Gumboro Disease : Evaluation of Serological and Anatomopathological Responses in Vaccinated Broiler Chickens Challenged with Very Virulent Virus Strain

This paper describes the effects of a strong type of vaccine Moulthrop G603  and an intermediate-plus type of vaccine 228E  in meat-type chickens challenged with a very virulent strain of infectious bursal disease virus (vvIBDV). Blood samples and bursa of Fabricius were taken weekly up to 42 days of age. It was concluded that bursa of Fabricius weight, bursa weight:body weight ratio, body weight and antibody titer evaluated before and after challenge with vvIBDV were not enough to consistently and conclusively differentiate or estimate the protection given by vaccination. Quantitative evaluation of injury intensity and the number of altered lymphoid follicles revealed that Moulthrop G603 caused moderate microscopic lesions in one out of seven birds vaccinated at 14 days of age, while 228E vaccine did not induce IBD-typical microscopic lesions in the bursa of Fabricius. Good protection against bursal microscopic lesions was obtained when a strong type of vaccine was used before challenging with vvIBDV. It was concluded that quantitative evaluation of microscopic lesions might be useful to measure the injury induced by vaccinal IBDV, as well as the level of protection and/or immunosuppression induced by IBDV challenge in vaccinated and non-vaccinated chickens.


INTRODUCTION
Gumboro Disease (GD), also called infectious bursal disease (IBD), is a chicken disease caused by a Birnavirus from serotype 1.According to the virus virulence and pathogenicity, IBD cause more severe or less severe lesions on the bursa of Fabricius and other organs such as: spleen, thymus and kidneys, and may induce immunosuppression and mortality in birds (Lukert & Saif, 1997;McFerran, 1993).The disease was described in 1962, in the United States, and the first suspected occurrence in Brazil was reported by Nakano and confirmed by Saukas (1978).According to Lasher & Shane (1994), the occurence of a very virulent virus strain capable of inducing 100% mortality in chickens without specific antibodies was notified in Europe in 1989.Until then, the most virulent viruses reported caused at most 10 to 15% mortality in experimentally infected birds without specific antibodies, for instance, the reference sample 52/70 or Faragher.The first cases of high mortality due to Gumboro disease in Brazil were observed approximately in mid-1997(Di Fabio et al., 1999 a,b).The outbreak was an infection caused by a virus from the molecular group 11 (Gm 11), similar to the very virulent samples found in Europe and classified as very virulent virus (Ikuta et al., 2001).
Numerous vaccines and vaccination programs have been studied and proposed worldwide.This disease has been considered a defined nosologic entity able to cause economic losses because it induces immunosuppression and because of the appearance of new virus strains.Until the 1970s, during the period in which IBD viruses were being described and studied, prevention in the United States was done using a nonattenuated field strain of the virus with moderate pathogenicity.In the early 1970s, young birds were immunized with atennuated vaccinal virus propagated in embryonated eggs or cell cultures, or nonvirulent viral strains (Lasher & Shane, 1994).The concept of IBD prevention in very young birds arose in the mid-1980s, by passive immunization using emulsified vaccines in breeders (Lucio & Hitchner, 1979).Intermediate vaccine strains, cloned or with less passages in eggs or cells, were introduced with the objective of immunizing chicks with high maternal antibody titers.They substituted vaccines produced with very attenuated or nonvirulent strains, called mild vaccines.According to literature (Lukert & Saif, 1997;McFerran, 1993;Lasher & Shane, 1994;Van der Berg, 2000), oral, nasal or ocular mild vaccines were effective only in immunizing chicks that had passively acquired neutralizing antibody titers lower than 100.Nevertheless, chicks with this antibody levels and challenged with pathogenic field virus would be totally susceptible to infection.Thus, intermediate vaccines were introduced since they immunized chicks with circulating antibody titers equal or inferior to 250.It was known that flocks with antibody titers between 100 and 600 and vaccinated with mild vaccinal samples would be only 40% protected when exposed to field challenges.If intermediate strains were used, protection rate was better.According to McFerran (1993), IBD prevention by vaccination would be related to the appearance of circulating antibodies after immunization of Specific Pathogen Free birds or birds with no antibodies using attenuated samples, whereas chicks with higher circulating antibody levels should be vaccinated with less attenuated or more virulent vaccines.New control strategies of IBD were proposed when atypical viruses (serological escape variants) were described in the United States in 1985, Delaware A, D, E and G (Lasher & Shane, 1994) and strains of very virulent virus type were described between 1986 and 1987 in Europe ( Van der Berg, 2000).Vaccines against variant E and A were developed in the United States in 1989and 1992, respectively (Lasher & Shane, 1994).In Europe, it was proposed that less attenuated or strong vaccines should be used as a means to prevent IBD with high mortality.Van der Berg & Meulemans (1991) argued that intermediate