Isotype-specific antibody responses in sera and mucosal secretions of calves experimentally infected with bovine herpesvirus 1

Veterinary

immunology

Veterinary Immunology and Immunopathology 46(1995)267-283

ELSEVIER

and

immunopathology

Isotype-specific antibody responses in sera and mucosal secretions of calves experimentally infected with bovine herpesvirus 1 J. Madic’, J. Magdalena’,

J. Quak, J.T. van Oirschot*

Institutefor Animal Science and Health, Department of Virology,P.O. Box 65, 8200.4B Lelystad, Netherlands Accepted 14 July 1994

Abstract Enzyme-linked immunosorbent assays (ELISAs) were developed for studying the kinetics of isotype-specific antibody responses in sera, nasal, ocular and genital secretions of calves infected with bovine herpesvirus 1 (BHVl ). The BHVl-specific IgM and IgA antibodies were measured in antibody capture assays, and the IgG 1 and IgG2 antibodies in indirect double antibody sandwich assays. The ELISAs were shown to be isotype-specific, sensitive and reproducible. Antibodies of all isotypes were able to neutralise the virus in vitro. Calves were infected intranasally with one of seven BHVl field strains. Nine to 13 days after infection BHVl-specific antibodies of the IgM isotype appeared in serum, nasal and ocular secretions and these were detectable until four weeks after infection. The first IgA antibodies were detected a few days later than the IgM antibodies. In serum the IgA antibodies were no longer detectable after 3 weeks, but these did persist for prolonged periods in mucosal secretions. The calves developed a uniform IgGl response from 13 days after infection, but the IgG2 response was quite variable; both persisted until the end of the experiment. No antibody responses were detected in genital secretions. There were no marked differences in isotype responses between calves infected with different strains of BHVl. Abbreviations ACA = Antibody capture assay; BHV 1 = Bovine herpesvirus 1; EBTr = Embryonal bovine trachea; ELISA = Enzyme-linked immunosorbent assay; EMEM = Earle’s minimal essential medium; IDAS = Indirect double antibody sandwich assay; MAbs = Monoclonal antibodies; PBS = Phosphate buffered saline; PID = Postinoculation day; SPF= Specified pathogen free.

* Corresponding

author.

’ Present address: Department of Microbiology and Infectious Diseases, Veterinary Faculty, University of Zagreb, 41001 Zagreb, Heinzelova 55, P.0.B 190, Croatia. ’ Present address: Institut de Chimie B6, UniversitC de Lihge, Sart Tilman, B-4000 Liege, Belgium. 0165-2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI0165-2427(94)05363-4

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1. Introduction

Bovine herpesvirus 1 (BHVl ) is an economically significant pathogen of cattle, which can cause rhinotracheitis, pustular vulvovaginitis, endometritis, oopho&is, abortions, encephalitis, conjunctivitis and systemic infections (Kahrs, 1977; Gibbs and Rweyemamu, 1977; Yates, 1982; Miller, 199 1). A BHV 1 infection or vaccination induces a broad spectrum of immune responses, among which isotype- and virus-specific antibody responses. There are several reports on isotype-specific antibody responses against BHV 1 after vaccination (Mukkur et al., 1975; Gerber et al., 1978; Ungar-Waron and Abraham, 199 1; Israel et al., 1992; Van Drunen Littel-Van Den Hurk et al., 1993) and after infection. Rodak et al. ( 1983) followed the isotype responses in serum and nasal secretions for 18 days after infection only. Guy and Potgieter (1985), Osorio et al. (1989), UngarWaron and Abraham ( 199 1) and Edwards et al. ( 1991) described the isotype responses in serum, but not in mucosal secretions, after a BHVl infection. The objective of our study, therefore, was to systematically monitor the isotype responses against BHVl in nasal, ocular and genital secretions and in sera of calves for several months after experimental infection with one of seven BHVl strains. For this purpose, we first developed enzyme-linked immunosorbent assays (ELISAs) to specifically detect IgM, IgA, IgGl or IgG2 antibodies directed against BHVl.

