Effect of Maternally-Derived Antibodies on The Performance and Immunity of Broilers Induced by in Ovo or Post-Hatching Immunizations with a Live Vaccine Against Infectious Bursal Disease

The interference of low or high maternal antibodies titers on the attenuated infectious bursal disease (IBD) virus (IBDV) vaccine infection and its effects on the performance of broilers vaccinated at the 18th day of incubation (in ovo), at one day of age (subcutaneously-SC), or at 15 days of age (drinking water-DW) were investigated. After a series of three live vaccinations, breeders were given or not an IBD oil emulsion vaccine (IBD-OEV) prior to sexual maturity. At day 18 of incubation (in ovo), a commercial vaccine containing HVT and an intermediate IBDV strain or the single HVT vaccine was given. An intermediate IBDV vaccine was given SC at one day of age, or at 15 days of age via DW. The progeny of unvaccinated breeders presented higher neutralizing IBDVspecific antibody (IBDVab) titers at 25 and 40 days of age than those of the progeny of IBD-OEV breeders (p<0.05) at any broilers vaccination age and route. The lower IBDV RNA detection by RT-PCR in the bursa of Fabricius (BF) and the lower IBDV antibody titers in the serum of the groups vaccinated at one and 15 days of age derived from IBD-OEV breeders may indicate antibody-mediated IBDV neutralization. The inovo and one-day vaccinations did not interfere with performance, both in low and high antibody-titered progenies. The in-ovo vaccination against IBD is considered convenient and safe for industrial chickens, irrespective their maternal antibody levels.


INTRODUCTION
Infectious bursal disease (IBD) is one the most common diseases of poultry worldwide, and it is an important cause of immunodepression or immunosuppression in chickens.Biosafety measures and vaccination have been attempted to reduce the risk of infection and disease.For less than two decades, progeny protection was successfully achieved based on the transference of IgG (IgY) antibodies from the female breeder.As passive immunity in chickens is mediated by a single antibody class, it may have eventually caused the selection of progressively more virulent IBDV, as occurred in the mid-1980s, a risk which presently challenges the passive protection strategy.In addition, passive protection may prevent adequate vaccinal IBD virus (IBDV) infection, and therefore hinder effective protective immune response (Wyeth and Cullen, 1976;Van der Berg and Meulemans, 1991;Sharma, 1985;Wyeth and Chettle, 1990;Knoblich et al., 2000;Kumar et al., 2000;Tessari et al., 2000;Alam et al., 2002;Ahmed et al., 2003;Ahmed e Akhter, 2003;Bolis et al., 2003;and Hair-Bejo et al., 2004).The challenge is to determine the exact early timeframe of susceptibility for inducing protection with the minimum risk of wild IBDV infection.This problem has been indirectly solved by monitoring breeder flocks for antibody levels and carefully establishing the best date for progeny vaccination with least vaccine IBDV neutralization and higher resistance to field IBDV.The in-ovo vaccination enables early infection, and thus early immune response and protection against Marek's disease (Sharma, 1985;Whitfill et al., 2002), in addition, of not interfering with chick hatching or livability; however, it does not induce immune response and protection against IBDV (Coletti et al., 2001;Corley et al., 2002).
The objective of the present study was to evaluate the effects of maternal antibodies on IBDV vaccinal infection and specific antibody response in broilers, using three conventional vaccination strategies.

MATERIALS AND METHODS
Two thousand and eight hundred (2,800) Avian Cobb broiler breeders (Rio Branco Alimentos, Brazil) were separated into two groups: one group (1,400) was vaccinated at 18 weeks of age with a trivalent oil-emulsion inactivated vaccine containing IBD-Newcastle disease and infectious bronchitis viruses (IBD-OEV) and the second group (1,400) was vaccinated with a bivalent vaccine only with Newcastle diseaseinfectious bronchitis viruses (non-IBD-OEV).A sufficient number of eggs (4,320) for incubation were collected when breeders were 32 weeks of age, 2,160 from each group.Breeders from both groups had received three live intermediate IBDV vaccines at 5, 10, and 15 weeks of age, prior to the oil-based vaccine.The progenies were distributed into the following experimental groups: A)

Serology Passive Maternal Humoral Immunity
In order to evaluate IBDV-specific serum neutralizing (SN) antibody levels in the sera of breeders given or not the IBDV-oil based vaccine at the time of egg collection (IBD-OEV), at 32 weeks of age, blood samples were collected from the ulnar superficial vein.Thirty hatchlings from each breeder experimental group (vaccinated or not the oil-emulsion vaccine) were sampled 24 hours post-hatching for antibody levels.