live vaccines were not protecting against vvIBD challenges because maternal antibodies would be affecting its ability to populate lymphoid organs.In Brazil, a vaccine denominated intermediate plus (Bursine plus ® Fort Dodge and Nobilis 228-E ® Intervet) was released in 1999 for the control of IBD caused by type Gm 11 virus.Strong or hot vaccines became available in 2000: sample Moulthrop G603 (Avimmune -F ® -Coopers), sample V 877 (Poulvac ® -F -Fort Dodge) and sample Winterfield 2512 (Cevac ® IBD L Ceva).According to Simon & Ishizuka (2000), vaccines might be used to establish immunity levels to limit IBD outbreaks in endemic areas.Maas et al. (2001) reported that two different vaccinal strains in oil emulsion were used in breeders and induced passive immunity with different ability to prevent mortality caused by vvIBD and vIDB 52/70.Nevertheless, severe lesions in the bursa of Fabricius were not prevented if birds were challenged with more virulent virus (Maas et al., 2001).According to Abdel-Alim & Saif (2001), very virulent IBD virus (vvIBDV) are antigenically related to attenuated or mild vaccine SAL type virus (70 to 80% homology), and the immunity induced by vvIBDV protects 100% against challenge with standard vIBDV, sample STC.Reference strain STC is a standard virulent type virus that belongs to the same molecular group as the vaccinal sample Moulthrop (Gm3) (Ikuta et al., 2001).Therefore, it is possible that such vaccine protects adequately against vvIDBV.
Histopathological examination of the bursa of Fabricius identifies the occurence of IBD because very typical microscopic alterations are present after infection (Cheville, 1977;Winterfield et al., 1972;Riddell, 1987).Evaluation of lesion type and intensity permits to identify different pathogenicity among the different serotype 1 IBDV (Muskett et al., 1979;Sharma et al., 1989).Generally, lesions are scored from 0 to 4 or 5, based on the presence of degenerative alterations, necrosis, follicular atrophy in the bursa of Fabricius and the percentage of altered lymphoid follicles.The intensity of microscopic alterations in the bursa of Fabricius may also be quantified to evaluate the level of immune protection (Abdel-Alim & Saif, 2000;Maas et al., 2001) or immune modulation of the infection (Poonia & Charan, 2001).Nielsen et al. (1998) evaluated the lymphoid depletion or necrosis degree present in the bursa of Fabricius and observed that layers and broilers had equally severe lesions after infection with an IBD 52/70 isolate.McFerran (1993) described that chronic and/or subacute infection by IBDV would be related with microscopic alterations in the bursa of Fabricius, i.e., vacuolar degeneration of lymphoepithelial cells and area reduction of the medular regions.In the newly hatched chick, three or more primary pre-bursal cells develop into 8 to 12x10 3 bursal follicles which accomodate long or short halflife lymphocytes that proliferate rapidly (Glick, 1991) and originate approximately twenty generations of cells with surface IgG from clones expressing IgM (Hammer, 1974).Lymphoid proliferation and maintenance that occurs in lymphoid follicles depend entirely on bursal microenvironment, given by non-lymphoid dendritic cells or secretory cells.B-dependent immune response in chicks treated with ciclophosphamide was restored with cell adoptive transference because secretory cells had not been destroyed (Glick, 1991).Histologically, lymphocyte loss and alterations in the structure of bursa of Fabricius may be observed when the chicken is infected with IBDV; nevertheless, it is very important to evaluate the stage and extension of the injury.Some IBDV isolates cause small or no inflammation in the bursa of Fabricius, whereas others cause intense loss of the bursal stroma and of the follicular microenvironment that sustains cell B differentiation, which explains the consequent severe immunodepresison.IBDV isolates that induce partial follicular lymphocyte loss in the acute phase of infection will show follicles with extensive lymphocytic regeneration areas during the recovery phase (Pope, 1991).Bursal lymphoid follicles lose their regenerative ability probably because there are no reticular or dendritic cells that sustain the maturation of lymphocytes and no epithelial cells from the corticalmedullary cord that contribute as germinative cores for the medullary region of the bursa of Fabricius.According to Glick (1991), blasts and mitosis can be seen in the medular region of a normal bursa, very close to the cortical-medullary cord.
This work was conducted with the objective of evaluating the protection given by vaccination of bird chicks with maternal antibodies against vvIBDV using an intermediate plus vaccine, 228E, or a strong vaccine, Moulthrop G603.The effect of the immune response induced by the two vaccines was evaluated before and after vaccination and after challenging with a vvIBDV isolate (2050/97), using serological tests and macro and microscopical evaluations of the bursa of Fabricius.
Quantitative evaluation of the microscopic alterations present in the bursa of Fabricius was performed in the present study considering both lesion types (acute and recovery phases) and percentage of lymphoid follicles with loss of the follicular parenchyma (total loss of lymphoepithelial cells), with the objective of obtaining values or scores that could better express the protection rate of vaccinated birds challenged with vvIBDV.

