Print version ISSN 1516-635X
Rev. Bras. Cienc. Avic. vol.11 no.1 Campinas Jan./Mar. 2009
Michell BCI; Gomes ADII; Baião NCI; Resende MII; Lara LJCI; Martins NRSI
IEscola de Veterinária - UFMG
IIInstituto de Ciências Biológicas - UFMG
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.
Keywords: Attenuated IBDV, attenuated IBDV neutralization, broiler chicken, infectious bursal disease, infectious bursal disease virus, IBDV, in-ovo vaccination, maternal antibody, RT-PCR, serum neutralization, vaccination versus performance.
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) Progeny of IBD-OEV breeders; day-old chicks given the IBDV intermediate attenuated vaccine strain subcutaneously.
B) Progeny of non-IBD-OEV breeders given the IBDV intermediate attenuated vaccine strain via drinking water at 15 days of age.
C) Progeny of IBD-OEV breeders given IBDV intermediate attenuated vaccine strain via drinking water at 15 days of age.
D) Progeny of non-IBD-OEV breeders; day-old chicks given IBDV intermediate attenuated vaccine strain subcutaneously.
E) Progeny of non-IBD-OEV breeders vaccinated in ovo with IBD-HVT bivalent intermediate attenuated vaccine.
F) Progeny of IBD-OEV breeders vaccinated in ovo with IBD-HVT bivalent intermediate attenuated vaccine.
The experiment was designed to evaluate the progenies of breeders with low or high antibody titers, vaccinated at the most common ages and through the most frequent routes of IBD vaccination. As to in-ovo vaccination, eggs from groups A, B, C and D were vaccinated at 18 days of incubation with a commercially available HVT FC-126 strain (at least 1,000 PFU/dose) and eggs from groups E and F with a commercial combination of HVT FC-126 (9,600 PFU/dose) and intermediate IBDV attenuated vaccine (103,50 TCID50/ dose) using Inovoject® equipment (Embrex, The Netherlands). Chicks from treatments A and D were subcutaneously vaccinated at hatch with an intermediate IBDV attenuated vaccine (104,1TCID50/ dose), and those from treatments B and C received the same vaccine via drinking water at 15 days of age. Chicks vaccinated for IBDV or not were housed in separate houses at the experimental farm "Prof. Hélio Barbosa" of Universidade Federal de Minas Gerais (Igarapé, Minas Gerais, Brazil). Feed and water were provided ad libitum. Two different feeds were formulated: a starter feed was fed from 1 to 21 days, and a grower feed, from 22 to 40 days of age.
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.
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).
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 X2 tests.
RESULTS AND DISCUSSION
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.
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 posthatching, 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).
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.
The IBDV RT-PCR results determined in the progeny cloacal bursa 96 hours post-vaccination are presented in Table 2. Unvaccinated breeders produced progenies that were more susceptible to vaccinal IBDV infection, as detected by PCR of the reversely-transcribed bursal RNA as compared to the chicks derived from breeders with high IBDV-specific antibodies. Vaccinal IBDV may have been inactivated by maternal antibodies in IBD-OEV breeder progenies vaccinated at hatching or at 15 days of age. However, in progenies with high IBDVspecific antibody titers, the in-ovo vaccination resulted in a higher rate of bursal infection (58%) as compared to the other vaccination routes and ages (36.6% subcutaneous at hatching and 22% via drinking water at day 15). The higher susceptibility of chicks vaccinated at 18 days of incubation day embryos to vaccine infection may be due to the fact that the passively acquired antibodies are not yet available in the embryonic circulation and are still retained in the yolk. Similar findings of vaccinal IBDV post-vaccination with live attenuated IBDV were previously reported (Coletti et al., 2001; Corley et al., 2002), although these authors tested SPF-breeder progenies for the absence of passive antibodies.
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 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.
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.
The authors would like to thank Rio Branco Alimentos, Brazil, for the experimental chickens and incubation. This research study was also funded by CNPq, FAPEMIG and Fundação de Ensino e Pesquisa em Medicina Veterinária e Zootecnia (Escola de Veterinária da UFMG).
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Nelson Rodrigo S. Martins