2. Materials aad methods 2. I. Viruses

The following seven field strains of BHVl were used for experimental infection: Babiuk (Dr. L.A. Babiuk, Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Canada), Cooper (Vr-864, American Type Culture Collection, Rockville, MD), Edwards (Dr. S. Edwards, Central Veterinary Laboratory, New Haw, Weybridge, UK), Espuna (Dr. E. Espuna, Laboratorios De Sanidad Veterinaria, Amer, Spain), Iowa (Dr. J.M. Miller, National Disease Center, Ames, IA ), Lam and Harberink. The latter two strains are Dutch strains, isolated in 1973 and 1987, respectively. All strains were cloned by three limiting dilutions in an embryonic bovine trachea (EBTr) cell strain. Thereafter they were passaged once more on the same cell strain. When the monolayers were affected by the cytopathic effect for more than 90%, the cultures were freeze-thawed, clarified by centrifugation and titrated. The virus pools were free of bovine viral diarrhoea virus. 2.2. Experimental animals and experimental design Thirty-two Friesian calves were used in this study. These calves were delivered by caesarean section and then immediately placed in isolation facilities, where

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they remained throughout the study. They were randomly divided into eight groups of four animals. Each group was housed in separate isolation rooms of the same size and with identical climatic conditions. For the first 6 weeks a commercial milk preparation was given and thereafter dried grass pellets and concentrated feed. At the age of 3 weeks, the calves of seven groups were intranasally spray-inoculated, 4 ml into each nostril, with an inoculum containing a total of lo* TCIDsO of BHVl . Each group received one of seven BHV-1 strains. Calves 42, 43, 44 and 45 were given the Babiuk strain; 66, 68, 69 and 70 the Cooper strain; 55,57,58 and 61 the Edwards strain; 52,53,54 and 56 the Espuna strain; 71, 73, 93 and 98 the Iowa strain; 48, 49, 50 and 51 the Lam strain; 59, 60, 65 and 67 the Harberink strain. The titre of the inoculum was verified by virus titration on the day of inoculation. The four remaining calves (46, 47, 62 and 92) were intranasally spray-inoculated with culture medium used to dilute the inocula and served as control group. All calves, except the mock-infected controls, developed severe clinical signs typical for BHVl infection and two calves infected with the Iowa strain died on post-inoculation days (PID) 8 and 10. 2.3. Sampling procedures Blood samples were collected on the day of inoculation (PID 0) and PID 6, 7, 8,9, 10, 13, 15, 17,20,28,49,77 and 112. Serum was obtained from whole blood by centrifugation and stored at - 20’ C until tested. Nasal, ocular and genital secretions were collected on PID 0,6, 8, 10, 13, 15, 17,20,28, 35,42,49, 56, 63, 77,9 1 and 104. The mucosal secretions were collected with cotton tips (Medical Wire and Equipment Co. (Bath) Ltd., Potley, Corsham, UK) and suspended in 3 ml of phosphate buffered saline solution (PBS), pH 7.2, clarified by centrifugation and stored at - 70°C until tested. Between 100 and 200 ~1 of nasal or ocular secretions and 50-200 ~1 of genital secretions were absorbed by a cotton tip. Thus, the dilution ( “log) factor for nasal or ocular secretions was between 1.2 and 1.5, and for genital secretions between 1.2 and 1.8. Blood contamination of secretions was checked with the Sangur-test (Boehringer GmbH, Mannheim, Germany ) . 2.4. Serum neutralisation test The serum neutralisation test was performed using microtitre plates (Greiner, Labortechnik, Netherlands). Twofold serial dilutions of heat-inactivated (56°C for 30 min) samples in Earle’s minimal essential medium (EMEM) containing 2% heat-inactivated foetal calf serum, 100 U penicillin, 100 pg gentamicin and 100 M mycostatin ml-‘, were mixed with an equal volume (25 ~1) of BHVl strain Lab containing 100 TCIDSo. The mixtures were incubated for 24 h at 37°C. A 24 h test is at least ten times more sensitive than the conventional 1 h neutralisation test (Bitsch, 1978). Thirty minutes before the cells were added, guinea

