r i n f e c t i o n (Fig. 3 B ) . T h i s f e a t u r e of n a s a l w a s h IgA is c o n s i s t e n t w i t h t h e s t u d i e s of B u t l e r et al. (1970) on h u m a n r h i n o v i r u s infections. I t is difficult to assess t h e relative i m p o r t a n c e of n a s o p h a r y n g e a l a n t i b o d y in p r o t e c t i o n f r o m c h a l l e n g e w i t h equine i n f l u e n z a in t h e p r e s e n t e x p e r i m e n t s b e c a u s e of t h e p r e s e n c e of high levels of c i r c u l a t i n g IgGab. H o w ever, p r e v i o u s studies h a v e s h o w n t h a t p o n i e s w e r e p r o t e c t e d f r o m r e c h a l l e n g e w i t h i n f l u e n z a in t h e a b s e n c e of d e t e c t a b l e levels of c i r c u l a t i n g a n t i b o d y ( H a n n a n t et al., 1988) if first e x p o s u r e to virus was b y i n f e c t i o n u p to 62 w e e k s previously. T h i s c o n t r a s t s w i t h v a c c i n e - i n d u c e d i m m u n i t y w h i c h c o r r e l a t e d s t r o n g l y w i t h levels of c i r c u l a t i n g a n t i b o d y ( M u m f o r d et al., 1983). ACKNOWLEDGEMENTS T h i s s t u d y was s u p p o r t e d b y t h e H o r s e r a c e B e t t i n g L e v y B o a r d a n d t h e A n i m a l H e a l t h T r u s t . W e t h a n k H. S a w y e r a n d his s t a f f for e x c e l l e n t m a n a g e m e n t of t h e ponies, M. P r i c e for skilled t e c h n i c a l a s s i s t a n c e a n d D. B u r k e t t for p r e p a r a t i o n of t h e m a n u s c r i p t . S o m e of t h e results in t h i s p a p e r were p r e s e n t e d at t h e 5 t h I n t e r n a t i o n a l C o n f e r e n c e on E q u i n e I n f e c t i o u s Diseases, K e n t u c k y , 1987.
REFERENCES Akerstrom, B., Brodin, T., Reis, K. and Kjorck, L., 1985. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol., 135: 2589-2592. Burnett, D., Crocker, J. and Stockley, R.A., 1987. Cells containing IgA subclasses in bronchi of subjects with and without obstructive lung disease. J. Clin. Pathol., 40: 1217-1220. Burrell, M.H., Mackintosh, M.E. and Taylor, C.E.D., 1986. Isolation of Streptococcus pneumoniae from the respiratory tract of horses. Equine Vet. J., 18: 183-186. Butler, W.T., Rossen, R.D. and Waldmann, T.A., 1967. The mechanism of appearance of immunoglobulin A in nasal secretions in man. J. Clin. Invest., 46: 1883-1893. Butler, W.T., Waldmann, T.A., Rosseu, R.D., Douglas, R.G. and Couch, R.B., 1970. Changes in IgA and IgG concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man. J. Immunol., 105: 584-591. Galan, J.E. and Timoney, J.F., 1985. Mucosal nasopharyngeal immune responses of horses to protein antigens of Streptococcus equi. Infect. Immun., 47: 623-628. Goudswaard, J., van der Donk, J.A., Noordzij, A., van Dam, R.H. and Vaernman, J.-P., 1978. Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol., 8: 21-28. Hannant, D., Donaldson, K. and Bolton, R.E., 1985. Immunomodulatory effects of mineral dust. I. Effects of intraperitoneal dust inoculation on splenic lymphocyte function and humoral immune responses in vivo. J. Clin. Lab. Immunol., 16: 81-85. Hannant, D., Mumford, J.A. and Jessett, D.M., 1988. Duration of circulating antibody and immunity following infection with equine influenza virus. Vet. Rec., 122: 125-128. Johnson, P.R., Feldman, S., Thompson, J.M., Mahoney, J.D. and Wright, P.F., 1985. Comparison of long-term systemic and secretory antibody responses in children given live attenuated, or inactivated influenza A vaccine. J. Med. Virol., 17: 325-335.
