FOUR LAYER ENZYME-IMMUNOASSAY ( EIA ) DEMONSTRATION OF A SPECIFIC IgG AGAINST THE AUJESZKY ' S DISEASE VIRUS IN BLOOD SERA , FOLLICULAR FLUIDS AND OVA OF VACCINATED SOWS

In five sows vaccinated and revaccinated against Aujeszky's disease (AD) with an inactivated AD vaccine, specific IgG to the AD virus was detected by means of four-layer enzyme-immunoassay (EIA) in blood sera and disintegrated ova, and in three of these animals also in ovarian follicular fluids. In all sows under study, titres of antiviral antibody of the IgG class ·occurred and increased after vaccination and revaccination. The specific IgG was also detected in disintegrated ova from 4 of these sows. Its calculated amounts surpassed considerably those found in simultaneously collected blood sera of these animals.

In five sows vaccinated and revaccinated against Aujeszky's disease (AD) with an inactivated AD vaccine, specific IgG to the AD virus was detected by means of four-layer enzyme-immunoassay (EIA) in blood sera and disintegrated ova, and in three of these animals also in ovarian follicular fluids.In all sows under study, titres of antiviral antibody of the IgG class •occurred and increased after vaccination and revaccination.The specific IgG was also detected in disintegrated ova from 4 of these sows.Its calculated amounts surpassed considerably those found in simultaneously collected blood sera of these animals.
Interaction of viruses with unfertilized and fertilized ova in different periods of development has been studied by a number of authors.Porcine ~was shown to adhere to the zona pellucida of porcine ovum but dose not penetrate it (W r a t h a l l and Men gel i n g 1979).Interaction of the porcine ova and embryos with Aujeszky'~ disease virus has been studied by B 0 1 in et al. (1981).The ova and embryos originated from sows free of neutralizing antibodies against the AD virus and the semen used for insemination was from serologically negative boars.After cocultivation with the AD virus on a cell line from porcine kidney no AD virus was demonstrated either in embryonic cells or in zona pellucida.Further electronmicroscopic study (B 0 1 i n et al. 1983) revealed no productive infection with AD virus, porcine ~rus or porcine enteroviruses.B 0 1 i nand B 0 1 i n (1984) d..onstrated the AD virus in porcine embryos harvested 6 and 10 d after intrauterine inoculation of bred gilts with AD virus.However.from available literature sources no information was obtained that could be employed as a basis for i..unoglobulin demonstration in porcine ova.
In context with our previous work (J e f abe k et al. 1987) the aim of this study was to find whether specific antibodies against AD virus (porcine herpesvirus 1 -S zan t 0 et al. 1979;Roc k and R e e d 1980;S abo 1981) can be detected not only in the blood serum and in follicular fluids but also in porcine ova.

Experimental animals
Five sows after their first farrowing were used.three of them Duroc (No.3, 4 and 7), 2 crosses of the Large White and Landrace (No. 25 and 26).The sows were from AD-free farms.
The ova were recovered during natural (No.3) or hormonallv stimulated heat.The hormonal stimulation was performed in the second half of expected cycle by administration of 1500 1. U. PMSG followed by 1000 I. U. HeG (Praedyn, Lel:iva) 56 h later.Twenty four hours after Praedyn administration laparotomies were carried out estimating that 30 -50 h after HCG application ovulation occurred (K 0 z u m p I i k and K u d I a l: 1980).In this way sow No.25 was prepared for the first and second ova collection and sows No.4, 7 and 26 for the second collection (in these animals the first collection was carried out during a natural ovulation).

Vaccination
The sows were vaccinated and revaccinated with 5 ml in inactivated vaccine against AD i.m. (according to the manufacturer's instructions -Bioveta, Ivanovice na Hane).They were vaccinated after the first sample collection (d 0) and revaccinated on d 16 (No.7 and 25) or 21 (No.3,4 and 26) after the first vaccination.

