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Studies on the serological detection of antibodies toavian encephalomyelitis virusA.A.S. Ahmed a , I.M. Abou EL‐Azm a , N.N.K. Ayoub b & B.I.M. EL‐Toukhi b

a Department of Avian and Aquatic Animal Medicine, Faculty of Veterinary Medicine ,Alexandria University , Edfina, Behera, Egyptb Animal Health Research Institute , Dokki, Cairo, EgyptPublished online: 02 Jan 2008.

To cite this article: A.A.S. Ahmed , I.M. Abou EL‐Azm , N.N.K. Ayoub & B.I.M. EL‐Toukhi (1982) Studies on the serologicaldetection of antibodies to avian encephalomyelitis virus, Avian Pathology, 11:2, 253-262, DOI: 10.1080/03079458208436099

To link to this article: http://dx.doi.org/10.1080/03079458208436099

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Avian Pathology, 11: 253-262, 1982

STUDIES ON THE SEROLOGICAL DETECTION OFANTIBODIES TO AVIAN ENCEPHALOMYELITIS VIRUS

A.A.S. AHMED1, I.M. ABOU EL-AZM1, N.N.K. AYOUB2

and B.I.M. EL-TOUKHI2

1 Department of Avian and Aquatic Animal Medicine, Faculty of VeterinaryMedicine, Alexandria University, Edfina, Behera, Egypt

2Animal Health Research Institute, Dokki, Cairo, Egypt

SUMMARY

An avian encephalomyelitis virus (AEV) antigen, prepared from the gas-trointestinal tract of infected chick embryos and partially purified andconcentrated by chloroform and polyethylene glycol treatments, exhibitedthe highest reactivity in the agar-gel precipitin test (AGPT). Antigen usedin the passive haemagglutination test (PHAT) that was purified and con-centrated yielded higher antibody titres than when untreated crude anti-gens were used.

The use of the AGPT, PHAT and embryo susceptibility test (EST) onchicken breeding flocks with and without a history of previous vaccinationagainst AE revealed that the PHAT was more sensitive in detecting AEVantibodies than the AGPT. The sensitivity of the PHAT was nearly equalto the EST. No correlation was found between the results of the AGPTand the immune status of a flock judged by the EST.

INTRODUCTION

The methods widely adopted for the detection of antibodies to avian encephalómye-litis virus (AEV) are based on the examination of serum or fertile eggs by the virusneutralisation test and the embryo susceptibility test respectively. Although both testsare well-established and reliable, they are time-consuming requiring 10 to 12 daysfor their completion. In addition, the restricted availability of susceptible eggs hasdeterred the wider usage of the virus neutralisation test.

On the other hand, the complement fixation test (Sato et al, 1969) and the indirectfluorescent antibody technique (Choi and Miura, 1972) have had limited applicationdue to the complexity of the procedure and/or the unreliability of the results. Thepurpose of the present work was to evaluate and compare the relative merits of theagar-gel precipitin test (Lukert and Davis, 1971) and the passive haemagglutinationtest (Heider et al, 1973) with the embryo susceptibility test in detecting antibodiesto AEV.

Received 9 June 1981Accepted 6 October 1981

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254 A.A.S. Ahmed étal

MATERIALS AND METHODS

Fertile eggsFertile chicken eggs for virus propagation,.virus titration, antigen preparation andvirus neutralisation tests came from susceptible breeding flocks. They were inoculatedvia the yolk sac after incubation for 5 to 8 days.

VirusThe egg-adapted Van Roekel strain (A 37020) of AEV was used throughout this study.Its median embryo infective dose (EIDS0) was estimated to be 106-S per ml.

Antigen preparationsInfected antigens were prepared from 18-day-old chick embryos which were inocu-lated with 102-S EID50 of AEV. Brains, gastrointestinal tracts (proventriculus, ventri-culus and intestine) and eviscerated whole bodies (minus heads and feet) wereseparately collected, and 20% extracts were prepared in phosphate buffered saline(PBS), pH 7.2. These preparations were designated as crude antigens. Portions of thelatter were partially purified by chloroform treatment (20%) after the method ofFeldman and Wang (1961). Other portions were further concentrated either by preci-pitation with ammonium sulphate (60%) as described by Nairn (1976), or by dehydra-tion with polyethylene glycol (PEG, mol. wt. 4000) overnight at refrigeratortemperature. Antigens were concentrated by these methods approximately two and20 times respectively.