Birds
Two hundred Ross broiler chicks with one day of age were vaccinated subcutaneously with one dose of the vaccine Mark-GumborÒ BioVet (lot 794/99, Nov/ 99), containing the HVT vaccinal sample of Mareks disease at the concentration of 1,179 PFU per dose and the BV8 sample of IBDV virus at 10 3.83 infective doses 50% in culture of chicken embryo fibroblasts (ID 50 CF) per dose.Twelve Specific Pathogen Free chicks (SPF) were obtained from BioVet, Vargem Grande Paulista, SP.

Experimental groups
The birds were distributed in four experimental groups of 50 chicks each and selected at random.Each group was housed in separated isolation unity at Centro de Pesquisas da BioVet (Ibiuna, SP) and designated as T1, T2, T3 and T4.All birds were reared at isolation unities until the end of the experiment, and were fed disinfected pelleted diet (fumigated with paraformaldehyde) and chlorine-treated filtered water.

Experimental Procedure
Group T1 was not vaccinated or challenged.At 21 days of age, non-vaccinated birds from group T2, but challenged, and vaccinated birds from groups T3 (Moulthrop G603) and T4 (Moulthrop 228E) were given 50 µl of the homogenate produced with bursa of Fabricius containing vvIBDV 2050/97.At 1, 7, 14, 21, 28, 35 and 42 days of age, 15 birds from each group were individually identified and blood was taken for seroneutralization test.From 7 to 42 days of age, seven birds from each experimental group were sacrificed and necropsied at seven-day intervals.At the end of the experiment (42 d), 10 birds were killed per experimental group.Birds were individually weighed, necropsied and the bursas of Fabricius were collected, weighed and fixed in formol (10%) for histopathology.

Virus
Isolate 2050/97 virus was used in the challenge test.This isolate was identified as a serotype 1 Gumboro disease virus of molecular group 11 (Simbios, Canoas, RS) and characterized as a very virulent virus strain able to induce 100% mortality of SPF birds in 48 to 96 hours after inoculation.Bursa of Fabricius were collected from SPF birds that had been experimentally infected, macerated and resuspended in 10% phosphate tryptose broth (Difco) and cleared by centrifugation at 1,500 g for 30 min.The cleared homogenate was used to infect 21-day-old birds of the experimental groups.Inoculum potential was confirmed by inoculating 20 SPF birds at the same age.
Birds from group 3 (T3) were inoculated with a Moulthrop G603 sample (Avimmune F ® Coopers, lot 005/00 produced in Nov/00).Birds from T4 were inoculated with the sample 228E (Nobilis 228EÒ Intervet, lot 007/00 produced in Sep/00).Birds were vaccinated at 14 days of age using the eye drop method, with a single vaccine dose resuspended in the diluent provided by the manufacturer.

