PRODUCTION OF EGG YOLK - BLUETONGE SPECIFIC ANTIBODIES (IGY) FOR THE DIAGNOSTIC PURPOSES OF BLUETONGUE VIRUS INFECTION

Department of Virology,

Animal Health Research Institute,

Agricultural Research Center, Egypt.

Production of egg yolk - bluetonge  specific antibodies (IgY) for the diagnostic purposes of bluetongue

virus infection

(With One Table, 3 Figures and 2 Photos)

By

A.R.A. Habashi; I.A. Karim and H.B. Hassan*

*Faculty of Veterinary Medicine, Tanta University Kafr-El-Sheikh Branch

(Received at 18/8/2005)

انتاج الأجسام المناعية المضادة لفيروس مرض اللسان الازرق

 فى مح البيض وإستخدامها فى تشخيص المرض

أحمد رفعت احمد حبشى ، اکرام عبد الکريم اسماعيل ، حسن بدر حسن

يمثل هذا البحث دراسة تجريبية لتقييم إنتاج الأجسام المناعية المضاده لفيروس اللسان الأزرق فى الدجاج المحصن والمحقون باللقاح المتعدد ضد فيروس اللسان الأزرق وکفاءة استخدامها فى أغراض تشخيص المرض: تم الکشف على تکون الأجسام المناعية المضادة لفيروس السان الأزرق في سيرم الدجاج المحصن بأستخدام اختبار الأجار الترسيبى. تم تجميع عينات سيرم وبيض خلال فتره التجربة (21 اسبوع) وکذلک فصل الجلوبيولين المناعى (IgY) من مح البيض بترسيبه بسلفات الأمونيوم. بإستخدام اختبار الأليزا ثم مقارنة بين الأجسام المناعية فى السيرم و تلک المستخرجة من مح البيض حيث وجد ان مستوى الأجسام المناعية بهما متساويآ ويستمر مستوى الأجسام المناعية عاليآ فى مح البيض لفترة أطول بالمقارنة بمستواه فى الدم مما يتيح لنا انتاج کمية کبيرة من الأجسام المناعية المضاده للفيروس من  کل دجاجة محصنة ضده معتمدا علي حقن جرعات منشطة من الفيروس واستمرارية انتاج البيض. تم ايضا إثبات کفاءة استخدام هذه الأجسام المناعية المستخرجه من مح البيض فى الکشف عن انتيجين فيروس اللسان ألأزرق وتشخيص المرض بإستخدمها فى اختبار الفلوسنت المناعى المشع للتعرف على 13 معزلا تمثل     5 عترات من الفيروس.

Summary

An experimental study was carried out to evaluate a new simple method for massive production of bluetongue virus (BTV) specific antibodies by specific IgY extraction from the egg yolk of immunized hens with BTV polyvalent vaccine. After detection of the development of the serum precipitating antibodies, serum samples and egg batches were collected from the immunized hens at regular intervals then titrated by the indirect ELISA. The serum antibody titres were compared with the extracted egg yolk IgY antibody titres during the period of the study which elapsed 21 weeks and during this period 3 doses were injected. The results revealed that the egg BTV specific antibody titres were nearly similar to the serum antibody titres providing a higher yield of specific antibodies can be collected from the immunized hens depending on the availability egg production and occasional posturing. The diagnostic efficiency of the prepared egg yolk antibodies was also proved by successful detection of 13 BTV isolates using it in the procedures of indirect fluorescence antibody technique.

Key words: Virology, blue tongue, antibodies.

Introduction

The use of chicken as alternative labanimal for polyclonal antibodies production has described by (Polson et al., 1980).Hens could be the antibody factories of the futureand the production of specific polycolonal antibodies in the chicken egg yolk may be used for diagnosis of various diseases of poultry, livestock and human (Anonymous 1994).The egg yolk of chicken is recognized as a rich source of specific antibodies (Arshad et al., 1996). These include the advantages of producing more specific antibodies against mammalian antigens in birds compared to mammals because of the phylogenic distance between birds and mammals, low cost of production, convenience and what is becoming important, compatibility with regulation for modern animal production and animal welfare. Relatively small amount of highly conserved mammalian protein are sufficient to induce a sustained immune response. Chicken egg- yolk antibodies are also phylogenetically distinct from mammalian antibodies. Therefore they can be used in a multiple antibody assay with mammalian antibodies with minimal cross-reactivity (Sim and Nakai 1994).