Controls materials were prepared in a similar way from uninfected embryos.

SeraHyperimmune sera were prepared in five adult susceptible chickens by the intravenousinjection of AEV infected brain tissue extract (106-S EIDS0/ml) followed by weeklyintramuscular injections for 2 to 3 weeks. The birds were bled 2 to 4 weeks after thelast injection and sera separated and examined for their neutralising antibody contentby the standard virus neutralisation test. Pre-inoculation sera served as negative serumcontrols. AU sera were used without heat inactivation. Other sera used included ade-novirus type 1 (CELO) and infectious bursal disease (IBD) antisera.

Determination of infectivity endpointsThis was carried out in groups of four to five embryos per dilution as outlined byHitchner et aL (1975). Inoculated embryos were examined for lesions 10 to 12 dayspostinoculation (p.i.) and the EID50 was estimated by the method of Reed andMuench(1938).

Methods of antibody detectionThe virus neutralisation test (VNT). The variable virus-constant serum procedure wasadopted (Hitchner et aL, 1975).

The embryo susceptibility test (EST). Samples of 30 to 50 fertile eggs taken frombreeding flocks were subjected to the EST (Luginbuhl and Helmboldt, 1978) using adose of 102 EIDso per egg of AEV. Four to five eggs from each sample were left asuninoculated controls. The test was read 10 to 12 days p.i. according to the criteriareported by Monreal (1963) and Sumner et aL (1957).

The passive haemagglutination test (PHAT). The microtitre procedure in V-shapedmicrotitre plates was adopted as described by Ayoub (1974). A 1% suspension of sen-sitised tanned sheep red cells in PBS (pH 7.2), containing 1% inactivated and well-absorbed normal rabbit serum, was used in the test. This suspension could be preservedfor up to 2 weeks at 4°C.

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Detection of antibodies to AE virus 255

Chicken sera to be examined were pre-absorbed with a 5% suspension of tanned sheepred cells in the ratio of one part serum to four parts red cell suspension. Endpointswere recorded after 6 to 8 hours incubation of the plates at refrigerator temperature.Titres of 1:10 and higher were considered as positive. Suitable antigen, serum and redcell controls were included.

The agar-gel precipitin test (AGPTJ. The micro titre test was adopted in Petri-dishes,9.0 cm in diameter. The medium consisted of 1% Nobel agar, 8%NaCl and 0.0001%sodium merthiolate, and the pH was adjusted to 7.2. The medium was poured whilestill hot into the dishes in 10 ml volumes. Five sets of wells, each consisting of sixouter wells surrounding a central one, were cut into the medium after solidification.The distance between the central and the peripheral wells as well as the diameter ofthe wells were 4 mm. Approximately 0.05 ml of serum or antigen could be containedin each well.

RESULTS

Examination of antigen preparations for their suitability for the A GPT and the PHA TResults of titrations of various antigen preparations for infectivity prior to their ex-amination by the AGPT and the PHAT are given in Table 1. It is evident that amongthe crude preparations examined the gastrointestinal tract (GI) antigen exhibited thehighest titre. Treatment with chloroform had no significant effect upon the titres.Concentration of chloroform-treated antigens by ammonium sulphate was unsuccess-ful with extracts from the brain and without marked effect on the titres of the othertissue extracts. On the other hand, PEG treatment increased the titre of all prepara-tions by 1 to 1.5 log io (proportional to the degree of concentration).

Table 1. Results of examination of various avian encephalomyelitis virusantigen preparations for infectivity.