303 Johnson, P.R., Feldman, S., Thompson, J.M., Mahoney, J.D. and Wright, P.F., 1986. Immunity to influenza A virus infection in young children: a comparison of natural infection, live coldadapted vaccine, and inactivated vaccine. J. Infect. Dis., 154:121-127. Kumanomido, T. and Akiyama, Y., 1975. Immuno-effect of serum and nasal antibody against experimental inoculation with influenza A-Equi-2 virus. Exp. Rep. Equine Health Lab., 12: 44-52. Mair, T.S., Batten, E.H., Stokes, C.R. and Bourne, F.J., 1987a. The histological features of the immune system of the equine respiratory tract. J. Comp. Pathol., 97: 575-586. Mair, T.S., Stokes, C.R. and Bourne, F.J., 1987b. Quantification of immunoglobulins in respiratory tract secretions of the horse. Vet. Immunol. Immunopathol., 14: 187-203. McGuire, T.C., Crawford, T.B. and Henson, J.B., 1973. The isolation, characterisation and functional properties of equine immunoglobulin classes and subclasses. In: J.T. Bryans and H. Gerber (Editors), Proceedings of the 3rd International Conference on Equine Infectious Diseases, Karger, Basel, pp. 364-381. Mumford, J.A., Wood, J.M., Scott, A.M., Folkers, C. and Schild, G.C., 1983. Studies with inactivated equine influenza vaccines. 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8). J. Hyg., 90: 385-395. Mumford, J.A., Wood, J.M., Folkers, C. and Schild, G.C., 1988a. Protection against experimental infection with influenza virus A/equine/Miami/63 (H3N8) provided by inactivated whole virus vaccines containing homologous virus. Epid. Infect., 100: 501-510. Mumford, J.A., Hannant, D., Jessett, D.M. and Scott, A.M., 1988b. Experimental infection of ponies with equine influenza (H3N8) virus by intranasal inoculation or exposure to aerosols. Equine Vet. J., in press. Murphy, B.R., Chalub, E.G., Nusinoff, S.R., Kasel, J. and Chanock, R.M., 1973. Temperaturesensitive mutants of influenza virus. III Further characterisation of the ts-1 [E] influenza A recombinant (H3N2) virus in man. J. Infect. Dis., 128: 479-487. Pahud, J.J. and Mach, J.P., 1972. Equine secretory IgA and secretory component. Int. Arch. Allergy Appl. Immunol., 42: 175-186. Rearden, T.P., Sprouse, R.F. and Garner, H.E., 1982. Radioimmunoassay for the detection of antigen-specific IgM, IgG and IgA in equine sera. Am. J. Vet. Res., 43: 294-298. Rouse, B.T. and Ditchfield, W.J.B., 1970. The response of ponies to Myxovirus influenzae AEqui-2. III. The protective effect of serum and nasal antibody against experimental challenge. Res. Vet. Sci., 11: 503-507. Stockley, R.A. and Burnett, D., 1980. Local IgA production in patients with chronic bronchitis: effect of acute respiratory infection. Thorax, 35: 202-206. Surolia, A., Pain, D. and Khan, M.I., 1982. Protein A: nature's universal anti-antibody. Trends Biochem. Sci., 7: 74-76. Wagner, D.K., Clements, M.L., Reimer, C.B., Snyder, M., Nelson, D.L. and Murphy, B.R., 1987. Analysis of immunoglobulin G antibody responses after administration of live and inactivated influenza A vaccine indicates that nasal wash immunoglobulin G is a transudate from serum. J. Clin. Microbiol., 25: 559-562. Wood, J.M., Mumford, J.A., Folkers, C., Scott, A.M. and Schild, G.C., 1983. Studies with inactivated equine influenza vaccines. 1. Serological responses of ponies to graded doses of vaccine. J. Hyg., 90: 371-384.