There is a prerequisite for full understanding and evaluation the efficiency of this system for production specific antibodies against important diseases and their use for diagnostic purposes. Bluetongue (BT) is an infectious non contagious insect-born viral disease of sheep, caused by an orbivirus. Severe disease usually occurs in certain breeds of sheep, particularly the fine-wool and mutton breeds. Cattle, goats and wild ruminants can be also infected, but usually with mild or no clinical signs (Howell, 1963; Erasmus, 1975; Mellor, 1996). The Disease is worldwide distributed and endemic in temperate climate areas(Gibbs et al. 1989). The importance of bluetongue virus became very clear especially after occurrence recent out breaks in many countries around the world since a long period of silent circulation of the virus without showing clinical disease conditions (OIE disease information 1999- 2001).

The aims of present study are:

1. Evaluation of a new simple method for massive production of specific antibodies against bluetongue virus by extraction of IgY from the egg yolk of hens immunized with BTV polyvalent vaccine.
2. Determination of the diagnostic efficiency of the experimentally prepared egg yolk antibodies.

Materials and methods

According to Hudson and Hay (1991), Immunization of laying hens with BTV was done by 1ml oil emultion of an equal volume of both the tissue culture bluetongue polyvalent vaccine was obtained from Serum and Vaccine production Institute, Abassia, Cairo, Egypt and Ferund’s complete adjuvant "Sigma" injected subcutaneously at both sides of the chest and the inoculation was repeated after 3 weeks and 3 months from the frist dose.

24 serum samples, and 17 egg batches were collected from the immunized hens at regular intervals from the zero day during the period of this study (21weeks) depending on the availability of laying eggs. Six normal chicken control sera (with no record of BTV antigen inoculation) were used as control negative.

According to the method described previously by Pearson and Jochim (1979),development of specific antibodies was detected by AGPT in the serum samples collected 2 weeks after the 2^nd and 3^rd imunization of the hens. 0.9% agarose was prepared in isotonic saline solution (0.85% NaCI in distilled water). A test pattern with a central well and 6 peripheral wells was used, with each well measuring 4 mm in diameter and separated by a distance of 2.4 mm between each well and positive-control serum was placed in 3 peripheral wells alternately with 3 undiluted test sera; and the central well was filled with the Bluetongue AGID reference antigen "Pirbright England" Each well contained approximately 0.03 ml. Three samples were tested with each pattern. Test plates were held at room temperature in a humidified chamber for 24 hours.Serum was recorded negative when the positive-control precipitin line went into the test serum well and was recorded as positive when the precipitin lines formed lines of identity with the positive-control serum or when they caused the positive control precipitin line to bend toward the antigen well (weak positives).

Extraction of egg yolk IgY:

Ammonium sulfate precipitation method(Hadsun and hay 1991) was usedfor extraction of the egg yolk IgY as following: 

            After careful separation of the egg yolk from the egg white, the yolk was rolled on a filter paper in order to free it completely from albumen material. The membrane of the egg yolk was perforated, so that the content (approximately 10 - 15 ml) may drip into a 100 ml measuring cylinder. Distilled water was added to make a dilution of 1:5. The mixture was stirred for homogenizing and then frozen at -20ºC for a minimum of 72 hours. The frozen suspension was thawed slowly at 4^˚C and centrifuged at 2800 x g for 20 minutes. For each ml of supernatant 0.5ml of ammonium sulfate saturated salt solution was added (2:1). After stirring for two hours at room temperature the mixture was centrifuged at 2800 x g for 20 minutes and then the supernatant was discarded and the precipitate was dissolved in DDW to half of its original volume. 0.5 ml of ammonium sulfate solution was added for each 1ml of the solution. The solution was stirred at room temperature for 2h. , and then centrifuged at 2800 x g for 15 mm. The supernatant was discarded; the precipitate was dissolved in 2.5ml of PBS per egg yolk and dialysed against PBS at 4ºC for 48 hours. After filtration (0.45μm) the immunoglobulin suspension was stored at -20 ºC.

