Sero-prevalence of Infectious Bursal Disease Virus (IBDV) Specific Antibody in Chicken

MATERIALS AND METHODS

This experiment was conducted at Virology laboratory of Animal Health Research Division (AHRD) and Poultry Production Research Division (PPRD) of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka during June 2001 to May 2002.

Vaccination of the chickens: A total number of 10 broilers (10 day old) were collected from the commercial hatchery. These young chicks were reared by maintaining all the hygienic measures in well-ventilated poultry house of AHRD, BLRI, Savar, Dhaka. Later on, these chicks were divided into two groups. Group one consisted of 6 chicks which was vaccinated and group two consisted of 4 chicks, which was without vaccination. As these chickens attained the age of 14 days, 6 chickens were vaccinated while remaining 4 chickens were kept without vaccination. Before vaccination, the route of vaccination, time and dose of vaccination were considered. Intermediate strain GUMBORO D78 live vaccine (Intervet Company, Netherland) was administered through intra ocular route (eye drop) at day 14 in 6 chickens.

Inoculation of IBDV suspension in chickens: Naturally occurring IBDV isolates were collected from the bursa and spleen of sick and dead chickens of 33-37 age group from experimental flock of AHRD, BLRI, Savar, Dhaka. The samples were triturated in a pestle and mortar. A required amount of phosphate buffer saline (PBS) was added to the tissue homogenate (bursa) to make a 20% weight /volume (w/v) virus suspension. The suspension was centrifuged at 4000 rpm for 15 min. The supernants was collected and diluted in proper ratio, which was then used as an inoculum. Five days after vaccination i.e. on day 19, a total of 10 chickens (both vaccinated and non-vaccinated) were challenged by inoculating IBDV suspension to infect the chickens. Virus suspension was inoculated orally or through intra ocular route (eye drop) in the form of droplets (2-3 drops). The challenged chickens were kept in the separate room to observe the signs and characteristics lesions of IBD as well as to prevent the spread of disease to other flocks. Normally the incubation period of IBD was 2-3 days, but the chickens were observed for 10 days so that there would be a better immune response.

Preparation of Sera: Blood samples were collected from vaccinated and non-vaccinated chickens on day 10 post-inoculation of IBDV suspension (i.e. on day 29). One ml or 2.5 ml sterile disposable syringes were used to collect blood samples aseptically directly from the heart or wing vein. Soon after the collection of blood, the syringes with blood were kept at 4-8 ^o C overnight for clotting of blood in one side of the syringe. Clotted blood was removed carefully with sterile needle and sera were transferred into sterilized eppendorf tubes. Separate needles were used for each syringe. The sera in eppendorf tubes were subjected to centrifugation at 1000 rpm for 10 min for clarification. Finally, the clarified sera were stored at –20 ^o C until tested. This serum was used as a test sample for the detection of IBDV specific antibody level in the chicken using ELISA. Also, a total of 49 blood samples from the chickens of different breed and age groups were collected from the different poultry farms of Savar area. These collected blood samples were studied to determine the level of MDA in different breed and age of chickens from vaccinated parentstocks (PS).

Application of ELISA for IBD: ELISA at a single dilution (1: 500) of serum was applied for the detection of the IBDV specific antibody. ELISA kit for IBD manufactured by IDEXX laboratory, Inc. West brook, Maine 04092, USA was used in the study. Serum was diluted 500 folds (1: 500) with sample diluent, provided in the ELISA kit for IBD, prior to assay (i.e. by diluting 1 μl of the sample with 500 ul of sample diluent). Antigen coated 96-well plate was taken and marked for positive controls, negative controls and samples. 100 ul of undiluted negative control and positive control were dispensed into wells A1, B1, C1 and D1 respectively. Then 100 ul of diluted sample was dispensed into remaining 92 wells of the plate. Multiwell plate was incubated at room temperature for 30 min. Each well was washed with approximately 350 ul of distilled or deionized water (3-5 times). 100 ul of Anti-chicken (Goat): Horseradish peroxidase conjugate was added into each well. Then Plate was incubated at room temperature for 30 minutes and again washed with approximately 350 ul of distilled water (3-5 times). Then 100 ul of tetra methylene blue (TMB) substrate was added into each well and the plate was incubated at room temperature for 15 minutes. 100 ul of the stop solution was added into each well to stop the reaction. Finally reading was taken by putting the plate on the ELISA reader using 650 nm filter.