Seroneutralization
Sera were diluted 1:10 in M209 medium (Difco) with penicilin (1,000 IU/mL) and dihydrostreptomycin (1.0 mg/mL), loaded to wells of a flat-bottom cell-culture microplate and serially diluted in 50mL of medium previously loaded to the wells from 1:20 to 1:20,480.To each serum dilution, 100 ID 50 CF of the isolate GBV-8 of IBDV was added, obtained from the vaccine Gumbor Vet ® BioVet Laboratórios, lot 530/00, which is registered at the Ministério da Agricultura as a Lukert type.Serum and antigen were allowed to react for 40 min and 200 mL of suspension containing 6.5 x 10 6 SPF chicken embryo fibroblasts per mililiter was added to each well of the microplate.In each microplate, wells containing only fibroblast suspension (cell control) and antigen plus cells (virus control) were included.Microplates were kept at 37ºC in 5% CO 2 incubators and the presence of cythopatic effect was evaluated 4 days later using an inverted microscope (Olympus, Tokyo, Japan).Neutralizing antibody titer was the reciprocal of the highest serum dilution that was able to completely neutralize the cytopathic effect induced by 100 ID 50 CF of antigen.Log 2 of the values relative to the reciprocal of dilution able to completely neutralize the antigen was used to calculate the geometric mean titer (GMT) and the coefficient of variation (CV%) for each experimental group.

Histopathology
Bursas were included in paraffin and fine cuts of 3 to 8µ were prepared, mounted between glass slides and coverslips and stained with hematoxilin and eosin.All cuts were examined in a light microscope and scored from 0 to 6 based on lesion characteristics in the acute phase of infection as described by Cheville (1977), Lukert & Saif (1991) and Poonia & Charan (2001) for standard serotype 1 virus (STC, Faragher 52/70).Readings were standardized as follows: numbers from 1 to 6 were given according to the histological changes on the lymphoid follicle, interstitial conjunctive tissue and bursa of Fabricius follicles.Therefore, scores for tissue alteration were: 0 = no alteration; 1 = cells with picnotic nuclei in the medullary region of the lymphoid follicles and discrete dissociation of the intersticial conjunctive tissue, or lymphoid rarefaction in the medullary zone and/or roughening of the corticomedullary cord and/or central epithelization foci on the medullary region; 2 = hypotrophy of medullary and cortical regions of the lymphoid follicle due to degeneration of lymphoepithelial cells, and/or lymphocytes and/or macrophage-or plasmocyte-type cell aflux, associated to a discrete dissociation and infiltration of inflammatory cells on the intersticial conjunctive tissue.During the recovery phase, roughed epithelium and presence of mitosis or sparse lymphoid cells in the medullary zone of the lymphoid follicles; 3 = lymphoid depletion in the cortical and medullary zones and/or intense degeneration of lymphoepithelial cells in the lymphoid follicle, associated to a discrete edema and intersticial infiltration of inflammatory cells, besides roughening and/or discrete epithelial invagination and perivascular lymphoid hyperplasia at the septum of the conjunctive tissue; 4 = degeneration of cells from the medullary and cortical regions, with moderate infiltration of inflmmatory cells in the lymphoid follicle, intersticium and epithelial layer, or lymphoid follicles depleted from lymphoid cells, with sparse lymphoepithelial cells, intersticial fibroplasia and many foci of epithelial invagination; 5 = medullary necrosis with cyst formation, intense granulocyte infiltration in the parenchyma and/or necrosis with hemorragia in the intersticium or lymphoid follicles and/or lymphoid follicles with epithelial invagination or fibrosed and intense intersticial fibroplasia; 6 = absence of lymphoid follicles due to hemorrhagic necrosis, atrophy or fibrosis.Decimal values were added to such scores, according to the amount of injured follicles, as proposed by Sharma et al. (1989): 0.1 = less than 5% of lymphoid follicles with alterations; 0.2 = 5 to 25%; 0.4 = 26 to 50%; 0.6 = 51 to 80%; and 0.8 = more than 80% of altered lymphoid follicles.A numeric value was obtained for each bursa of Fabricius by adding up the scores given for tissue alteration type (1 to 6) and altered lymphoid tissue (0.1 to 0.8).For example, 1.1 or 1.2 or 1.3 were scores of bursas showing very discrete alterations.

Bursa weight and body weight
In each experimental group, body weight (g) and bursa weight (g) were evaluated for each individual and the ratio bursa weight:body weight (%) was calculated.The results were expressed for each experimental group as the arithmetic mean and standard deviation.

Statistical Analysis
Data were submitted to statistical analysis by using GLM of SAS, and treatment means were compared at 5% probability level (p<0.05).

General Comments
No mortality was observed during the experimental period in the groups vaccinated and non-vaccinated and challenged with the sample 2050/97.The homogenate of the bursa of Fabricius 2050/97 induced 100% mortality in SPF chicks at 21 days of age from the period of 56 to 80 hours post-inoculation.Before death, there was intense prostration, inapetence, watery feces or pasting vents and anorexia.At necropsy, bursas showed intense edema and deposition of serofibrinous material on the muscular wall.