Source ofantigen

Chick embryo Brc

Chick embryo WBd

Chick embryo GIe

Infectivity titre (logio EIDS0/ml)before and after treatment

Crude3

6.56.27.0

Chloroform(20%)

6.56.56.8

Amm. sul.b

(60%)

NPf

6.56.4

PEGb

7.87.28.5

a 20% extract in PBS.b Rate of concentration two and 20 times the original volume by ammonium sulphate

(Amm. suL) and polyethylene glycol (PEG) respectively.

c Brain

d Eviscerated whole body

e Gastrointestinal tract

f No precipitate

The examination of the AEV-infected antigen preparations against two-fold dilutions(1:2 to 1:32) of each of five hyperimmune sera by the AGPT (Table 2) revealed apositive reaction, consisting of one precipitin line, with all preparations except thosefrom the brain. GI preparations exhibited higher reactivity than whole body antigensand reacted, even in the crude form, with most of the hyperimmune sera. The highest

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Table 2. Results of examination of antigen preparations by the agar-gel precipitin test (AGPT).

Serum

H S h l

HS 2

HS 3

HS 4

HS 5

NSj

AGP titre

Infected Bre

a

li

li

lí

b d

-

NormalBre

a

-

Infected WBf

a

—

b

-

c

;

d

( 1 : 2 )

( 1 : 2 )

(con)

(con)

NormalWBf

a

—

Infected G1B

a

(con)

(con)

(con)

b

(con)

(con)

(con)

c

(1 :2)

(con)

(1:4)

(con)

d

(1 :8 )

(1:8)

(1 : 16)

(1 :4)

(1 : 16)

NormalGISa

-

a 20% crude extract

b crude extract purified with chloroform

c purified extract, concentrated by ammonium sulphate (2x)

d purified extract, concentrated by polyethylene glycol (20x)

e brain

f eviscerated whole body

g gastrointestinal tract

h hyperimmune serum

j normal serum

k negative reaction

I positive reaction with titre given in brackets

con = concentrated

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Detection of antibodies to AE virus 257

antibody titres (1:8 to 1:16) were exhibited against the chloroform- and PEG-treatedGI antigens.

The AGPT reaction proved to be specific because there was no reaction between:(1) normal serum and AEV-infected or uninfected embryonic tissues, (2) hyperimmuneAE serum and uninfected embryonic tissue or (3) CELO and IBD antisera and AEV-infected embryonic tissues. Moreover, AEV-infected antigen (chloroform- and PEG-treated GI), preabsorbed with homologous antiserum, exhibited no reaction whenexamined by the AGPT against the homologous antiserum.

Using the PHAT (Table 3), antibody titres of 1:80 up to 1:1280 were obtained withtanned red cells sensitised with AEV-infected antigens and 0 up to 1:10 with thosetreated with uninfected preparations. Normal serum reacted with titres up to 1:5 withtanned red cells treated with AEV-infected and uninfected antigen preparations. Hence,titres of 1:10 and higher were considered as positive. Generally, partially purified andconcentrated antigens gave higher antibody titres than crude antigens, and no markeddifference was noticed between antigens concentrated with ammonium sulphate andthose treated with PEG, regardless of the degree of concentration achieved.

Screening breeding flocks for antibodies to AEV by the AGPT and the PHA TThe results of screening chicken breeding flocks for antibodies to AEV by the AGPTand the PHAT, using a chloroform- and PEG-treated GI antigen, are shown in Tables4 and 5. In flocks known to adopt vaccination with a live AEV vaccine (TAD) at 14weeks of age (Table 4), precipitating antibodies were not detected in any of the bloodsamples taken just prior .to vaccination or at 2 and 4 weeks post-vaccination. At 6, 8and 45 weeks post-vaccination, only 1/17,1/14,1/10 samples revealed precipitinsrespectively. In these flocks, PHA antibodies were detected in serum levels of 1:5 upto 1:20 in samples taken just prior to vaccination and up to 1:80 in those taken atintervals post-vaccination. The percentage of positive samples, with titres of 1:10 andhigher, increased from 35% just prior to vaccination to above 80% at the 4th weekpost-vaccination. Subsequently, it dropped slightly during the 6th and 8th weeks toreach 50% at 45 weeks post-vaccination. The highest serum titres of 1:40 to 1:80 weredetected in samples taken at two weeks post-vaccination and onwards, with thehighest incidence of 81.1% relative to the total positives at week 8 post-vaccination.