293
Antibody Isotype Responses in the Serum and Respiratory Tract to Primary and Secondary Infections with Equine Influenza Virus (H3N8) D. HANNANT, D.M. JESSETT, T.O'NEILL and J.A. MUMFORD
Department of Infectious Diseases, Animal Health Trust, Lanwades Park, Kennett, Nr. Newmarket, Suffolk CB8 7PN (Gt. Britain) (Accepted for publication 9 November 1988)
ABSTRACT Hannant, D., Jessett, D.M., O'Neill, T. and Mumford, J.A., 1989. Antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (H3N8). Vet. Microbiol., 19: 293-303. Serum antibody (IgGab, IgM and IgA) responses to primary and secondary infection with influenza A/equine/Newmarket/79 (H3N8) by nebulised aerosol were compared with local (nasopharyngeal and tracheal) antibody responses in ponies. Circulating IgGab antibody was of long duration after primary infection, whereas IgM responses were short-lived after both primary and secondary infections. The antigenic stimulation of secondary infection with equine influenza was sufficient to induce elevations of serum IgM and IgA in the presence of high levels of circulating IgG,b. These results support the potential of virus-specific IgM measurement for the detection of recent exposure to virus in horses which have high levels of circulating IgGa~, Unlike serum IgG~,,,, nasal and tracheal wash antibody of this isotype did not show long duration after primary infection, but local antibody memory was demonstrated by anamnestic responses on rechallenge. Nasopharyngeal IgA developed later than IgGal~and IgM, and was more durable.
INTRODUCTION
Protective immunity from rechallenge with equine influenza virus does not correlate absolutely with the level of circulating antibody. For example, although it has been shown that there is a close relationship between immunity and the level of circulating antibody to haemagglutinin in horses vaccinated with inactivated whole virus vaccines (Mumford et al., 1983, 1988a), effective protection from rechallenge in the absence of detectable circulating antibody has been demonstrated in ponies where the first exposure to virus antigens was by respiratory infection (Hannant et al., 1988). The duration of immunity after vaccination is rather short lived (Wood et al., 1983; Mumford et al., 1983 ), whereas partial clinical immunity (absence of coughing, reduction in pyrexic 0378-1135/89/$03.50
© 1989 Elsevier Science Publishers B.V.
294 responses) has been demonstrated in ponies rechallenged with equine influenza virus > 1 year after their first infection (Hannant et al., 1988). These studies emphasise that the duration of immunity to equine influenza may not be a function of the duration of serum antibody after infection or vaccination. Moreover, the kinetics of production and duration of circulating antibody in the latter study was shown to vary markedly depending upon the selection of serological tests. The short duration of circulating antibody to the haemagglutinin, as measured by single radial haemolysis (SRH), was a consistent feature. In contrast, antibody to whole virus proteins was of long duration and detected > 32 weeks after infection. The contribution of local (respiratory tract) antibody to protection from challenge with equine influenza is poorly understood, although studies have shown that nasopharyngeal antibody is stimulated (Rouse and Ditchfield, 1970; Kumanomido and Akiyama, 1975 ). The presence of a well developed mucosal immune system in horses has been described histologically (Mair et al., 1987a) and locally produced antibody has been detected in nasopharyngeal, tracheal and bronchial secretions of clinically normal horses (Mair et al., 1987b). Pahud and Mach ( 1972 ) showed that the predominant antibody in equine nasal secretions was secretory (dimeric) IgA and emphasised the homology between equine and human mucosal secretory immune systems. The importance of nasopharyngeal antibody in immunity to equine influenza may be inferred from human studies which showed that nasal IgA was associated with protection (Murphy et al., 1973; Johnson et al., 1985, 1986). The data on the kinetics of circulating antibody production (Hannant et al., 1988), and the possibility that local antibody responses may have contributed to protection from virus challenge, have highlighted the importance of studying antibody isotype responses to equine influenza infections. The purpose of the present study was to examine three serum antibody isotype responses (IgGab, IgM, IgA) to primary and secondary infections with equine influenza, and to compare these with local (nasopharyngeal and tracheal) antibody responses. A further objective was to assess the potential of antibody isotype in the differential diagnosis of influenza in animals primed by previous infection. MATERIALSAND METHODS
Challenge virus The equine influenza virus used for aerosol infection of Welsh mountain ponies, seronegative for H3N8 viruses, was the second egg passage of A/equine/ Newmarket/D55/79 (H3N8). Infection was by exposure to a standard nebulised aerosol of challenge virus as described previously (Mumford et al., 1988b).