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pig serum, as a complement source, in a final concentration of 10% was added to the mixtures. Then, 100 ~1 of EBTr cells in EMEM supplemented with 2% of heat-inactivated foetal calf serum were added to the wells and the plates were further incubated for 3 days. Titres were expressed as log,, of the reciprocal of the highest dilution of test sample that inhibited the cytopathic effect. Parallel test were carried out using the same amount of heat-inactivated guinea pig serum. 2.5. Monoclonal antibodies Monoclonal antibodies ( MAbs) against bovine immunoglobulin isotypes (antiIgG 1, no. 15.8; anti-IgG2, no. 12.5; anti-IgM, no. 17.4; anti-&A, no. 16.35) were those produced, characterised and selected by Van Zaane and IJzerman ( 1984). From a large panel of anti-BHVl MAbs, a MAb directed against gB was selected, because it gave the best results in the isotype-specific ELISAs. This MAb was used as conjugated antibody in IgA and IgM ELISAs and as coating material in IgGl and IgG2 ELISAs, and gave superior results (e.g. no background with control antigen) when compared with polyclonal rabbit anti-BHVl sera. The MAbs were purified from mouse ascites fluid by 50% ammonium sulphate precipitation, followed by dialysis overnight against PBS, pH 7.2, and conjugated with horseradish peroxidase according to Wilson and Nakane ( 1978 ) . 2.6. Preparation of antigen for ELISAs Monolayers of secondary bovine kidney cells, free of bovine viral diarrhoea virus, were prepared in roller bottles. The cells were grown in Hanks’ MEM (Gibco Ltd., Breda, Netherlands) with 10% of foetal calf serum, 100 U ml- ’ of penicillin, 100 ,ug ml- ’ of gentamicin and 100 pg ml-’ of mycostatin. The cultures were inoculated with lo3 TCIDSo of BHVl strain Iowa, and rolled for 1 h. Thereafter, 100 ml of maintenance medium consisting of EMEM with 2% foetal calf serum and antibiotics was added. After incubation for 48-72 h at 37°C when an extensive cytopathic effect was evident, the cultures were frozen at - 70°C and thawed. After two freeze-thaw cycles, cellular lysate was clarified by centrifugation (5000 rev min- ’ for 15 min ). Supematant was stored for use as antigen in IgGl and IgG2 ELISAs. For use in IgA and IgM ELISAs BHV 1 was precipitated from clarified lysate using saturated ammonium sulphate solution and pelleted by centrifugation. The pellet was suspended in PBS with 1 mM EDTA, pH 7.2, and dialysed against PBS overnight. The dialysate was stored in small aliquots at 70°C for use as antigen. Mock-infected bovine kidney cells were prepared in the same way to provide control antigen. 2.7. Isotype-specific BH VI ELISAs In a previous study, an antibody capture assay (ACA) was found optimal for the detection virus-specific IgA and IgM, and an indirect double antibody sandwich assay (IDAS) for the detection of virus-specific IgGl and IgG2 (Van Zaane

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and IJzerman, 1984). We therefore developed an ACA for detecting BHVl-specilic IgM and IgA, and an IDAS for detecting BHVl-specific IgGl and IgG2. Both assays were performed in micro-ELISA plates (Greiner, Labortechnik, Alphen a/d Rijn, Netherlands). To optimise ELISAs the following variables were tested: the influence of buffers on binding MAbs to microtitre plates, blocking of nonreactive sites after coating with ovalbumin, the influence of the sodium chloride concentration in the ELISA buffer on colour development and the possibility of background reduction by using different concentrations of bovine serum albumin, gelatin, ovalbumin and normal horse serum in ELISA buffers. Use of carbonate buffer, pH 9.6, for coating the MAbs in ACA showed high binding. In IDAS, high binding was recorded by using PBS, pH 7.2, as coating buffer. There was high colour development and no background by using ELISA buffer containing 0.35 M sodium chloride and 1% gelatin in ACA. In IDAS, colour development was optimal by using ELISA buffer containing 0.60 M sodium chloride. The background staining in IDAS with negative control sera obtained from SPF calves was eliminated by adding 5% normal horse serum or 2% ovalbumin to the ELISA buffers. For performing an ACA, the plates were coated with the MAb directed against bovine IgA or bovine IgM in 50 mM carbonate buffer, pH 9,6, by incubation at 4°C overnight. After coating, the plates were stored at -20°C until use. In subsequent steps, the plates were incubated with test sample, antigen and the enzyme-labelled MAb directed against BHVl at 37 “C for 1 h and with substrate solution at 4°C overnight. For IgA detection, the incubation with antigen was at room temperature overnight, instead of 37 “C for 1 h. The dilutions of all reagents were prepared in ELISA buffer (PBS, pH 7.2, 1 mM EDTA, 0.35 M NaCl, 0.05% (w/v) Tween 80, 1% gelatin). The conjugate was diluted in ELISA buffer containing 0.2% foetal calf serum. Optimum dilution for each reagent was determined by checkerboard titration. At each step, 100 ~1 of each reagent were added per well. For IDAS the plates were coated with the MAb directed against gB of BHV 1 in PBS, pH 7.2, at 4°C overnight and then were stored at -20°C. Subsequent incubation steps with antigen, test samples and enzyme-labelled MAb directed against bovine IgGl or IgG2 were performed for 1 h at 37°C. The incubation with substrate solution was at 4’ C overnight. The reagents were diluted in ELISA buffer (PBS, pH 7.2, 1 mM EDTA, 0.60 M NaCl, 0.05% (w/v) Tween 80, 5% horse serum). Optimal dilutions of all reagents were predetermined. In each step 100 ~1 of reagent were added per well. After each incubation step, plates were rinsed six times with deionised water containing 0.05% Tween 80 in both ACA and IDAS. As substrate solution, 0.1% recrystallised 5-aminosalicylic acid in 10 mM Naphosphate buffer, pH 6.8, containing 0.1 mM EDTA and freshly added 0.005% H202 was used (Ellens and Gielkens, 1980), Colour development was measured with a Titertek-multiskan spectrophotometer using a 450 nm filter. The sera were first screened in a dilution of 1: 10 in ACA and 1: 20 in IDAS and the mucosal secretions were screened undiluted. Positive sera were then two-