In breeding flocks without a history of vaccination against AE (Table 5), precipitinswere detected in the 25 to 30 blood samples from 8 out of 9 (88.8%) flocks. The percen-tage of AGPT reactors varied from 6.6% to as high as 52.0%. PHA antibodies were alsoalso detected in titres of 1:5 up to 1:320 in the same samples from all of the nineflocks. The percentage of PHAT positive samples ranged from 36.6% to 85.1% amongthese flocks. All of the nine flocks proved to be exposed to AEV as judged by theresults of the EST done on embryos collected at the time of blood sampling.

DISCUSSION

In a search for a simple and reliable means for the detection of antibodies to AEV,the AGPT and the PHAT have been tried by some workers (Ayoub, 1974; Heider etal, 1973; Ikeda, 1977a and b; Lukert and Davis, 1971) with encouraging results. Theantigens used for the AGPT were either 50% crude extracts of various tissues fromAEV-infected chick embryos, or 20% to 30% suspensions of infected chick embryobrain which were clarified by a fluorocarbon and concentrated 10 to 30 times bydehydration with PEG. For the PHAT, a 20% extract of infected whole chick embryo,submitted to three cycles of freezing and thawing, was used for the sensitisation of

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Table 3. Results of examination of antigen preparations I

Serum

H S k iHS 2HS 3HS 4HS 5NS1

NIe

(logio)

3.32.63.02.03.20.7

AGPtitre

1:81:81:161:41:160

InfectedBrK

a

1:1601:6401:3201:1601:1601:5

NormalBrS

a

1:101:5001:50

iy the passive haemagglutination test (PHAT)

Infected Wßh

a

1:801:3201:1601:801:80

0

c

1:3201:12801:6401:3201:3201:5

d

1:3201:6401:6401:1601:3201:5

PHA titre

Normal WBh

a

01:51:501:50'

. d

1:51:5001:51:5

Infected G1J

. a

1:1601:3201:3201:801:1601:5

c

1:6401:12801:6401:3201:6401:5

d .

1:6401:6401:3201:1601:3201:5

Normal GI

a

1:501:5000

d

1:51:51:51:51:51:5

a 20% crude extractc purified extract, concentrated by ammonium suiphate (2x)d purified extract, concentrated by polyethylene glycol(20x)

e Neutralisation index

f agar gel precipitin titre (chloroform- and PEG-treated antigen)

g brain

h eviscerated whole body

j gastrointestinal tract

k hyperimmune serum

1 normal serum

ps»

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Detection of antibodies to AE virus

Table 4. Results of screening vaccinated flocks for antibodies to avianencephalomyelitis (AE) by the agar-gel precipitin (AGPT) andpassive haemagglutination (PHAT) tests3.

259

FlockNo.

123456

Agewks.

141618202259

Weekspost-vaccina-tion

02468

45

No.sera

tested

201718171410

PHATb

0

922324

1:5

411111

1:10

663212

1:20

135412

1:40

057661

1:80

000130

% cpositive35.082.383.376.478.550.0

AGPTb

%positive

0005.87.1

10.0

a Chicken breeding flocks vaccinated with a live AE vaccine (TAD) at 14 weeks of age.b Using chloroform- and polyethylene glycol-treated gastrointestinal tract antigen,c Titres of 1:10 and higher were considered positive.

tanned red cells.

In the present studies, the most potent antigen for the AGPT was from the GI of AEV -infected chick embryos. Moreover, antigen concentration by PEG gave higher antibodytitres than concentration by ammonium sulphate, probably because of the differencein the degree of concentration by the two methods. Brain tissue extracts, either crudeor concentrated by PEG, exhibited no reactivity in the AGPT. These results are ingeneral agreement with those reported by Lukert and Davis (1971). However, contra-dictory results were reported by Ikeda (1977a), who obtained positive precipitationreactions with crude and PEG-concentrated infected embryonic brain tissue. Thefinding of this worker that a virus content of approximately 105 EID50 or more per0.1 g of tissue might be necessary for antigenicity to be manifested by the AGPTcould not be supported by the results of the present work (Tables 1 and 2).

With regard to the PHAT, although reactivity was demonstrated with all crude as wellas partially purified and concentrated preparations examined, higher antibody titreswere obtained with the latter preparations. Nevertheless, the titres obtained with con-centrated antigens were below our expectation. It was also remarkable that prepara-tions concentrated two times the original volume with ammonium sulphate exhibitedequal or even higher reactivity than those concentrated 20 times by PEG. Furtherwork appears necessary to determine the best method for antigen concentration forthe AGPT.