Indirect ELISA:

According to Voller et al., (1976), Indirect ELISA was used for monitoring of BTV antibodies in the serum and the extracted egg yolk IgY of the immunized hens:

- Cheak Board titration of BTV reference antigen:

BTV reference AGID stock antigen "Pirbright England" was titrated against mouse BTV reference positive serum "Pirbright England and normal negative mouse serum for detection of the proper dilution could be used in the plates coating for the indirect ELISA procedures was carried out as following:

50 µl carbonate buffer was placed into each well of a microtitre plate. 50 µl of antigen (Bluetongue AGID antigen) at a starting dilution 1:50 was placed into all wells of row A. After mixing, the antigen was serially diluted by 12 channel multichannel pipette in 50µl amounts from row A to G from where 50 µl are discarded. The plate was incubated on the shaker at 37ºC incubator for one hour then at 4 ºC over night. The antigen was removed by flicking the plate, and subsequently washed with PBS-T 3 times. 50 µl PBS-T with 6% FCS (Blocking buffer) was placed into each well of the plate then 50µl of reference positive BTV mouse serum  previously dilluted at 1:5 in the same blocking buffer and  placed into  wells of column 1 and 50µl of negative control serum (normal mouse serum previously dilluted 1:5)  placed in the wells of column 7, reaching a final starting dilutions at 1:10 for both positive and negative serum. After mixing the serum was serially diluted by 8 channel multichannel pipette in 50 µl amounts from column 1 to 6 from where 50 µl are discarded and from 7 to 11 after changing the tips. The plate was then incubated on a shaker at 37ºC for 1h. The plate was then washed and soaking with PBS-T 3 times. Anti-mouse Ig-HRPO-conjugate "Sigma" at working dilution in PBS-T (1:1000) was added in 50µl amounts to all wells. The plate was incubated in a moist chamber at 37ºC for 1h, and supsequtly washed with PBS-T 3 times. 50 µl of freshly prepared substrate indicator system (OPD) was added to all wells then incubated for 10 min at room temperature where the color reaction was stopped by addition of 50µl H₂SO₄stopping solution and finally the plate was read on ELISA reader (MCC Multiscan) at filter 492.

Fig. 1: Plate layout in cheak board titration of reference BT  antigen.

-Test procedure:

50 µl of BTV antigen at a predetermined dilution in carbonate-bicarbonate buffer was placed into all wells of the plate. The plate was placed on a shaker at 37 ºC incubator for 1 h. and then at 4 ºC over night. The antigen was removed by flicking the plate, and washed with PBS-T 3 times. Test and negative control sera were double fold diluted in PBS-T+6% FCS (Blocking buffer) in rows from A-H, 50 µl volume  and the plate was  incubated 1 h at 37ºC then washed with PBS-T 3 times. Anti-chicken Ig-HRPO-conjugate at working dilution (1:5000) in Blocking buffer was added in 50u1 amounts to all wells and the plate was incubated for 1h. at 37ºC then washed with PBS-T 5 times. 50 µl  of freshly prepared  OPD substrate indicator system was added to all wells and incubated for 10 min at room temperature then the color reaction was stopped by addition of 50 µl of H₂SO₄ stopping solution. The antibody titres of the serum samples, and egg batches were collected from the immunized hens during the study were estimated according to the OD of the negative control and the cut of was determined as following:

Statistical Analysis for determining cut of value:

 Limit for negative serum values was calculated for comparison of OD values of negative serum and weak-positive serum. One of the difficulties in identifying weak-positive reactions aroused from the fact that even the negative control sera exhibiting some degree of reaction. Moreover, this background reaction is not uniform, but differed from one control serum to another in a random fashion. The problem was then to establish a borderline which could separate weak-positive sera from true negative sera (Hubschel et al. 1980).This was done as following:

 - Six normal chicken control sera (with no record of BTV antigen inoculation) were used as controls. The OD of those sera were:

   0.218, 0.162, 0.183, 0.193, 0.221, and 0.226. by indirect ELISA

- The arithmetic mean (M) and the SD were calculated as M = 0.20 and SD = 0.0254, respectively. Then the cut off value (COV) was estimated as (Chris et al., 1993).

 Cut off value (COV) = M + 3SD

According to this rules the samples with an OD of 0.277 or higher was classified as positive; and any samples with an OD of less than 0.277 was considered negative. By this way we can easily estimate and compare the ELISA titre of different samples

Tissue Culture-Indirect Fluorescent Antibody Test:

According to Foster et al., (1972), this test was done for detection of (13) BTV local isolates constitute 5 serotypes (4, 10, 12, 13, and16) were isolated and identified during September–November 2000 (Habashi 2002).

