الفهرس 
Chapter I : IntroductionOnly 14 pages are availabe for public view
 |  | from | 230 |  |  |
| | | from | 230 | | |
Abstract
Newcastle disease (ND) is an economically important disease of poultry for which vaccination is applied as a preventive measure in many countries. Booster vaccinations of breeders during production are also required to maintain the hen’s hyperimmune status as soon as to boost the maternal antibody titres of their progeny, but there is some constraints such as the timing of vaccination, invasive sampling techniques and disadvantages of routine monitoring tools, which still affect the effectiveness of vaccinations, thus delay the control of the disease.
These complexities have led us to focus and search for possible solutions in this study to establish methods for indirect prediction of antibody titres in dam hens and day-old chicks with regard to the maternal antibodies transferred to egg yolks with optimization of a simple and rapid methodology for large scale extraction of anti ND IgY and evaluate a commercial ELISA kit when used to measure NDV antibody in eggs compared to HI method. Also, to evaluate the effect of the time between vaccinations and age on the immune statues of breeders, and on the level and duration of maternally derived immunity in their progeny.
To achieve these goals; a total of 112 healthy 20 week-old broiler breeder chickens of native breed (Balady) with known vaccination history have been sampled, (blood and eggs) for obtaining the base line titre of NDV antibody in serum and yolk. At the same time, they have been vaccinated intramuscularly with inactivated NDV oil emulsion vaccine and divided into three equal experimental groups which were submitted to three different revaccination schedules with live ND LaSota vaccine via drinking water (DW) throughout the study period. Birds of group 1: had received one dose simultaneously with inactivated vaccine and ten booster doses of LaSota vaccine had given regularly at three–four weeks interval (concurrently with the sampling time). Birds of group 2: had received six booster doses of LaSota vaccine with five – six weeks interval (at the 25th, 31st, 36th, 42nd, 47th and 53rd week of age), while birds of group 3: had received three booster doses of LaSota vaccine with ten weeks interval (at the 30th, 40th and 50th week of age). Five samples of blood for serum preparation, 20 eggs for saline and chloroform yolk extraction and 30 eggs for hatching, had been collected from each group at 11 occasions with approximately three weeks interval among 35 weeks started from the 23rd week and ended up in the 55th week of breeders’ age, at which birds submitted to challenge test intramuscularly with the field strain of vNDV. Representative field groups (FG1, FG2, FG3) of blood and egg samples had been collected from breeders of three different flocks aged 29, 40 and 55 week-old respectively (once from each farm) which almost have a vaccination history similar to that in experimental groups but vary in timings and frequency. They had been compared correspondingly to that nearest ages of experimental samples.
Two simple methods, saline and chloroform extraction of egg yolk had been compared for titration of IgY that related to the serum samples.
The hatching chicks had been numbered serially (cks1-cks11) depending on the age of their parents when sampling, the sera of those eleven hatching chicks from each group in addition to the three field group hatched chicks (CKsFG), have been bled within 8–12 hours after hatching for comparison of maternal antibody with that in sera of the parents and their egg yolks. At five different occasions, chicks (CKs2, CKs4, CKs6, CKs8 and CKs10) which referred to breeders at the, 26th, 33rd, 39th, 45th, and 51st week–old respectively, as soon as, those of CKsFG, had been divided into two subgroups of 5 chicks. One subgroup was bled for tracing the maternal antibody and its decline rate at five to ten days interval up to 30-35 day-old and the other subgroup had been challenged intramuscularly at the same day of bleeding with 0.1 ml containing 106 ELD50 of vNDV/chick.
The antibody titres in all collected samples were assessed by ELISA using NDV plus ELISA kits ( Synbiotics Corporation), and compared to beta HI micro titre method, then, the correlation between the two test have been evaluated. The data obtained from ELISA reader, HI test plates and challenge results were entered and managed by Microsoft Office Excel (2003) program and had statistically analyzed using the computer (SPSS 10, 1999) program.
Our obtained results were summarized as follow:
1. The mean Ab titres of breeders at the 20th week was relatively low that considered at the critical protective level (3.8 – 5.1 log2 HI and 1.3 – 4.6 × 103 ELISA titre) and significant increase (P<0.05) had been reported in Ab titres of vaccinated groups at the 23rd either in serum or in yolks. On contrast, no significant differences (P > 0.05) had shown between pre experimental breeders’ sera and field samples (FG).
2. from the 23rd week onwards, both HI and ELISA titres of breeder’s sera were relatively high with divergent in the immune responses between vaccinations and other. It was observed that the Ab titre curves of G1 and 2 had almost showed a reciprocal of rise and decline trends among study with some variances, particularly the falling down of group1 titre at the 26th and 30th week. Group 3 trend showed that the mean titres increased gradually and had a significant increase of titre at the period between the 33rd and 45th week. In the comparison to group 1 and 2, the titre curve of group 3 was more stable and reflects uniformity and significant increase (P<0.05) in serum antibody level both in HI and ELISA measures through the experimental period, however, no significant differences were found between group 1 and 2 (P > 0.05). In general, the experimental curves of different kinds showed a slight and slow decrease in overall HI and ELISA titres at the last ten weeks of breeders’ age.