When the AGPT and the PHAT were used to determine the AE antibody status ofbreeding ñocks, both tests gave a higher percentage of reactors in naturally infectedflocks than in vaccinated flocks. The PHA titres were also generally higher in thenaturally infected flocks. Ikeda (1977b) reported as high as 89 to 100% positivereactors using brain tissue in the AGPT in chickens 12 weeks after exposure to AEVby different routes. It is, however, doubtful that such high percentages of reactorscould be detected by the AGPT at any interval after exposure, regardless of the typeof antigen used in the test.

Our results suggest a higher sensitivity of the EST and the PHAT than the AGPT,with the sensitivity of the two former tests nearly equal. This is in agreement withAyoub (1974) and Heider et al (1973) who found a high correlation between theresults of the EST and the PHAT. However, Ikeda (1977b) reported that the

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Table 5. Results of screening unvaccinated flocks for antibodies to avian encephalomyelitis (AE) by the agar-gel precipitin(AGPT), passive haemagglutination (PHAT) and embryo susceptibility (EST) tests*.

FlockNo.

123456789

AGPTb

No. seratested

302530302727302530

%positive

10.00

13.313.333.340.7

6.652.0

6.6

PHATb

0

138

1472152

12

1:5

352726753

1:10

664346856

1:20

345563656

1:40

223555233

1:80

002353130

1:160

000022120

1:320

000011010

No. seratested

302530302727302630

%positive

36.648.046.653.385.174.060.073.050.0

ESTC

No.embryostested

402628292526272724

%protected

40.073.064.272.480.076.974.070.366.6

a Breeding chicken flocks aged 12 to 18 months.

b Using chloroform- and polyethylene glycol-treated gastrointestinal tract antigen.PHAT titres of 1:10 and higher were considered positive.

c Using 10 EID50 of egg-adapted AE virus per chick embryo.

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Detection of antibodies to AE virus 261

sensitivity of the AGPT was nearly equal to that of the VNT. Results of our study arein conflict with this worker. Although the VNT was not done with the sera of theflocks in this investigation, the results of the EST and the AGPT (Table 5) did notcorrelate well. This is particularly evident in flock No.2 which proved to be immuneby the EST, although none of 25 blood samples examined from this flock revealedprecipitating antibodies.

No correlation was found between the results of the AGPT and the immune status ofa flock as judged by the EST. The AGPT has to be interpreted as a flock test wherebypositive results indicate exposure but the opposite is not true.

It was concluded that the use of the PHAT appears very promising in the serodiagno-sis of AEV infection and in determining antibody responses to vaccination. It has theadvantages of being more rapid, less expensive and applicable to all age groups whencompared with the EST or the AGPT. Confirmatory investigations are still neededand standardisation of test antigen and procedure is required.

REFERENCES

Ayoub, N.N.K. (1974). Grundlagen und Ergebnisse beim Aufbau und Erhaltung einer SPF-Huehnerzucht zur Erzeugung kontaminanten-freier Bruteier. D.Sc.Med Vet. Thesis.Humboldt Universität, Berlin.

Choi, W.P. and Miura, S. (1972). Indirect fluorescent antibody technique for the detection ofavian encephalomyelitis antibody in chickens. Avian Diseases, 16: 949-951.

Feldman, H.A. and Wang, S.S. (1961). Sensitivity of various viruses to chloroform.Proceedings of the Society of Experimental Biology and Medicine, 106: 737-738.

Heider, G., Ebner, D. and Kobilke, H. (1973). The indirect (passive) haemagglutination inmonitoring the industrial poultry production. Proceedings of the Vth InternationalCongress of the World Veterinary Poultry Association, Munich, Germany.

Hitchner, S.B., Domermuth, C.H., Purchase, H.G. and Williams, J.E. (1975). Isolation andidentification of avian pathogens. American Association of Avian Pathologists.Arnold Printing Corporation, Ithaca, New York.