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fold serially diluted, starting at 1: 10 in ACA and 1: 20 in IDAS. Twofold serial dilutions, starting at a dilution of 1: 2 were made of mucosal secretions. In each plate a twofold dilution series of a strong-positive standard sample (ranging from positive to negative) and a negative sample were included. In the IgM, IgG 1 and IgG2 ELISAs the strong-positive sample was serum and in the IgA ELISA it was nasal secretion. The strong-positive sample had a high titre of specific antibodies and a high maximum E450 nm absorbance value. The standard negative control serum was obtained from an SPF calf free of BHV 1 antibodies. To check for nonspecific reactions, all samples in the lowest dilution were incubated with control antigen. A sample was scored positive if it scored one matrix unit above the value obtained with the negative control sample. One matrix unit corresponded to 1/ 10 of the maximum optical density (OD) value obtained with the standard positive serum (OD between 1.5 and 2.0). The antibody titre of the sample was expressed as the log,, of the reciprocal value of the highest dilution scoring one matrix unit above the value obtained with the negative control sample. The matrix was set per plate. In expressing the titres in mucosal secretions, no correction was made for the dilutions that were introduced by preparing the samples. In calculating the geometric mean titre per group, negative scores were included as well. The two calves that were infected with the Iowa strain and died were not included in calculating the geometric mean titre. 2.8. Immunoafinity chromatography of BHVI speci$c isotypes To study the neutralising capacity of isotypes, samples taken from calves infected with the Lam strain having high titres of a particular isotype were pooled and deprived of BHVl antibodies of other isotypes by precipitation with 50% saturated ammonium sulphate and subsequent immunoaffinity chromatography (Van Zaane and IJzerman, 1984).

3. Results 3.1. Specificity, sensitivity and reproducibility of isotype-speci’c ELISAs Table 1 shows that the IDAS and ACA ELISAs were highly specific. Selected samples with a high titre for one isotype and those samples enriched for antibodies of one isotype did not react in ELISAs for the other isotypes. The samples containing more than one isotype did not react uniformly in the various ELISAs. In addition, sera with antibodies to BHVl did not react with control antigen and all negative sera were scored negative (data not shown). To assess the relative sensitivity of the isotype-specific ELISAs, selected samples were titrated in a complement-dependent and a complement-independent 24 h neutralisation test. The titres scored in ELISAs were, with a few exceptions, higher than those in the neutralisation test (Table 1). All isotypes were able to

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J. Madic et al. / Veterinary Immunology and Immunopathology 46 (1995) 267-283 Table I Isotype specificity and sensitivity of isotype-specific with and without the addition of complement

ELISAs compared with a 24 h neutralisation

Neutralisation

ELBA

I Serum” 2 Serum 3 Serum 4 Serum 5 Serum” 6 Serum 7 Seruma 8 Nasal secretion 9 Eye secretion IO Nasal secretion I 1 Eye secretion 12Nasal secretion

IgG,

IgGz

IgM

2.4b 1.9 4.3 3.1

_ 3.1 2.5 2.1

_

_ -

3.4 3.1 3.4 1.8 1.2 1.5

-

1.5

_ 1.3 _ 2.4 2.4 1.8

t&1

test

+ compl.