295
Study design A pilot experiment was carried out in which four ponies were infected with influenza virus and rechallenged 8 weeks later, and serological assays were carried out at weekly intervals. The results (data not shown) confirmed earlier findings on the duration of circulating antibody of the IgGab isotype ( H a n n a n t et al., 1988). The experiment was repeated with three ponies also seronegative for H 3 N 8 viruses at the first infection. The sampling frequency was increased to daily intervals over the first week of primary infection and after rechallenge at 62 days later. The findings in both experiments were essentially the same and those of the second experiment are presented in detail.
Virological and clinical observations Regular sampling by nasopharyngeal swabs was carried out for the isolation of live virus as described by Mumford et al. (1983). Rectal temperatures were recorded at ~ 15.00 h daily in order to minimise diurnal variations and temperatures > 38.9 ° C were regarded as abnormal. Clinical records of nasal/ocular discharges, coughing, etc., were made for each animal throughout the observation period.
Nasal and tracheal wash samples Nasal washes were collected by flushing the nasopharynx with ~ 100 ml sterile phosphate-buffered saline, p H 7.2, using a flexible polythene tubing attached to a large syringe. Tracheal washes were collected from conscious ponies with the aid of a bronchoendoscope as described (Burrell et al., 1986). Samples were stored on ice and frozen at - 2 0 ° C after the addition of 0.02% NAN3. Before antibody measurements were carried out, samples were concentrated ~ 10-fold by dialysis against Aquacide (Calbiochem).
Antibody measurements All samples were measured by a radioisotopic antiglobulin binding assay (RABA) as described by H a n n a n t et al. ( 1988 ). Nasal and tracheal wash samples were also assayed for equine serum albumin (ESA) content by an inhibition radioimmunoassay using reagents prepared in this laboratory. Antibody measurements were expressed in terms of micrograms ESA in each sample. Antibody isotypes were identified using monospecific polyclonal antiglobulin reagents prepared in this laboratory. These were necessary to amplify the radioisotopic signal derived from binding of radiolabelled Staphylococcus aureus protein A (Protein A) or Protein G, prepared from Group G streptococci. Virus-specific equine IgGab was detected with a rabbit antiserum as revealed
296
by the subsequent uptake of ~2SI-labelled Protein A. The antiglobulin reagent was used in this case because preliminary tests confirmed the findings of Goudswaard et al. (1978) that Protein A had low or no affinity for equine IgG. Similarly, sheep antiglobulin reagents to equine IgA and IgM have been shown to have poor reactivity with Protein A (Surolia et al., 1982). Therefore, the binding of these reagents was detected using Protein G (Akerstrom et al., 1985 ). ~2SI-labelled Protein A was prepared as described ( H a n n a n t et al., 1985) and ~2SI-labelled Protein G was supplied by Amersham International plc.
Data analysis Antibody activity was recorded in terms of counts m i n - 1 ~__S.D. of triplicate samples and results were subjected to analysis of variance. Comparisons of antibody levels between sequential sera of individuals and between groups was made by Student's t-test. RESULTS
Serum antibody responses Titration of pre-infection and post-infection (Day 14) sera from the same individual showed that there was a considerable non-specific background activity at low dilutions (Fig. 1). In order to discriminate between control and antibody-positive sera, all samples were analysed at a dilution of 10 -2. Evidence for the long duration of the virus-specific IgGab isotype after primary infection is given in Fig. 2A, which shows that the initial rise in antibody was maintained until second challenge 62 days later. There was no evidence of a significant anamnestic increase in antibody after rechallenge, but increases in levels were detected at 8 days. The high levels of circulating IgGa~, were 20o x15. V 10-
O'
i
I
In
20
,
l
i
,
,
,
40 80 150 320 640 1280 RECIPROCAL DILIIT[ON OF SERUM
i
2560
Fig. 1. Titration of pre-infection and post-infection serum by RABA.
297 12lO-
A
x
8
4-
w
~ O--
!
I
I
I
I
I
I
O 4 8 12 FIRST INFECTION
!
I
I
16
0
[
I
I
!
I
4
-T-
I
8
42
I SECOND INFECTION
46 DAYS
12 +, 10 8-
x
7
z
6--
48 2-
0-
I I 0
I 4
FIRST
!
I I ! I I 8 12 16
INFECTION
i
I I I I I I T 0 q 8 42 i SECOND INFECTION
46 DAYS •
12
+~ 10
o × 7 £
¢
8 6
T
4
8
T7£
O-
I
;
' I ' ~ '1'2 ' 1~ FIRST INFECTION i
46 DAYS
!