Calculation of antibody titre using equation provided in ELISA kit: The presence or absence of antibody to IBDV was determined by relating A (650) value of the unknown to the positive control mean. The positive control has been standardized and represents the significant antibody level to IBDV in chicken serum. The relative level of antibody in the unknown can be determined by calculating the sample to positive (S/P) ratio.

The equation for calculation provided in the ELISA kit was used for the calculation of antibody titer, which is as follows:

Titer-relates S/P at a 1:500 dilution to an end point titer:

Log₁₀ Titer = 109 (Log₁₀ S/P) + 3.6

Calculation of the antibody titer using computer software program developed by AHRD of BLRI: This software program contained data (positive control, negative control, s/p ratio and OD value) which are adjusted in such a way, when OD value of the sample obtained from the ELISA reader is fed, the antibody titer of that sample can be obtained automatically. To get the antibody titer of all 92 samples in the particular plate, positive control and negative control should be kept fixed in the program only for that particular plate.

RESULTS

Determination of the persistence of IBDV specific MDA in chickens from vaccinated parent stock: For the determination of the persistence of IBDV specific MDA, a total of 49 chickens were used which included four breeds of chickens at different age. For detection of persistence of MDA in different breeds at different age, a total of 49 chickens of different breeds and ages were used. These chickens included 8 one days old, 8 five days old, 7 ten days old and 3 fifteen days old BV-300 breed and 8 one day old chicks of each breed (BV-300, Broiler Kasile, LBM and Hi-sex).

Determination of serological response in chickens: Serological response against IBDV in chickens was demonstrated in terms of antibody titer obtained through ELISA test. Blood samples were collected from the chickens at prescribed time and the sera were subjected to ELISA test.

Table 1:	Persistence of MDA in Broiler Kasile breed on day 1 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 2:	Persistence of MDA in LBM (flock 59) breed on day 1 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 3:	Persistence of MDA in Hisex breed on day 1 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 4:	Persistence of MDA in BV -300 breed on day 1 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 5:	Persistence of MDA in BV-300 breed on day 5 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 6:	Persistence of MDA in BV-300 breed on day 10 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 7:	Persistence of MDA in BV-300 breed on day 15 from vaccinated parent stock
PC= Positive Control; NC= Negative Control; OD= Optical Density; S/P ratio= Sample to positive ratio; Sl. No.= Serial Number

Table 8:	Antibody titer in vaccinated broiler serum (on day 29) after infection with IBD virus suspension
OD= Optical Density

Table 9:	Antibody titer in non-vaccinated broilers serum (on day 29) after infection with IBD virus suspension
OD= Optical Density

Fig. 1:	Comparative average MDA titer in different breeds of chicken on day 1 from vaccinated parent stock

The results of the ELISA test with antibody titer and S/P ratio are presented separately in the Table (1-7) and (Fig. 1 and 2) for each breed and at various days. According to the tables, all breeds of chickens from vaccinated parent stock contained high level of MDA at day1 and the level gradually declined below protection level within 15-20 days after hatching. The rate of declination was about half by every 5 days. According to the IBD antibody test kit manufacturing company, S/P ratio greater than 0.2 (titre 396) should be considered as protection level.

Fig. 2:	Average MDA titer in BV-300 on 1, 5, 10 and 15 day from vaccinated parent stock

Determination of the persistence of IBDV specific antibody in vaccinated and non-vaccinated broiler after inoculation with IBDV suspension: A total number of 10 broilers (6 vaccinated and 4 non-vaccinated) were used for the determination of IBDV specific antibody in chickens after inoculation with IBD virus suspension. Blood from both vaccinated and non-vaccinated chickens were collected on day 29 i.e. on day 10 after inoculation and the sera were subjected to ELISA test for the determination of antibody level in chickens. The results of ELISA test are presented separately in Table 8 and 9. According to the tables, vaccinated chickens contained average antibody level of 1489.50, which is much above the minimum protection level and which has the ability to protect the chickens for few days or weeks. Whereas non-vaccinated chickens contained zero level of antibody and may be 100% susceptible to disease.

DISCUSSION

The present study was aimed to monitor the persistence of MDA in different breed and age of chickens from vaccinated parentstock and to determine the IBDV specific antibody level in the sera of both vaccinated and non-vaccinated chickens after inoculation with IBD field virus suspension.