- compl.

1.2 2.1 > 3.6 2.4 1.2 I.5 0.3 _

0.9 0.9 > 3.6 2.1 I.2 0.6 _

1.2 1.2 1.5

0.9 0.9 0.3

‘Samples that were purified for antibodies of a particular isotype by immunoaffinity chromatography. “Titre in isotype-specific ELISA and neutralisation test expressed as log,,. -, Negative (i.e. < I for IgM and IgA, and < 1.3for IgG, and IgG, in serum and < 0.3 for all isotopyes in mucosal secretion in ELBA and ~0.3 in neutralisation test ).

neutralise BHV 1. Neutralising antibodies were not found in two mucosal secretion samples that contained only IgM antibodies. The titres in the neutralisation test were increased, particularly in samples containing IgM antibodies, by adding complement. In general, the ELISAs thus had a higher detection limit than the neutralisation test. The reproducibility of the assays was evaluated by testing positive reference samples in twofold dilution series on each plate in each assay. The range of titres obtained for the positive samples put on different plates or tested on different days usually did not exceed one dilution step. Coating different batches of plates with optimal dilutions of MAbs, prepared on different occasions also showed reproducible results. 3.2. Antibody responses after infection 3.3. &I4 antibody responses The IgM specific antibody response was the first isotype response to be detected. All infected calves developed IgM antibody in serum and nasal secretions. In ocular secretions, IgM antibodies were not detected in 7 of 28 infected calves. No antibodies were detected in genital secretions. Mock-infected calves remained negative. The means and ranges of PID of first detection, of reaching the peak titre and of last detection are given in Table 2. The IgM response was somewhat earlier

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Table 2 Isotype-specific

antibody responses in calves infected with BHVI

Isotype and specimen

Mean PID” of first detection (range )

Mean PID of reaching peak titre (range)

Mean PID of last detection (range )

8.9 (7-10) 10.2 (8-13) 13.0 (10-20)

12.6 (10-15) 14.0 (S-20) 14.3 (10-20)

$28) 22.6 (15-28) 17.8 (13-20)

12.0 (9-13) 12.5 (10-13) 14.2 (10-28)

14.6 (IO-I?) 21.4 (13-77) 19.7 ( 13-20)

21.5 (15-35) 100 (20-104) 62.2 (20-104)

12.8 (10-15)

29.4 (13-49)

112

23.8 (15-55)

56.6 (17-77)

112

I&f Serum

Nasal secretion Ocular secretion &A Serum Nasal secretion Ocular secretions IgGl Serum IgG2 Serum ‘Post-infection

day.

detectable in serum than in nasal secretions. Ocular secretions became positive usually three days later than nasal secretions and were earlier negative than nasal secretions. IgM antibodies were not detected after PID 28. The mean IgM peak titres in serum reached levels above 1000, in nasal secretions above 10 and in ocular secretions lower than 10 (Figs. 1,2 and 3 ). Some sera that had a high IgM titre had a low OD at 450 nm in the lower dilutions. There were no marked differences in IgM responses in calves given different BHV 1 strains (Figs. 1,2 and 3). 3.4. IgA antibody responses The appearance of the IgA specific antibody response could be detected later than the IgM response (Table 2 ) in serum and secretions. All calves, except one, had IgA antibodies in serum. The serum IgA antibodies were usually detected between PID 12 and 2 1. In all calves IgA antibodies were detectable in nasal and ocular secretions. The calf that developed no serum IgA response had a normal IgA response in nasal secretion and a short and low response in ocular secretion. All genital secretions and sera and secretions of mock-infected calves were negative. The means and ranges of PID of first detection, of reaching the peak titre and

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Fig. 1. The mean IgM antibody responses against BHVl in sera of calves infected with one of seven BHV 1 strains.