I
I
I
i
=r"
0 4 8 42 I SECOND INFECTION
Fig. 2. Serum antibody responses to equine influenza virus after primary and secondary infections. Results are mean 4- S.D. for three ponies. A -- IgGab; B = IgM; C -- IgA.
maintained at least until 32 days after rechallenge when sampling was stopped. Virus-specific IgM development is summarised in Fig. 2B. (The background, Day 0, counts are higher than for other isotypes in this assay which is a feature
298 of the antiglobulin reagents.) The data show that there was a rise in serum IgM on primary infection which had declined by ~ 50% at the time of rechallenge. There was a further stimulation of IgM after rechallenge and this was also of relatively short duration, showing a marked decline by 32 days after secondary infection. This contrasted markedly with the kinetics of serum IgA production (Fig. 2C), which increased much more slowly after primary infection. At the time of rechallenge, serum IgA levels had declined by ~ 20%. A strong and more rapid response was seen on rechallenge and the high levels were maintained at least for a further 32 days. Antibody responses in the respiratory tract In order to correct for variable sample dilution and serum transudation, antibody measurements in nasal and tracheal washes were expressed in terms of ESA content. The kinetics of virus-specific IgGab production in the nasopharynx after primary infection are shown in Fig. 3A and appeared to be similar to that of serum IgGab in primary infections (Fig. 2A). However, there was a marked decline in nasal wash antibody by the time of secondary infection. A typical anamnestic antibody response was observed on rechallenge, illustrated by a significant antibody rise at 4 days after secondary infection. In addition to the nasal wash samples, tracheal washes were available for virus-specific IgGab estimations and these also confirmed the secondary response on rechallenge (Fig. 3A). Therefore, virus-specific IgGab in the nasopharynx and trachea did not show the features of long duration after primary infection, as seen with serum antibody of this isotype. Nasopharyngeal IgM responses to primary infection with influenza virus were rather transient and had declined by ~ 50% by the time of secondary infection (Fig. 3B). In contrast to the serum IgM response, secondary infection did not induce a marked re-stimulation of virus-specific IgM in the nasopharynx. However, the level of nasal IgM following secondary infection seemed to be more durable and had not declined by 42 days. Nasal wash IgA responses to primary infection were in accordance with those of serum IgA, but virus rechallenge failed to stimulate a strong or rapid secondary response (Fig. 3C). The levels of nasopharyngeal IgA achieved at 14 days after rechallenge appeared to be maintained until the end of the sampling period 28 days later. Clinical responses Primary infection with equine influenza induced a severe, self-limiting respiratory disease characterised by pyrexia, dyspnoea and cough (Fig. 4). Infectious virus was recovered from nasopharyngeal swabs from 2 to 6 days after infection and all the animals coughed for > 8 days. In contrast, after secondary
299
~I0"
%8 #
.m 0
I
0
I
I
4 FIRST
I
I
I
I
-r-
I
0'
' 4' ' 8' ' 12' ' i SECOND INFECTION
I'6
8 12 INFECTION
I
42
46 DAYS
~1o +1
8 x
T
6
2-L S
2 I
I
0
I
I
4
|
I
8
FIRST
I
I
12
I
|
1~
0
|
INFECTION
I
I
I
4
I
l
8
SECOND
t
12
42
INFECTION
46 DAYS
~1o +1
o
8
T
6
z
¢
4
2 w
0
s
~)'
L~'
FIRST
8I
i
1'2
INFECTION
!
16
!
0 46 DAYS
|
i
1
1
1
I
i
4 8 12 SECOND INFECTION
"1""
42
Fig. 3. Nasopharyngeal antibody responses to equine influenza virus after primary and secondary infections. Symbols are as for Fig. 2. Secondary IgGab responses in the trachea are shown by the broken line in A. * = significant rise in antibody at Day 4 after secondary infection (P < 0.01 ).
infection, there were no clinical signs of influenza and no virus was isolated from the nasopharynx. These data indicate that clinical protection from rechallenge was stimulated by primary infection 62 days previously.