Active Humoral Immunity
Active IBDV-specific SN antibody response was evaluated for each experimental group by sampling 30 chicks on days 10, 25, and 40 post-hatching.The SN assay was performed in 96-well microtest plates with 100 TCID50 of an IBDV Moulthorp chicken embryo fibroblast (CEF)-adapted strain with monolayers evaluated at 72h post-infection.

Reverse-Transcriptase PCR
The vaccinal infection was verified in the cloacal bursa of embryos at day 21 of incubation or in chicks 96 hours post-vaccination using reverse-transcription polymerase chain reaction (RT-PCR), with specific primers for the VP1 gene, as previously described (Gomes et al., 2005).

Live Performance
Cumulative live performance parameters (mean weight, feed conversion and livability) were determined at 7, 21 and 40 days of age for a total of 1,080 chickens from all experimental groups.

Experimental Design and Analyses
In order to evaluate performance, a completely randomized experimental design with a 2x3 factorial arrangement of 6 treatments with six replicates of 30 birds each was applied.Differences were compared using the Student-Newman-Keuls (SNK) test or Kruskal-Wallis (livability).Serological and PCR results were statistically analyzed using the same experimental design, using one bird per replicate, and means were compared using Kruskal-Wallis, Mann-Whitney or χ 2 tests.

Chickens
The chicks used in this experiment were passively protected against Newcastle disease and infectious bronchitis viruses and derived from breeders free from Mycoplasma gallisepticum, M. synoviae, Salmonella gallinarum and S. pullorum.The experimental birds were raised in insect-and bird-proof houses, and did not show any sign of disease, being sent for normal processing at the end of the experiment.

Serology
Average antibody titers of breeders at time of egg collection were 11,434 for the IBD-OEV and 810 for the non-IBD-OEV and chicks 24 hours post-hatching were 16,816 for IBD-OEV and 547 for the non-IBD-OEV.At 32 weeks of age, mean IBDV-specific SN titer of breeders vaccinated with the combined oil-emulsion IBDV vaccine at the 18-week of age was much higher than the unvaccinated group (p<0.05),suggesting that the progeny of the vaccinated breeders would have high titers of passive antibody at hatching.These results are consistent with previous reports Wood et al. (1983) and Van der Berg and Meulemans (1991).At hatching, passive IBDV-specific SN antibody titers were higher in the progeny of breeders given the IBDV-containing oilemulsion vaccine, as shown in literature (Naqi et al. (1982); Knoblich et al. (2000).Progeny mean IBDVspecific SN antibody titers at 10, 25, and 40 days of age are shown in Table 1.At 10 days post-hatching, chicks from IBD-OEV breeders presented the highest (p<0.05)IBDV-specific antibody titers.At 25 days post-In-ovo vaccination titers were similar to those previously described (Coletti et al., 2001).In-ovo vaccinated IBD-OEV breeders progenies had lower responses (p<0.05) at 25 and 40 days of age as compared to unvaccinated breeders progenies, which agrees with previous findings (Coletti et al., 2001), and suggests that high titers of IBDV-specific passive antibodies negatively interfere with intermediatelyattenuated IBDV vaccine strain replication.

RT/PCR
The IBDV RT-PCR results determined in the progeny cloacal bursa 96 hours post-vaccination are presented hatching, the chicks from unvaccinated breeders and vaccinated in ovo had higher antibody titers as compared to chicks from IBD-OEV vaccinated breeders (p<0.05).These findings are in agreement with previous reports evaluating the interference of maternal antibodies on vaccinal IBDV infection (Van der Berg and Meulemans, 1991;Knoblich et al., 2000;Alan et al., 2002;Rautenschlein et al., 2005).Indeed, Sharma (1985) found similar in ovo responses, with low passive antibody-titered progenies at hatching presenting low response to day-old subcutaneous vaccination and progenies with no IBDV-specific antibody titers presenting better protection to vvIBDV challenge.At 40 days of age, chicks from unvaccinated breeders responded with higher titers (p<0.05),irrespective of vaccination route (in ovo, subcutaneous, or drinking water).The results of the progeny derived from oilemulsion vaccine vaccinated breeders and vaccinated at hatching are consistent with those of Naqi et al. (1982), Knoblich et al. (2000), Kumar et al. (2000), Bolis et al. (2003), and Ahmed et al. (2003).(Coletti et al., 2001;Corley et al., 2002), although these authors tested SPF-breeder progenies for the absence of passive antibodies.