of last detection are given in Table 2. On average, the IgA response appeared at approximately the same time in serum and nasal secretions and somewhat later in ocular secretions. The nasal secretions were longer positive than the ocular secretions. The IgA response was detectable on PID 104 in nasal secretions of 23 of the 26 calves; in nine calves the response was transiently undetectable. In nine of the 26 calves the ocular secretions were positive on PID 104; in seven of these the response was intermittently detectable. The mean peak IgA titres in serum were around 100, in nasal secretions slightly above 10 and in ocular secretions somewhat below 10 (Figs. 4, 5 and 6). The mean IgA titres peaked at a lower level than those of serum IgM. In nasal and ocular secretions mean peak IgA and IgM titres were comparable. There were no clear differences between the groups of calves (Figs. 4, 5 and 6). 3.5. IgGl antibody responses All calves produced serum IgGl antibodies. The IgGl antibodies were first detectable in sera around PID 13 (Table 2 ) . The titres rose and reached a peak around four weeks post infection, after which they remained at a stable level until the end of the experiment (Fig. 7 ). In nasal secretions of 12 calves, IgG 1 antibodies titres were irregularly detected usually between PID 15 and 28. In six calves

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Fin. 2. The mean IgM antibody responses against BHVl in nasal secretions of calves infected with one of seven BHV 1 strains. 2

OcularIgM

Days after infection

Fig. 3. The mean IgM antibody responses against BHV I ocular secretions of calves infected with one of seven BHV I strains.

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217

Serum IgA 25.

Days after infection

Fig. 4. The mean IgA antibody responses against BHVl in sera of calves infected with one of seven BHV 1 strains.

r

Nasal IgA

I

Days after infection

Fig. 5. The mean IgA antibody responses against BHVl in nasal secretions of calves infected with one of seven BHVl strains.

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J. Madic et al. / VeterinaryImmunology and Immunopathology 46 (1995) 267-283 OcularIgA 2, Bal&k CO_geW Harkink

Fig. 6. The mean IgA antibody responses against BHVl in ocular secretions of calves infected with one of seven BHVl strains. Serum lgG1

Fig. 7. The mean IgGl antibody responses against BHVl in sera of calves infected with one of seven BHVl strains.

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279

IgGl antibodies were also irregularly detectable in ocular secretions; usually between PID 28 and 56. IgGl antibodies were virtually always found in undiluted or 1: 2 diluted secretions and not in higher dilutions. All IgG 1-positive nasal secretions contained 1O-50 erythrocytes per microlitre (except from one calf ) ; the ocular secretions were not contaminated with erythrocytes. The genital secretions of infected calves and sera and secretions of mock-infected calves were negative for IgGl antibodies. The groups of infected calves showed similar IgGl antibody patterns in sera (Fig. 7). 3.6. IgG2 antibody responses All calves elicited IgG2 antibodies in serum. They appeared on average 12 days later than the IgG 1 antibodies. The individual variation of day of first detection was great. The mean peak titres were reached much later than those of IgG 1 antibodies (Table 2). In general, the serum IgG2 antibody titres steadily rose until the end of the experiment and remained lower than the serum IgG 1 titres (Fig. 8 ) . Calves given the Espuna strain had a low mean IgG2 antibody titre during the first seven weeks post infection (Fig. 8). All nasal, ocular and genital secretions of infected calves and sera and secretions of mock-infected calves were negative. Serum

lgG2

41

Fig. 8. The mean IgG2 antibody responses against BHVI in sera of calves infected with one of seven BHV 1 strains.