Bursa weight
According to the results shown in Table 1, birds that were not vaccinated at 14 days of age (T1 and T2) had bursa weights of 0.950 and 1.341 g at 21 days, respectively.There was an increase of 0.610 and 0.799 g from the weights obtained at 14 days of age (0.340 g and 0.542 g, respectively).Group T3, vaccinated with Moulthrop G603, had an increase of 0.351 g in bursa weight from 14 to 21 days (0.442 g at 14 days and 0.793 g at 21 days), whereas T4, vaccinated with 228E, had an increase of 0.528 g (0.420 g at 14 days and 0.948 g at 21 days).Therefore, birds from T3 group had the lowest increase in bursa weight after vaccination.
Considering the weights of the bursa of Fabricius from 14 to 42 days of age (Table 1), birds from group T1 (non-vaccinated and not challenged with sample 2050/97 at 21 days) showed an increase of 2.594 g (762.9%).Birds from group T2, non-vaccinated and challenged, showed a reduction of 0.094 g in bursa weight, or 17.43% in the period from 14 to 42 days of age, whereas those vaccinated with the strong strain Moulthrop 603 showed an increase of 0.229 g (51.8%).Chicks from group T4, vaccinated with the sample intermediate plus 228E, had an increase of 0.028g or 6.9% in bursa weight.Thus, birds from groups T2, T3 and T4 showed a reduction in the size of the bursa when compared to birds from group T1.

Body Weight
Broiler chicks within the isolators had lower body weight than the expected for Ross strain from 7 to 42 days of age (Table 2), independent of the experimental group.Mean body weights for each week and between experimental groups were not different (P>0.05).

142
Bursa weight:body weight ratio (BuW/BW) Table 3 data revealed that birds from groups T2, T3 and T4, challenged with sample 2050/97, had a significant reduction (p<0.05) in BuW/BW ratio after 21 days of age when compared to the control group (T1).BuW/BW ratio at 42 days of age was higher in group T3 than in T2 and T4 (p<0.05).an increase in the neutralizing antibody titers after 28 days, when compared to the data from group T1 (Table 4).In the birds of groups vaccinated at 14 days of age, there was no significant difference in antibody titers until 21 days when compared to birds from groups T1 and T2.On the other hand, at 42 days, birds vaccinated with the strong vaccinal sample Moulthrop G603 (T3) showed a higher geometric mean titer (GMT) than groups T1, T4 and T2, respectively.Such difference was significant (p<0.05)only when T3 was compared to T1.

Histopathology
Based on lesion distribution and scores (Tables 5 and  6) from 7 to 14 days of age, it was not found degenerative or inflammatory alterations in the bursa of Fabricius.At 21 days of age, one bird from group T3 (Table 6) showed lymphoid follicles with cellular degeneration and discrete mononuclear cell afflux.In the birds from groups T2 and T4, one of seven bursas of Fabricius had discrete lymphoid rarefaction in the medullary zone of some lymphoid follicles (Table 6).According to Table 6, 100% of the non-vaccinated birds challenged with sample 2050/97 (T2) had severe lesions in the lymphoid follicles from 28 to 42 days of age.Histological evaluation at 42 days showed that 100% of the birds had fibrosed bursas of Fabricius and complete loss of lymphoid follicles, similar to that found in SPF birds.In birds from group T4, which were vaccinated at 28 days with intemediate plus vaccine type, six of the seven birds showed inflammatory changes of the bursa of Fabricius.Lesion intensity in 1 -T1 = non-vaccinated, not challenged; T2 = non-vaccinated, challenged; T3 = vaccinated with Moulthrop G603 and challenged; T4 = vaccinated with 228E and challenged.
Table 6 Distribution of birds at 42 days of age, according to the lesion score of the bursa of Fabricius.
1 -T1 = non-vaccinated, not challenged; T2 = non-vaccinated, challenged; T3 = vaccinated with Moulthrop G603 and challenged; T4 = vaccinated with 228E and challenged group T4 was comparable to those observed in the non-vaccinated challenged birds (T2), whereas in group T3, only two of the seven birds showed similar lesions.
From 28 to 42 days, lesion score of group T3 was lower than those observed in groups T4 and T2, respectively (Table 5).According to Table 6, birds from group T3 had bursas of Fabricius with score 3 at 42 days of age whereas birds from group T4 had scores equal or higher than 4.