Live performance
Feed intake, weight gain and livability from 1-to 40-day-old chicks are presented in Tables 3, 4, and 5, respectively.
No significant differences were observed in feed, feed conversion ratio or intakelivability among the treatments.Gagig et al. (1999) found similar results in SPF chickens vaccinated with an intermediatelyattenuated IBDV vaccine strain at 18 days of incubation, which did not affect livability at 7 days post hatching.
At 7 days post-vaccination, the weight gain of IBD-OEV breeder chickens was not higher (p<0.05)than that of the other groups (Table 3).A possible explanation is that the detrimental effects of vaccinal infection may have been prevented by the higher titers of IBDV-specific passive antibodies in the OEV breeder progenies.
Vaccination at 15 days of age resulted in lower weight gain, lower feed intake, but better feed conversion ratio (p<0.05), as evaluated in the period of 1 to 21 days of age (Table 4).The lower intakefeed intake may have resulted from vaccinal virus replication

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in the chickens, as observed in other vaccinal infections, such as those with infectious bronchitis virus vaccine strains (Talebi et al., 2005).
The in-ovo vaccination of the progeny from unvaccinated breeders may have impaired feed conversion ratio (p<0.05).Their lower body weight as compared to the vaccinated breeder progenies may be related to a lower protection against vaccinal infection.
No significant differences in livability were observed up to 21 days of age among the experimental groups (Table 4).These results are different from those of Giambrone et al. (2001), who observed lower livability at 21 days of age in SPF chickens as compared to commercial ones, when using in-ovo vaccination.SPF chickens are different from commercial chickens in several performance aspects and the type of chicken may account for the observed differences.The experimental breeders and progenies used in the present experiment were managed and fed according to the high standards of a chicken-produce exporting company; no deviation from the regular management was observed, and the breeders continued to produce until culling at the end of the production cycle.
The results shown in Table 5 do not indicate any significant differences in body weight and livability up to day 40.However, chickens vaccinated at 15 days of age presented higher feed intake and better feed conversion (p<0.05) as compared to the other treatments.At day 15, the IBD-OEV progeny has a lower rate of vaccinal infection, as detected by RT-PCR.

CONCLUSIONS
The vaccination of broiler breeders at puberty (18 weeks of age) with an IBD inactivated oil-emulsion vaccine, inducing high titers of circulating antibodies, may negatively affect the infection of the progeny by an intermediate attenuated vaccinal IBDV strain due to the presence of high titers of passive antibodies.Paradoxically, in more susceptible birds, such as the progeny of breeders that did not receive the oil-based IBD vaccine, the vaccinal infection by an intermediate attenuated strain may result in a significant performance loss.The vaccination with an intermediate vaccine strain in ovo or at hatching did not affect feed intake, feed conversion ratio or weight gain up to the 40 days of age of the progenies derived from breeders given or not the oil-emulsion vaccine.Although the induction of high levels of circulating antibodies in breeders has been a gold standard for passively protecting progenies, the results of the present experiment show that there is a negative effect on subsequent vaccine uptake by the progeny.Hence, paradoxically, when 15-day-old broilers are vaccinated with an intermediately attenuated IBDV strain, the protective titers produced by vaccinal IBDV infections in the progeny may be necessary to prevent performance losses.
Michell BC,Gomes  AD, Baião NC, Resende M, Lara LJC, Martins NRS ffect of Maternally-Derived Antibodies on The Performance and Immunity of Broilers Induced by in Ovo or Post-Hatching Immunizations with a Live Vaccine Against Infectious Bursal Disease

Table 2 -
IBDV reverse transcription PCR of bursal RNA extracts 96 hours post-vaccination in ovo, from 1 to 15 days of age.

Table 3 -
Live performance of broiler chickens from 1 to 7 days of age.

Table 4 -
Live performance of broiler chickens from 1 to 21 days of age.Live intermediate IBDV vaccinations, at 5, 10 and 15 weeks of age; **Coefficient of variation; ***IBD-OEV breeders: given the IBD oil emulsion vaccine at 18 weeks of age; ****non-IBD-OEV: not given the oil emulsion vaccine; Means followed by different lower-case letters (row) or capital letters (column) are different (p<0,05) by the SNK test or Kruskal-Wallis test (livability). *

Table 5 -
Live performance of broiler chickens from 1 to 40 days of age.Michell BC, Gomes AD, Baião NC, Resende M, Lara LJC, Martins NRS ffect of Maternally-Derived Antibodies on The Performance and Immunity of Broilers Induced by in Ovo or Post-Hatching Immunizations with a Live Vaccine Against Infectious Bursal Disease * Live intermediate IBDV vaccinations, at 5, 10 and 15 weeks of age; **Coefficient of variation; ***IBD-OEV breeders: given the IBD oil emulsion vaccine at 18 weeks of age; ****non-IBD-OEV: not given the oil emulsion vaccine; Means followed by different lower-case letters (row) or capital letters (column) are different (p<0,05) by the SNK test or Kruskal-Wallis test (livability).