One of the major problems in the development of poultry industry in developing countries like Bangladesh is the outbreak of various diseases that cause about 30% mortality of chicken in every year (Ali, 1994). Among these diseases, IBD in chicken is the most important and severe one. In Bangladesh, this disease is prevailing since March 1992 with a very high morbidity and mortality (Rahman, 1994).

To render the poultry industry, emphasis should be given first in the prevention and control measures of diseases that cause heavy mortality. Anjum et al. (1993) mentioned that Gumboro was the second highest cause of mortality of the chicken in Pakistan. The prevalence of diseases in the particular area depends on various factors like geo-climatical conditions, biological barriers, age, breed, sex of the chickens, immune status and social awareness. Mass vaccination against a particular disease without knowing its effects to immune system cause not only economic loss in terms of vaccination but also stress to the chicken making them more susceptible to other diseases. It is noted that immunization by vaccination could not give 100% protection against IBD. The possible causes of outbreak in immunized flock were maternal antibody interference, poor husbandry and improper vaccination, antigenic variation among the vaccine strain and field strains and timing of vaccination. Timing of vaccination of chickens depends upon the persistence of maternal antibody level and also their response to immune system after vaccination.

In Bangladesh, there is no vaccine of local isolates of IBDV. To control IBD and other diseases, different types of vaccine is being imported from different manufacturing companies. Usually, they have their own instruction about dose, route and age of administration of vaccine to the chicken. Without concern about the maternal antibody in offspring, farmers are utilizing Gumboro vaccine from day old to onward. The optimum vaccination time could be estimated by titration of MDA against IBDV in day old chicks by an ELISA test (Tsukamoto et al., 1995).

For the detection of the persistence of MDA in progeny of different breed and age from vaccinated parent stock, blood samples were collected from day old boiler Kasile, LBM, Hisex and BV-300. Blood samples of day old, five days old, ten days old and fifteen days old BV-300 chickens were also collected. After separation of the sera, the testing samples were subjected to ELISA test. Among four breeds, broiler (Kasile) contained high level of antibody titer (average of 5877.15) followed by layers (Hisex; average antibody titer of 5581.55, BV-300; average antibody titer of 5320.79 and LBM; average antibody titer of 3676.24).

MDA level in day old, day 5, day 10 and day 15 BV-300 was also observed. The day old chickens contained high level of antibody with an average of 5320.79, 5 days old with an average of 5848.57, 10 days old with an average of 2615.53 and 15 days old with an average of 580.88. The level of antibody gradually declined and persisted up to 15 to 20 days after hatching. The rate of declination of MDA was about half by every 5 days. According to the manual provided in the IBD antibody test kit, the protection level of antibody is considered when S/P ratio is greater than 0.2 (titer 396). Cao-Youngchang et al. (1995) evaluated immunological efficiency of IBDV by ELISA and found MDA level was high at day one. The half-life of the MDA to IBD in chicken was 3.46 day according to Saijo and Higashihara, (1998). The variations of the persistence of MDA might be due to use of different types of vaccine and vaccination schedules for parent stock.

The above results showed that the level of antibody in all four breeds of chickens is above the minimum protection level and there is no significant difference in the level of antibody among 4 breeds at day 1. As the MDA persists up to 15-20 days, the vaccine should be given at day 14 at when the chickens have the ability to resist the virus attack. If vaccine is given at that time, it can adapt to immune system and can show response before antibody level drops to minimum protection level.

For the detection of antibody level in vaccinated and non-vaccinated chickens, a total of 10 broilers (6 vaccinated on day 14 and 4 non-vaccinated) were used. Blood samples were collected on day 29 following inoculation with IBDV suspension on day 19. Sera were then subjected to ELISA for the determination of antibody level in both vaccinated and non-vaccinated chickens. The results showed that the serum of the vaccinated chickens contained average antibody level of 1489.50, which is much above the minimum protection level and ensures protection against virus attack for few more days or weeks but there is zero level of antibody in the serum of non-vaccinated chickens and may be 100% susceptible to IBD. Therefore, the chicks must be vaccinated at around day 14, at the time when MDA level tends to reach the minimum protection level. The vaccine triggers the immune system and there is a subsequent production of antibody against IBD.