300 +
+
+
+
+
+
+
+
+
+
+
I 7
• g
I 9
C,
+
© 40-
39"
38-
Ld 37"
I 0
I 1
I 2
! 3
FIRST
! 4
I 5
I 6
I 0
INFECTION
I 1
I ?
! 3
SECOND
i
46 DAYS
I 4
I 5
f (,
I 7
I 8
I 9
INFECTION
I
Fig. 4. Mean rectal temperatures, virus isolation and coughing responses in ponies after primary and secondary infections with equine influenza virus. V.I., virus isolation from nasal swabs: + =positive; - =negative. C, coughing recorded during sampling: + =positive; - =negative.
DISCUSSION
The RABA proved to be a very sensitive method for measuring antibody to equine influenza virus in horses. Rearden et al. (1982) used a similar assay for equine serum antibody to Escherichia coli, but employed a fixed dilution of 10 -1 test serum. In the present study, there was considerable non-specific binding of pre-infection horse serum at low dilution to the antigen-coated plates (Fig. 1). It was necessary to use a dilution of 10 -2 before it was possible to discriminate between pre-infection and convalescent samples or to detect increases in antibody levels in sequential samples. The present study has confirmed earlier findings on the long duration of circulating IgGab antibody after infection with equine influenza virus (Hannant et al., 1988). The serum IgM responses were short-lived after both primary and secondary infections (Fig. 2B ), and showed features of duration consistent with those observed using the S R H technique ( H a n n a n t et al., 1988). It is likely that equine IgM would function in a test based on complementmediated lysis (McGuire et al., 1973 ) such as SRH, in addition to IgGal, However, it is not known if the transient IgM response during primary and secondary infections showed similar specificity to the antibodies defined by SRH. In contrast to IgGab and IgM, serum IgA development was rather slow after primary infection. There was a more rapid rise in serum IgA on rechallenge and the high levels of antibody which resulted showed features of long duration. Therefore, the antigenic stimulation of a secondary infection of equine influenza was sufficient to induce elevations of serum IgM and IgA under the cover of high levels of circulating IgGab. The high levels of IgM stimulated by primary infection had declined by > 50% after ~ 50 days (Fig. 2B), which suggests that
301
the measurement of virus-specific IgM may be useful in the detection of recent exposure to virus in horses which have high levels of circulating IgGab (e.g., heavily vaccinated animals ). In order for a secondary stimulation of antibody to occur, it must be assumed that some virus infection/replication took place in previously infected ponies. Occasional low levels of virus recovery have been described in experiments similar to those described here (Hannant et al., 1988). Moreover, it has been shown that seroconversions did not occur in ponies exposed to inactivated virus of the same antigenic mass used for nebulised aerosol infection with live virus (D. Hannant and J.A. Mumford, unpublished results, 1988). Unlike serum IgGab, nasal and tracheal wash antibody of this isotype did not show long duration after primary infection (Fig. 3A). On rechallenge, local antibody memory was demonstrated by a rapid production of antibody at Day 4. This response was consistent with the anamnestic serum antibody responses described in a previous report (Hannant et al., 1988). The decline in local IgGab and its anamnestic stimulation in the presence of high levels of circulating antibody contrasts with the suggestion that nasal wash IgG was a transudate from serum in human influenza infections (Wagner et al., 1987). Some evidence in support of local antibody production in the nasopharynx and trachea after equine influenza infection was obtained by applying the formulae described by Stockley and Burnett (1980). The proportion of locally produced antibody for randomly selected samples (data not shown) varied from 15 to 60%. However, estimations of the degree of local antibody production in the presence of an inflammatory response after influenza infection is liable to error unless it is possible to measure components of the antibody molecules that are unique to the secretory immune system. Therefore, studies are in progress to measure secretory IgA in the horse based on two-site assays specific for the secretory component and alpha chains of immunoglobulin. These components have been described for equine secretory IgA (Pahud and Mach, 1972 ). Butler et al. (1967) studied the mechanism of appearance of antibodies in the nasal secretions