3. Both in HI and ELISA results, the three FG samples had had critical protective titres which were significantly lower than those in the corresponding ages of experimental samples except FG1 compared to group 1 at the 30th week of age, when group 1 had the lowest titre at that age.
4. The mean antibody titres measured by both ELISA and HI in SEY method showed higher level than those extracted by CEY method, there is a significantly positive correlation (P<0.01) between the two compared methods with correlation coefficients of r = 65 and r = 46 in HI test and ELISA respectively. Nevertheless, the correlation between egg yolk and serum titres of both breeders and day old chicks was mostly higher in SEY method than in CEY method in the two applied measurement assays.
5. The overall means of HI and ELISA antibody titres in experimental groups, both in SEY and CEY tended to be higher than those found in breeders’ sera. On the other hand, both of maternal sera and yolks had higher levels than those of day old chicks through the study period.
6. The titres of HI and ELISA antibody in the two kinds of extracted yolks were more constantly in group3 and significantly higher than those of group 1 and 2 (P< 0.05), and revealed a positive correlation with the titres in the hens, but it was relatively higher in SEY ( r = 0.31 & r = 0.37 ) than those of CEY (r = 0.23 & r = 0.22 ) of HI and ELISA respectively.
7. The correlation between the Ab titres of yolks and day-old chicks were significant (P< 0.05) with correlation coefficient of r = 0.65 & r = 0.41 by HI test and r = 0.48 & r = 0.68, with ELISA results in SEY and CEY respectively. However, the correlation between the Ab titres of day-old chicks and breeders was significant (P< 0.05) with correlation coefficient of r = 0.55 by HI test and weak (r = 0.20) by ELISA.
8. The mean HI and ELISA titres of the three FG samples appeared at similar pattern of the highest titre in one day-old chicks followed by serum of breeders, SEY and CEY respectively
9. In all of the experimental hatched chicks, the maternal antibody titres against NDV measured by both HI and ELISA assays were the highest in day one of their life, but this titre was significantly higher (P<0.05) in chicks 2, 4 and 6 than in chicks 8 and 10. The curve of the decline rate was divided into two periods, the first was in 1- to 10-day-old chicks in which the slop of titre diminution appeared more rapidly (3 days) of two-fold (1 log2) than those of second remained period (10- to 30 days) that which was slower (4.5 -5 days). In the latest chicks (CKs 8 and 10), it was uniformly among the tested period that estimated to be 5 and 5.5 days respectively. The decline rate measured by ELISA was estimated to be about 3 log10 titre every 3 days in CKs 2, 4 and 6, however, it was 4 days in CKs 8 and 7.5 days in CKs 10. By day 20 of age onward, the Ab levels in the experimental chicks were almost under the protective level and by day 30, the Ab levels had disappeared.
10. In CKsFG, values of the three groups were under 3 log2 at the 20th day without significant differences and no Abs had been detected at the 30th day. As shown in the experimental chicks, the decline rate of Ab titres in CKsFG was more rapidly in chicks that had taken from younger breeders than those from older, approximately 4 - 5 days in CKsFG1, 4.5 – 6 days in CKsFG2, and 6.5 -7 days in CKsFG3, that had sampled from 29, 40, and 54-week old breeders respectively.
11. The challenge result of breeders with the field VNDV showed that groups of vaccinated birds had had well protected, whereas the unvaccinated control hens failed to protect against the challenge. The protection of challenged day-old chicks had ranged between 40 – 60 %. Almost 100% of chicks were protected in the 5th and 10th day of age, then waned gradually from 80-100% at the 15th day and diminished to only 40% by 25 days of age reaching zero at the 30th day of age onwards. Statistically, there was a good positive correlation between the period elapsed after hatching and the degree of protection obtained up to the 15th day (r = 0.77) and a high negative correlation during subsequently period (r = - 0.93). The linear regression of antibody levels and mortality in experimental chicks was in negative direction and higher with ELISA titres y = - 0.01x + 70.7, and r = - 0.57 than that with HI titres y = - 7.7x + 75.4, and r = - 0.63. Also in CKsFG, the linear regression was higher in ELISA titres (y = - 0.01x + 82.06 and r = -0.64) than in HI titres (y = - 8.34x + 89.85 and r = -0.79), moreover, ELISA titre values were always higher than those of HI with high degree of correlation between mean ELISA and mean HI titres (r = 0.79). We discuss the implications of our findings for the design vaccination schedules with monitoring system aimed at protection of poultry flocks against outbreaks of ND.