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4. Discussion The ELISAs we developed were based on MAbs against bovine immunoglobulin isotypes and on a MAb against BHVI . The isotype specificity of these ELISAs relies on the specificity of the MAbs directed against bovine immunoglobulin isotypes. The chosen MAbs reacted specifically with IgG 1, IgG2 or IgA, while the anti-IgM MAb showed a weak cross-reaction with IgA (the titre against IgA was a lO.OOO-fold lower than that against IgM) (Van Zaane and IJzerman, 1984). The weak cross-reactivity of this MAb did not raise problems in our assays, because samples with a high BHV 1-specific IgM antibody titre were negative in the IgA assay and vice versa. The absence of cross-reactivity in our ELISAs (Table 1) underlines the high specificity of tests that are based on the MAbs directed against bovine immunoglobulin isotypes (Van Zaane and IJzerman, 1984; Van Zaane et al., 1986; Kimman et al., 1987a; Mulcahy et al., 1990). The specificity of the tests was further established by using a MAb against gB of BHVl instead of using polyclonal anti-BHV 1 sera. Our isotype-specific BHVl ELISAs are easier to perform and more suitable for testing large numbers of samples than previously described tests, such as indirect immunofluorescence test (Gerber et al., 1978), indirect radioimmunoassay (Rodak et al., 1983 ), or tests that use isolated immunoglobulin isotypes (Mukkur et al., 1975; Guy and Potgieter, 1985). The specificity of the above tests may be questioned, because conventional polyclonal antisera were used as isotype detecting antibody, which often appear to cross react (Butler, 1983 ) . All isotypes were able to neutralise the virus in the absence of complement, as has been reported previously (Mukkur et al., 1975). The addition of complement enhanced the neutralising antibody titres, but not in the case of isolated IgG2 isotype. This may be caused by the inability of bovine IgG2 to activate complement (Sellei, 1984). The most likely explanation for absence of neutralising activity in two mucosal samples containing only IgM isotype, is the difference in sensitivity of the IgM-ELISA and the neutralisation test. IgM antibodies could be detected a little earlier in serum than in nasal secretions, but the difference in starting dilution of the samples ( 15-30 for nasal secretion and 10 for serum) may also account for this difference in first appearance. Because IgM and IgA antibodies were detectable only up to PID 28, their presence indicates a recent BHV 1 infection. The detection of BHVl -specific IgM (Osorio et al., 1989; Ungar-Waron and Abraham, 199 1) or IgA antibodies, therefore, can be used in routine serodiagnosis of BHVl infections. Our results on serum IgM and IgG isotype responses are generally in keeping with those of others (Rodak et al., 1983; Osorio et al., 1989; Ungar-Waron and Abraham, 199 1). However, they do not fully agree with those of Guy and Potgieter ( 1985 ). The latter authors detected low neutralising activity against BHV 1 in the isolated IgM serum fraction until PID 90, whereas we detected IgM antibody no later than PID 28. In addition, they detected neutralising activity in the IgG fraction by PID 7. These contrasting findings might be explained by the lower starting dilutions ( 1: 2) they used in the test, by methodological differences or by

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a slight contamination of the IgM fraction by IgG and vice versa. In contrast with Rodak et al. ( 1983) and Edwards et al. ( 199 1 ), we could detect serum IgA responses against BHV 1. Specific systemic IgA responses have been earlier demonstrated in bovine respiratory syncytial virus infections (Kimman et al., 1987b). We found that the serum IgG2 response was the most variable. This finding agrees with that of others (Rodak et al., 1983; Guy and Potgieter, 1985; Edwards etal., 1991). The IgM response in nasal and ocular secretions was only transiently detectable, whereas the IgA responses (intermittently) persisted for many weeks or until the end of the experiment. In ocular secretions the isotype response was shorter and lower than in nasal secretions. The lower rate of BHVl replication in ocular than in nasal mucosae (data not shown) may account for the difference in responses in nasal and ocular secretions. Persistence of IgA antibodies in nasal secretions has also been demonstrated after multiple experimental vaccinations against BHVl (Israel et al., 1992) and after bovine respiratory syncytial infections (Kimman et al., 1987b). BHV 1-specific antibodies of the IgG 1 isotype were found in trace amounts in the blood-contaminated nasal secretions, suggesting that these IgGl may have been leaked from the blood as a consequence of mucosal damage. However, IgG 1 antibodies were also detected in nasal secretions of one calf and in ocular secretions of six calves that were not contaminated with blood. This latter phenomenon suggests some form of selective transport of IgGl from serum into nasal secretions, or the presence of a minute local production of IgG 1 at mucosal surfaces (Butler, 1983 ). The complete absence of IgG2 in secretions is also supporting the concept of selective IgG 1 transport or local production rather than transudation or leakage of IgGl from the blood. We detected IgGl in nasal secretions mainly from PID 15-28, whereas Rodak et al. ( 1983) found nasal IgGl antibodies between PID 11 and 14. This may be due to methodological or experimental differences. Strains of BHVl can vary in virulence for cattle, which may result in differences in immune responses. Edwards et al. ( 199 1) found an overall higher IgG 1 and IgG2 response in calves given BHV 1 subtype 1 than in calves given BHV 1 subtype 2b. We followed the isotype responses in 28 cattle that were infected with one out of seven strains of BHV 1 subtype 1. Although we noticed clear differences in virulence between these strains (Kaashoek et al., unpublished observations, 1990) there were no marked differences in isotype responses between the groups given different strains. We therefore may consider the results of this study to represent the isotype responses in young calves after infection with BHV 1 subtype 1.

Acknowledgements The authors thank H. Paal and K. Weerdmeester

for skilful technical assistance.

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