of man and suggested that IgA was derived in part from circulating monomeric IgA as well as being synthesised de novo as dimeric secretory IgA in the tissues of the upper respiratory tract. Rhinovirus-specific secretory IgA was also shown to develop in the nasal secretions after the acute phase of respiratory disease (Butler et al., 1970). Increased levels of secretory IgA were also a feature of chronic obstructive pulmonary disease in humans where there was no defined aetiological agent (Burnett et al., 1987). These studies and those carried out in horses (Galan and Timoney, 1985; Mair et al., 1987a, 1987b ) highlight the potential importance of locally produced antibody in the protection and recovery from viral and bacterial infections of the respiratory tract. The present study suggested that nasopharyngeal IgA developed later than
302 IgGab a n d I g M , a n d w a s also m o r e d u r a b l e in t h a t it h a d n o t declined to p r e c h a l l e n g e levels 62 d a y s a f t e r i n f e c t i o n (Fig. 3 B ) . T h i s f e a t u r e of n a s a l w a s h IgA is c o n s i s t e n t w i t h t h e s t u d i e s of B u t l e r et al. (1970) on h u m a n r h i n o v i r u s infections. I t is difficult to assess t h e relative i m p o r t a n c e of n a s o p h a r y n g e a l a n t i b o d y in p r o t e c t i o n f r o m c h a l l e n g e w i t h equine i n f l u e n z a in t h e p r e s e n t e x p e r i m e n t s b e c a u s e of t h e p r e s e n c e of high levels of c i r c u l a t i n g IgGab. H o w ever, p r e v i o u s studies h a v e s h o w n t h a t p o n i e s w e r e p r o t e c t e d f r o m r e c h a l l e n g e w i t h i n f l u e n z a in t h e a b s e n c e of d e t e c t a b l e levels of c i r c u l a t i n g a n t i b o d y ( H a n n a n t et al., 1988) if first e x p o s u r e to virus was b y i n f e c t i o n u p to 62 w e e k s previously. T h i s c o n t r a s t s w i t h v a c c i n e - i n d u c e d i m m u n i t y w h i c h c o r r e l a t e d s t r o n g l y w i t h levels of c i r c u l a t i n g a n t i b o d y ( M u m f o r d et al., 1983). ACKNOWLEDGEMENTS T h i s s t u d y was s u p p o r t e d b y t h e H o r s e r a c e B e t t i n g L e v y B o a r d a n d t h e A n i m a l H e a l t h T r u s t . W e t h a n k H. S a w y e r a n d his s t a f f for e x c e l l e n t m a n a g e m e n t of t h e ponies, M. P r i c e for skilled t e c h n i c a l a s s i s t a n c e a n d D. B u r k e t t for p r e p a r a t i o n of t h e m a n u s c r i p t . S o m e of t h e results in t h i s p a p e r were p r e s e n t e d at t h e 5 t h I n t e r n a t i o n a l C o n f e r e n c e on E q u i n e I n f e c t i o u s Diseases, K e n t u c k y , 1987.
REFERENCES Akerstrom, B., Brodin, T., Reis, K. and Kjorck, L., 1985. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol., 135: 2589-2592. Burnett, D., Crocker, J. and Stockley, R.A., 1987. Cells containing IgA subclasses in bronchi of subjects with and without obstructive lung disease. J. Clin. Pathol., 40: 1217-1220. Burrell, M.H., Mackintosh, M.E. and Taylor, C.E.D., 1986. Isolation of Streptococcus pneumoniae from the respiratory tract of horses. Equine Vet. J., 18: 183-186. Butler, W.T., Rossen, R.D. and Waldmann, T.A., 1967. The mechanism of appearance of immunoglobulin A in nasal secretions in man. J. Clin. Invest., 46: 1883-1893. Butler, W.T., Waldmann, T.A., Rosseu, R.D., Douglas, R.G. and Couch, R.B., 1970. Changes in IgA and IgG concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man. J. Immunol., 105: 584-591. Galan, J.E. and Timoney, J.F., 1985. Mucosal nasopharyngeal immune responses of horses to protein antigens of Streptococcus equi. Infect. Immun., 47: 623-628. Goudswaard, J., van der Donk, J.A., Noordzij, A., van Dam, R.H. and Vaernman, J.-P., 1978. Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol., 8: 21-28. Hannant, D., Donaldson, K. and Bolton, R.E., 1985. Immunomodulatory effects of mineral dust. I. Effects of intraperitoneal dust inoculation on splenic lymphocyte function and humoral immune responses in vivo. J. Clin. Lab. Immunol., 16: 81-85. Hannant, D., Mumford, J.A. and Jessett, D.M., 1988. Duration of circulating antibody and immunity following infection with equine influenza virus. Vet. Rec., 122: 125-128. Johnson, P.R., Feldman, S., Thompson, J.M., Mahoney, J.D. and Wright, P.F., 1985. Comparison of long-term systemic and secretory antibody responses in children given live attenuated, or inactivated influenza A vaccine. J. Med. Virol., 17: 325-335.