DISCUSSION
Whilst 100% of the SPF chicks inoculated with virus 2050/97 died, birds with maternal antibodies and vaccinated at one day of age with attenuated vaccine associated to Marekd Disease virus (Mark-Gumbor ® Biovet) showed no mortality (group T2).Nevertheless, severe alterations associated to irreversible loss of the lymphoid follicles in the bursa of Fabricius were observed at 42 days of age (Table 6).Previous studies were performed with sample 2050/97 in broiler chicks non-vaccinated at one day-old and SPF birds.Whereas 10 out of 10 SPF birds without antibodies died, only one out of ten birds with passive immunity died after the challenge at 21 days.The residual passive immunity from the birds at 21 days of age might have contributed to control mortality after challenge with vvIBDV.According to Maas et al. (2001), circulating antibody levels up to 7 (log 2 ) or 1092 may change the mortality caused by vvIBD viruses.The authors reported that the reduction in mortality after the challenge would vary according to the vaccinal sample.In the present study, the distribution of the passively acquired neutralizing antibody titers (NAT log 2 ) at 21 days in birds nonvaccinated at 14 days (T1 and T2) was between 80 and £ 1280 (Table 7).Therefore, such antibody titers might have been enough to protect the birds to a certain extent, depending on the vaccine type used in the breeders.Yet, the absence of mortality in group T2 after challenge might also be related to the fact that the birds were vaccinated at one day of age.According to Lukert & Saif (1997), birds with antibody titers lower than 1000 or 10 (log 2 ) allowed colonization of mild-type virus inoculated by parenteral via at one day of age and, thus, immunization.From the 60 serum samples collected at one day in the present study, seven showed titers equal or lower than 1280 in the Evaluation of bursa weight and determination of bursa weight:body weight ratio is the most used model to estimate protection rate given by vaccines against IBD.In the present study, the two vaccines induced discrete reduction in bursa weight after inoculation at 14 days of age (Tables 1 and 3).Such discrete reduction in bursa size, more evident in a sample from group T3, was related to the bursa with a score 3.6 at histological evaluation.According to McFerran (1993), this score would be related to chronic or subacute bursal infection because not all lymphoid follicles were injured.The presence of some bursa alteration with such characteristics suggests that there was influence of the residual passive immunity in the propagation of Moulthrop G603 isolate.At 14 days of age, although sera with titers lower than 640 were predominant (43/ 53 samples), changes were not seen in 100% of the chicks from group T3.Only very virulent or mildly attenuated viruses are known to overcome titers of neutralizing antibodies of 500 (Lukert & Saif, 1997).Considering that only one bird showed bursal lesion and that at 14 days of age antibody titers from group T3 varied from 80 (log 2 = 8.32) to 640 (log 2 = 9.32) (Table 7), it might be concluded that the sample Moulthrop G 603 would be propagating in the birds with titers of 80. Birds from group T4, vaccinated with sample 228E, did not show relevant histological alterations in the bursa of Fabricius.Concluding, histopathology performed 7 days after vaccine inoculation may be a good method to evaluate the occurrence of vaccine virus replication and injury intensity in the bursa of Fabricius and, consequently, to estimate the ability of protection against field challenges.
The analysis of serology data (Table 7) shows that before challenge, no difference was observed between the neutralizing antibody titers in the different experimental groups.Similarly, after challenge, although higher geometric mean titer (GMT) was seen at 42 days in group T3 after challenge, when compared to groups T2 and T4, it was not possible to infer protection rate by titer evaluation, because no significant differences were detected.
After challenge with vvIBDV at 21 days, there was a significant decrease in the weight of bursa of Fabricius (Table 1), and in the bursa weight:body weight ratio (Table 3) in groups T2, T3 and T4 when compared to group T1 (control).Since no significant differences in body weight were seen between ages or groups (Table 2), we believe that the most important consequence of IBD virus in recovering birds is that bursa of Fabricius was atrophied or depleted from lymphoid cells.It is highly possible that differences in body weight were not detected among the experimental groups inoculated or non-inoculated with vvIBDV because of the absence of opportunistic infections.From the experimental observations, it was concluded that IBDV per se does not cause reduction in body weight.It is worth noting the fact that the observed weight gain was lower than the expected for broiler chicks of this strain in all experimental groups (Table 2), which was related to the fact that the birds were kept in isolators and blood samples were taken weekly.