 -Test procedures:

Vero cells were cultivated in Leighton tubes with cover slips (3 tubes for each sample) were inoculated with 0.2 ml of each isolate. Negative controls were also included. Adsorption of virus was allowed for 90 min at 37˚C. Cells were washed and maintenance medium was added (MEM without serum). Forty-eight hours later cells were fixed on the coverslips with cold acetone at 4ºC for 15 min. The indirect immunofluorescent test was then performed at room temperature:

Initially the cells were covered with the pre-diluted egg yolk antibodies diluted (1/10), (1/20) or (1/40 ) in PBS at pH 7.2 and incubated at 37ºC for 1h in a moist  champer then washed three with PBS for 5min. Antichicken fluorescence conjugate (1:40) was added and incubated for1 h and subsequently washed 3 times with PBS, for 5 min and Then the cover slips were dried and examined under fluorescent microscope (Under dark filed with X10, X25, and X40) for the presence of the intracytoplasmic fluorescent inclusions characteristic of BTV after adding a drop of mounting glycerol buffer.

Results

Detection of serum bluetongue specific antibody development in immunized Hens:

  The developments of bluetongue specific antibodies were detected in the serum samples that collected from the polyvalent BTV vaccine immunized hens, 2weeks after the 2^nd and 3^rd immunization by AGPT. The test was done against reference antigen with complete line of identity with the positive control reference BTV serum. While serum samples collected at 0 day were recorded negative.

Indirect ELISA for comparison of BTV antibody titres in the serum and the extracted egg yolk IgY of the immunized hens:

The pattern of specific BTV antibody production was studied on both of the serum samples collected from the immunized hens and the extracted immunoglobulin from the egg yolk (IgY) by using indirect ELISA.

- Cheak Board titration of BTV reference antigen:

 The results of titration standard reference BTV antigen, against reference positive antisera and negative serum to determine the optimal dilution that could be used for coating the ELISA plates during the test procedures as follow in the further steps.

            As presented in Fig. (2), from which it is clear that: Decreasing the antigen concentration used for coating of the plates led to reducing the absorbance reading for the same serum. This shift was even more pronounced at lower concentration, whereby the optimum antigen concentration could be judged by the comparison between both negative and positive serum values. On the basis of this result, 1:200 dilution of the purified antigen was considered optimal with lowest background of the negative serum and was used for coating the polystyrene micro-titration plates in all further steps of the  experiment.

-ELISA antibody titres in serum and egg yolk extracted IgY of the BTV immunized hens:

Results were shown in (Table 1, Fig. 3).

Specific BTV antibodies in the serum sample collected from the 2 immunized hens were detected in the serum samples collected at the 3^rd week post inoculation (PI) at low end point titre (20), and much more antibodies were detected after the 2^nd inoculation reaching the first peak value at week 5 and 6 in both hens respectively with end point titre (80), followed by gradual decreasing after 8 weeks (PI). The highest antibodies levels were detected after the 3^rd dose of immunization getting the second beak at week 14 (PI) with end point titre of (640 and 320) in both hens respectively. High level and long sanding antibodies platue were recognized after the 3^rd dose of immunization to the last week (week 21) of investigation when the experiment was terminated.

Compared with the serum antibodies levels the pattern of antibodies extracted from the egg yolk were nearly the similar (homologous) to that of serum but during the antibodies decreasing period the egg yolk antibodies titre were higher and longer standing. Therefore the production of BT antibodies through extraction of these specific antibodies from the egg yolk of immunized hens has the advantages of giving very large amount of antibodies from a single immunized hen depending on regular laying of the hens and occasional immunization.

Testing the diagnostic efficiency of the prepared egg yolk BTV specific IgY by Fluorescent antibody technique.

            An indirect Fluorescent antibody technique was used for the detection the BTV antigen in infected cells using the prepared egg yolk bluetongue specific IgY at different dilution (1:10, 1:20, and 1:40) for each BTV isolates.

            The results revealed that all the 13 BTV isolates were detected and specific fluorescence was observed in the cytoplasm of the infected cells and the most consistent results with brightest specific fluorescence throughout the experiment at dilution 1/20 in PBS, while no specific reaction was observed in both control non infected cells and infected cells with normal negative BTV egg yolk IgY (Photos 1&2).