Sample collection
Samples of blood, follicular fluids and ova were collected before vaccination (d 0), blood samples before revaccinatination, and blood, follicular fluids and ova on d 22 -27.after revaccination.Blood sera and follicular fluids were stored at -20•C pending analysis.
The ova were collected from ovarian follicles, by flushing the.oviducts or uterine horns.From sows No. 4 and 26 the ova were collected twice from t~e ovarian follicles, from sow No. 3 at the first collection by flushing, at the second collection from the follicles, from sow No.7 at both samplings by flushing, from sow No. 25 at the first collection by flushing and at the second by both flushing and from the follicles.A fivefold washing of all ova in PBS after Dulbecco (B 0 n i n 1973) followed.The ova were then individually placed in test tubes containing 0.1 ml PBS.They were stored at -20'C and three times thawn and frozen prior to examination.
All samples collected from the individual sows were always examined simultaneously.For the four-layer EIA, they ~ere diluted using a diluting and conserving solution (R 0 d a k et aI.1985): PBS supplemented with 0.5 % bovine serum albumin (BSA), 0.3 % Tween 20, 0.1 % EDTA-Na, 0.02 %

EIA reagents Viral and control antigen
The preparation of both antigens was described in detail earlier (R 0d a k et al. 1985).Virus antigen (V-Ag) was obtained by growing AD virus in the RK 13 cell line.In the same way the control antigen (C-Ag) was prepared from non-infected cell cultures.After concentration, both antigens were stored at -70•C.For use i,.nEIA, the antigens were diluted 1:200 in 0.05 M carbonate-bicarbonate buffer, pH 9.6.
Class specific antiserum to porcine immunoglobuline Class specific antiserum to porcine immunoglobuline was prepared by repeated immunization of rabbits with heavy chains of porcine IgG.The method of preparation "nd specifity checks were described earlier (R 0 d a k 1984).

POD-SwARIgG conjugate
A pig weighing 50 kg was injected i.m. with 3 consecutive 10 mg doses of purified rabbit IgG (RIgG) in AI-Span Oil adjuvant (SEVAC, Czechoslovakia) administered at 3-week intervals.Specific antibodies to rabbit IgG (SwARIgG) were separated from hyperimmune porcine serum collected 10 d after the last injec+;)n by affinity chromatography (IgG covalently bound to CNBr Sepharose 48, Pharmacia).These antibodies were conjugated with horse-radish peroxidase (POD; RZ = 3.0, Boehringer, Mannheim) using the periodate method (W i Ison and N a k a n e 1978).The stock solution of the POD-SwARIgG conjugate contained 2 mg of specific antibodies with addition of 1% LAH in 1 ml.For the four-layer EIA the stock solution was diluted 1: 1000 with PBST containing 1 % LAH.

Substrate solution
A solution of 8 mg of 5-aminosalicylic acid (FLUKA AG, Switzerland) in 10 ml 0.05 M citrate-phosphate buffer, pH 5.8, was supplemented with 1 ml 0.05% HzOz immediately before use.

EIA method
All samples were examined by the four-layer EIA for specific IgG to AD virus.Microtitre plates with flat-bottom wells were used (Koh-I-Noor, Czechoslovakia).Each well contained 100 III of all reagents.The wells were always washed three times using PBS.
Four-layer EIA Vertical rows of wells in microtitre plates were alternatively filled with viral -V (rows 1, 3, 5 etc) and control -C (rows 2, 4, 6 etc.) antigens.After adsorption of antigens overnight at 20• C and washing the wells with bound V and C antigens were filled with threefold diluted samples.The blood sera were examined in dilution range 1: 100 to 1: 218 700, follicular fluids in the range of 1: 9 to 1: 19 683 and PBS with ovum (presumably disintegrated due to alternate freezing and thawing) in the range of 1: 5 to 1: 10 035.Examined samples were always diluted in the same way in wells coated with viral and control antigen and left in a moist chamber at 20• C overnight.The next day the wells were washed and filled with a solution of class-specific rabbit antiserum against the porcine immunoglobulin (RASwIgG).After incubation for 90 minutes at 37• C and washing another incubation with  the conjugate peroxidase-antibody to rabbit immunoglobulin (POD-SwARIgG) followed under the same conditions.After this incuba.tionand washing the wells were filled with the substrate solution and the reaction was evaluated 60 min later spectrofotometrically at 492 nm.

Evaluation of EIA results
The results were evaluated by comparing the absorbance of the substrate solution in "the wells containing V-Ag with tQatfound for C-Ag incubated with the same sample dilution.k sample was considered positive if the V:C ratio was it 2.0.Besides it was required that after incubation without serum or with a negative serum the ratio V: C be. 1. 0 ± 0.2.
The antibody titres are given in reciprocal values.

Calculation of the IgG levels in porcine ova
Calculation of the IgG amount in the porcine ovum was based on its diameter 165 lJm (K 0 z u m p 1 f k and K u d 1 Ii I: 1980), its volume then being 0.00235 mm 3 giving thus a 42 554fold dilution of one disintegrated ovum in 0.1 ml PBS.The EIA result (i.e. the IgG titre of a disintegrated ovum in PBS) was multiplied by this value (e. g.EIA titre of IgG 405 x 42 554 = 17 2s4 370 = the IgG titre in one ovum).