As discussed previously, vaccination efficiency should not rely on bursa weight evaluations in IBDV challenges.Results from 7 to 14 days after challenge (Table 1) showed that bursa mean weight in T4 was slightly higher than in T2 and T3.On the other hand, T3 showed higher weight 21 days after infection.Score evaluation for histological lesions together with the evaluation of lesion type and percentage of altered lymphoid follicles (Table 5) showed that the challenge with vvIBDV in group T3 induced less severe alterations than in groups T2 and T4 at 7, 14 and 21 days after challenge, which might indicate better protection.Considering the distribution of lesion scores at 42 days of age (Table 6) and the number of bursas with fibrosis, the sample Moulthrop G603 gave better protection than sample 228E.Such observations obtained after the challenge with vvIBDV in vaccinated birds are very important because birds without possibility of lymphoid repopulation of the bursa would be immunodefficient.In the case of breeders or commercial layers, immunodepression would interfere on the performance of other vaccines.
Broiler strains are known to be more resistant to the mortality induced by IBDV than commercial layers.According to Nielsen et al. (1998), higher susceptibility in layers would be related to the differences between the two types of birds in the major histocompatibility complex (MHC) from allele B. The authors reported that MHC modulates the occurrence of lesions in the thymus and liver and modifications in the sedimentation coefficient of erytrocytes, and do not interfere on the induction of bursa morbid alterations in birds infected with IBDV.The occurrence of 100% mortality in SPF birds inoculated with vvIBDV 2050/97 might be attibuted not only to the fact that they were not immunized, but also because they are genetically similar to commercial layer strains.Therefore, the absence of mortality in the group that was not vaccinated and challenged with vvIBDV (T2) might be attributed to the influence of the MHC preventing severe injury of thymus, liver and erythrocytes, although permanent atrophy of the bursa of Fabricius was seen (scores 5.66 at 42 days, Table 5).According to Van Der Berg (2000), the reason for mortality in chickens infected with vvIBDV would be related to the excessive release of cytokines and fever induction, independent of the presence of intense injury in the bursa of Fabricius.Thus, in broilers, the main benefit of vaccination against vvIBDV would be to control the occurrence of irreversible atrophy of the bursa of Fabricius, and the consequent immunodefficiency caused by very virulent virus infection.
Vaccines are useful in preventing Gumboro disease because they might induce humoral and cellular immune responses.For example, Maas et al. (2001) showed that maternal antibodies present in White Leghorn chicks originated from breeders immunized with distinct vaccinal virus resulted in different incidence of mortality and bursal lesions after challenge with standard type virus, 52/70, and with a very virulent virus type, D6948.Histology performed in surviving birds 10 days after challenge showed that there was a significant difference in the number of convalescent birds with severe or intermediate lesions or absence of lesions in the bursa of Fabricius in the groups of birds with maternal antibodies generated by two distinct vaccines.In the present study, microscopic alterations in the bursa were quantified in scores, based on evaluation of typical lesions caused by IBD in the lymphoid follicles.The method used to score follicular changes evidenced that relative protection response of the bursa of Fabricius after challenging with vvIBDV was better in birds given strong vaccines.It was also possible to observe that, no matter macroscopic alterations were present after vaccination at 14 days of age in group T3, 100% of the birds vaccinated with the sample Moulthrop G603 (Table 6) had bursas histopathologically more preserved at 42 days of age than the birds vaccinated with 228E.Such observation permitted to conclude that the strong vaccine strain might induce better protection to the bursa, independent of causing more severe bursal injury.

CONCLUSION
The findings of this study showed that the strong vaccine strain gave better protection to the bursa, irrespective of the damage in the bursa tissue.

Table 1
Mean bursa weight (g±SD)in vaccinated and nonvaccinated broilers challenged with Gumboro Disease virus.

Table 2
Body weight (g±SD) of vaccinated and non-vaccinated birds challenged or not with virulent virus of Gumboro Disease.

Table 3
Mean bursa weight:body weight ratio of the experimental groups expressed in percentage (x10) ± SD at different ages.

Table 4
Titers of neutralizing antibodies against the virus of Gumboro Disease in broilers of different ages and experimental groups.

Table 5
Quantitative histological analysis of bursa of Fabricius expressed in lesion scores.

Table 7
Neutralizing antibody distribution before and after vaccination (14 and 21 days of age).