303 Johnson, P.R., Feldman, S., Thompson, J.M., Mahoney, J.D. and Wright, P.F., 1986. Immunity to influenza A virus infection in young children: a comparison of natural infection, live coldadapted vaccine, and inactivated vaccine. J. Infect. Dis., 154:121-127. Kumanomido, T. and Akiyama, Y., 1975. Immuno-effect of serum and nasal antibody against experimental inoculation with influenza A-Equi-2 virus. Exp. Rep. Equine Health Lab., 12: 44-52. Mair, T.S., Batten, E.H., Stokes, C.R. and Bourne, F.J., 1987a. The histological features of the immune system of the equine respiratory tract. J. Comp. Pathol., 97: 575-586. Mair, T.S., Stokes, C.R. and Bourne, F.J., 1987b. Quantification of immunoglobulins in respiratory tract secretions of the horse. Vet. Immunol. Immunopathol., 14: 187-203. McGuire, T.C., Crawford, T.B. and Henson, J.B., 1973. The isolation, characterisation and functional properties of equine immunoglobulin classes and subclasses. In: J.T. Bryans and H. Gerber (Editors), Proceedings of the 3rd International Conference on Equine Infectious Diseases, Karger, Basel, pp. 364-381. Mumford, J.A., Wood, J.M., Scott, A.M., Folkers, C. and Schild, G.C., 1983. Studies with inactivated equine influenza vaccines. 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8). J. Hyg., 90: 385-395. Mumford, J.A., Wood, J.M., Folkers, C. and Schild, G.C., 1988a. Protection against experimental infection with influenza virus A/equine/Miami/63 (H3N8) provided by inactivated whole virus vaccines containing homologous virus. Epid. Infect., 100: 501-510. Mumford, J.A., Hannant, D., Jessett, D.M. and Scott, A.M., 1988b. Experimental infection of ponies with equine influenza (H3N8) virus by intranasal inoculation or exposure to aerosols. Equine Vet. J., in press. Murphy, B.R., Chalub, E.G., Nusinoff, S.R., Kasel, J. and Chanock, R.M., 1973. Temperaturesensitive mutants of influenza virus. III Further characterisation of the ts-1 [E] influenza A recombinant (H3N2) virus in man. J. Infect. Dis., 128: 479-487. Pahud, J.J. and Mach, J.P., 1972. Equine secretory IgA and secretory component. Int. Arch. Allergy Appl. Immunol., 42: 175-186. Rearden, T.P., Sprouse, R.F. and Garner, H.E., 1982. Radioimmunoassay for the detection of antigen-specific IgM, IgG and IgA in equine sera. Am. J. Vet. Res., 43: 294-298. Rouse, B.T. and Ditchfield, W.J.B., 1970. The response of ponies to Myxovirus influenzae AEqui-2. III. The protective effect of serum and nasal antibody against experimental challenge. Res. Vet. Sci., 11: 503-507. Stockley, R.A. and Burnett, D., 1980. Local IgA production in patients with chronic bronchitis: effect of acute respiratory infection. Thorax, 35: 202-206. Surolia, A., Pain, D. and Khan, M.I., 1982. Protein A: nature's universal anti-antibody. Trends Biochem. Sci., 7: 74-76. Wagner, D.K., Clements, M.L., Reimer, C.B., Snyder, M., Nelson, D.L. and Murphy, B.R., 1987. Analysis of immunoglobulin G antibody responses after administration of live and inactivated influenza A vaccine indicates that nasal wash immunoglobulin G is a transudate from serum. J. Clin. Microbiol., 25: 559-562. Wood, J.M., Mumford, J.A., Folkers, C., Scott, A.M. and Schild, G.C., 1983. Studies with inactivated equine influenza vaccines. 1. Serological responses of ponies to graded doses of vaccine. J. Hyg., 90: 371-384.