Results
The EIA examination results in the individual sows are given in Tables 1 -5 and schematically in Fig. 1..They indicate that the specific IgG was found in the blood serum of all examined sows after their vaccination and revaccination.Comparison of the IgG concentrations in follicular fluids in two sows (No.4, see Table 2, and No. 26, see Table 5) prior to and after vaccination and revaccination shows that these treatments resulted also in occurrence of the specific IgG in follicular fluids.IgG was also found in fluids from two follicles of sow No. 25 (Table 4), however, without sample collection before her vaccination.In 17 ova disintegrated in PBS collected from 5 experimental sows prior to vaccination, no specific IgG was demonstrated using the EIA test.In one animal (No.7, Table 3) no IgG was detected by this method in 4 ova collected after revaccination.Fr(!m the remaining 4 sows 17 ova were collected after revaccination.Among them 4 (1 from sow No. 25 and 3 from sow No. 26) yielded negative results whereas in 13 diSintegrated ova (Tables 1, 2, 4, 5 and Fig. 1) the IgG titres were substantially higher than in simultaneously collected blood sera.

Discussion
The observation of virus neutralizing antibodies in follicular fluids of sows  Prior to vaccination (day 0) no IgG was detected in any of the 5 collected blood sera, 22 follicular fluids or 17 ova.In 5 follicular fluids collected on day 22 after revaccination, an IgG titre> 19 683 was detected using the EIA method.In 13 of 21 ova collected after revaccination, the' IgG titres were substantially higher than IgG titres in simultaneously collected blood serum samples.
as in the ova of sows vaccinated and revacCinated against AD.As no antibody de1ectDt in ova disintegrated in PBS could be expected using the virus neutralization method, a much more sensitive method was selected, namely the EIA, in our experiment the four-layer EIA.
Using this method, the specific antiviral IgG was found in blood sera of all vaccinated and revaccinated sows.Comparison of the results obtained from 2 sows before vaccination and after revaccination revealed that these treatments resulted in occurrence of the IgG also in their follicular fluids.Surprisingly, substantiaDy higher IgG titres (calculated 8.8fold to 70~fold) were found in 13 of the 17 collected and disintegrated ova from 4 sows than in their sera collected at the same time.In one sow (No.7), no IgG was found in her ova.The present data give no answer to the question why in this sow's four ova no IgG was detected despite a high IgG titre of her blood serum (72 900).Similarly, in sow No. 25 (see Table 4) after revaccination no IgG was found in one ovum but was present in 7 other ova, and in sow No. 26 (Table 5) EIA yielded negative results in 3 ova whereas in 4 other ova from the same sampling, a positive result was obtained.Nevertheless, the data obtained in present work are valuable and stimulating for further research that may enrichour knowledge not only about interaction -Virus-ovum-embryo (B 0 1 i n et al. 1981; B 0 1 i n et al. 1983; B 0 1 i nand B 0 1 i n 1984) but also provide information on immunity at the earliest stage of ontogenesis.
(J e f Ii b e k et al. 1987) was an impulse for our further study with the aim to find whether and in what amounts specific antibodies are present in ova of sows vaccinated and revaccinated against AD.Virus neutralizing antibodies to the virus of mucosal disease and to the virus of infectious rhinotracheitis of cattle found in blood sera of cows contained predominantly IgG (W hit m 0 r e and Arc h b aId 1977) that was also detected in their follicular fluids.This fact along with the observation of exclusively• IgG class antibodies in blood sera and oropharyngeal swabs occurring 3 weeks after vaccination of pigs against AD (R 0 d a k et al. 1986) directed our effort to demonstration of the IgG in blood sera and follicular fluids as well Fig.1.A schematic presentation of the specific IgG in blood sera, follicular fluids and ova of sows vaccinated and revaccinated against .AD.Prior to vaccination (day 0) no IgG was detected in any of the 5 collected blood sera, 22 follicular fluids or 17 ova.In 5 follicular fluids collected on day 22 after revaccination, an IgG titre> 19 683 was detected using the EIA method.In 13 of 21 ova collected after revaccination, the' IgG titres were substantially higher than IgG titres in simultaneously collected blood serum samples.

Table I
EIA examination results in BOW

Table 2
EIA examination results in sow No. 4