Ikeda, S. (1977a). Immunodiffusion test in avian encephalomyelitis. I. Standardization ofprocedure and detection of antigen in infected chickens and embryos. National Instituteof Animal Health, Tokyo, 17: 81-87.

Ikeda, S. (1977b). Immunodiffusion test in avian encephalomyelitis. II. Detection of precipita-ting antibody in infected chickens in comparison with neutralizing antibody. NationalInstitute of Animal Health, Tokyo, 17: 88-94.

Lukert, P.D. and Davis, R.B. (1971). A new method for the preparation of an antigen used inthe agar-gel precipitin reaction to detect avian encephalomyelitis virus antibodies.Avian Diseases, 15: 935-938.

Luginbuhl, R.E. and Helmboldt, C.F. (19 78). Avian encephalomyelitis (Epidemic tremor).In: Diseases of Poultry. Edited by Hofstad et al. VIIth edition, Iowa State UniversityPress, Ames, Iowa.

Monreal, G. (1963). The distribution of avian encephalomyelitis by an immunity test.Proceedings of the XVIIth World Veterinary Congress. Hannover, Germany.

Nairn, R.C.C. (1976). Fluorescent antibody methods. IVth edition. Churchill, Livingstone,Edinburgh-London and New York.

Reed, L.J. and Muench, H. (1938). A simple method for estimating fifty percent end-point.American Journal of Hygiene, 27: 493-497.

Sato, G., Watenabe, H. and Miura, S. (1969). An attempt to produce complement-fixationantigen for the avian encephalomyelitis virus from infected chick embryo brains.Avian Diseases, 13: 461-469.

Sumner, F.W., Luginbuhl, R.E. and Jungherr, E.L. (1957). Studies on avian encephalomyelitis.II. Flock survey for embryo susceptibility to the virus. American Journal of VeterinaryResearch, 18: 720-723.

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262 A.A.S. Ahmed et al

RESUME

Etude de la détection sérologique des anticorpsdu viras de l'encéphalomyélite aviaire

Un antigène viral de l'encéphalomyélite aviaire (AEV) préparé à partir du tractusgastro-intestinal d'embryons de poulets infectés et partiellement purifié et concentrépar traitement au chloroforme et au polyéthylene glycol, a montré une haute réac-tivité au test de précipitation en gélose (AGPT). L'antigène utilisé dans le testd'hémagglutination passive (PHAT) après avoir été purifié et concentré a permisd'obtenir des titres en anticorps plus élevés qu'avec l'antigène brut non traité.

L'utilisation de l'AGPT, du PHAT et du test de sensibilité de l'embryon (EST) sur destroupeaux de reproducteurs chair qui avaient subi ou non antérieurement une vaccina-tion contre l'encéphalomyélite a révélé que le PHAT était plus sensible pour la détec-tion des anticorps AEV que l'AGPT. La sensibilité du PHAT était à peu près égale acelle du EST. Aucune corrélation n'a été trouvée entre les résultats de l'AGPT et lestatut immunitaire d'un troupeau évalué par le EST.

ZUSAMMENFASSUNG

Untersuchungen über der serologischen Nachweis vonAntikörpern gegen das Virus der Aviaren Encephalomyelitis

Im Agargelpräzipitationstest (AGPT) zeigte ein Antigen des Virus der AviarenEncephalomyelitis die stärkste Reaktivität, das aus dem Gastrointestinaltrakt von in-fizierten Hühnerembryonen präpariert und durch Behandlung mit Chloroform undPolyaethylenglycol teilweise gereinigt und konzentriert wurde. Wenn für den passivenHaemagglutinationstest (PHAT) ein gereinigtes und konzentriertes Antigen verwendetwurde, wurden höhere Antikörpertiter festgestellt als bei Verwendung von unbehan-delten Antigen.

Untersuchungen von geimpften und nicht gegen AE geimpften Hühnerzuchtherdenmittels AGPT, PHAT und dem Embryoempfänglichkeitstest (EST) ergaben, daß derPHAT für den Nachweis von AEV Antikörpern empfindlicher ist als AGPT. Die Emp-findlichkeit von PHAT entspricht etwa dem EST. Es bestand keine Korrelationzwischen den Ergebnissen des AGPT und dem durch den EST ermittelten Immun-status einer Herde.

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