ORIGINAL ARTICLE

Comparative susceptibility of chickens, turkeysand ducks to infectious bursal disease virususing immunohistochemistry

O. A. Oladele & D. F. Adene & T. U. Obi & H. O. Nottidge

Accepted: 3 July 2008 /Published online: 29 July 2008# Springer Science + Business Media B.V. 2008

Abstract Twenty chicks, 12 turkey poults and 10 ducklings, all 5 weeks old were infectedwith 2×103.5 chick LD50 IBD virus to determine the course of the virus in the 3 poultryspecies. Uninfected control birds were kept separately. Two infected and 2 control birds/species were euthanized at time intervals between 3 and 168 hours post infection (pi).Sections of thymus, bursa of Fabricius, spleen, liver, kidney, proventriculus and ceacaltonsil were stained for the detection of IBD virus antigen using immunoperoxidasetechnique. IBD virus antigen positive cells stained reddish-brown and the amount of suchcells in tissue sections were noted and scored. Stained cells were present in all organsexamined for up to 168 hours pi in the 3 poultry species except ceacal tonsils of ducks at 72and 120 hours pi. Antigen score was highest in chickens and least in ducks as reflected byaverage of total scores/sampling time of 12, 10.8 and 8 in chickens, turkeys and ducksrespectively. Total antigen score/sampling time in infected chickens peaked twice; 24/48and 144 hours pi, whereas such bi-phasic peaks were absent in turkeys and ducks. Range oftotal antigen score at different sampling times was 7–17.5 in chickens, 10–13 in turkeys and7–10 in ducks indicative of marked viral replication in chickens. Presence of IBD viralantigen in organs of all 3 poultry species is indicative of infections. The innate ability ofturkeys and ducks to prevent appreciable replication of IBD virus after infection requiresfurther investigation.

Keywords Infectious bursal disease . Differential susceptibility . Immunohistochemistry .

Chicken . Turkey . Duck

AbbreviationsIBD Infectious bursal diseaseIBDV Infectious bursal disease virusLD 50 Lethal dose 50DAB-C 3,3’ diaminobenzidine

Vet Res Commun (2009) 33:111–121DOI 10.1007/s11259-008-9078-2

O. A. Oladele (*) :D. F. Adene : T. U. Obi : H. O. NottidgeDepartment of Veterinary Medicine, University of Ibadan, Ibadan, Nigeriae-mail: lade_ed@yahoo.co.uk

Introduction

Infectious bursal disease is an immunosuppressive viral disease of chickens sometimescausing high morbidity and mortality. Clinical disease is usually not observed in otherpoultry species like turkeys and ducks in spite of experimental and natural exposures, butIBD virus antibodies have been detected in turkeys. Natural IBD infections in turkeys(serotype 2) evident by viral isolation (McNulty et al. 1979; McFerran et al. 1980; Reddyand Silim 1991) and detection of antibody (Barnes et al. 1982; Sander 1995) have beenreported. Experimental infections of turkeys with serotype 1 IBD virus have yieldedsubclinical effects with production of neutralizing antibodies (Giambrone et al. 1978; Weissand Kaufer-Weiss 1994). In addition, Giambrone et al. (1978) detected microscopic lesionsand IBD virus antigen in bursa of Fabricius of turkeys and were able to re-isolate the virusafter 5 serial passages.

Reports on natural and experimental infections of ducks and duck embryos by variousworkers have produced inconsistent results. Although Karunakaran et al. (1992) havereported isolating IBD virus of unspecified serotype from bursae of Fabricius of 5 to16 days old ducklings recording high mortality while Karadas and Metin (1994) reportedimpaired development in 13-day old Perkin duck embryos experimentally infected withIBD virus serotype 1, Okoye et al. (1990) on the contrary, observed no clinical, pathologicand immunologic response in 6-weeks old Nigerian ducks. Also, Eddy (1990) in anexperimental IBD virus infection in 3-weeks old ducklings, reported antibody responses toboth serotypes 1 and 2. A serosurvey also reported by Eddy (1990), revealed that 6 out of14 duck flocks sampled for IBD virus (serotype 2) antibody were positive and that maternalantibody was detected in sera of ducklings from 3 out of 6 flocks sampled in anotherinvestigation.

It is therefore evident that clinical IBD is yet to be established in either turkey or duck.Proper understanding of the course of the virus infection in these 2 poultry species,compared with the chicken host is therefore important. In this regard, immunohistochem-istry has been found useful in the rapid and accurate diagnosis of IBD (Cho et al. 1987;Cruz-Coy et al. 1993; Patin-Jackwood and Brown 2003; Liu and Vakharia 2004), as well asin pathogenicity studies (Nunoya et al. 1992; Tanimura et al. 1995). Muller et al. (1979)used immunohistochemistry to determine the sequence of appearance of IBD viral antigenwithin various organs (i.e. pathogenesis) in chicken. Tanimura et al.( 1995) relatedpathogenicity of IBDV with viral antigen distribution using immunohistochemistry.

This study was therefore designed to investigate and compare the chronologicaldistribution of IBD virus antigen in tissues of chicken, turkey and duck against thebackground of their susceptibility differential, using immunoperoxidase technique.

Materials and methods

Experimental birds Twenty cockrels, 12 turkey poults and 10 ducklings (all 5 weeks ofage), were infected with 2×103.5LD50 IBD virus (serotype 1) in 80ul PBS. The virusinoculum used was derived from bursal homogenate (clarified by centrifugation) obtainedfrom an IBD outbreak with a flock mortality rate of 42%. The IBD virus isolate was titratedby the chick lethal dose method as described by Van den Berg et al. (1991) using the Reedand Muench formula. Uninfected control birds of the same number were kept separately.Two infected and 2 uninfected birds per species were euthanased in chloroform chambers at

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time intervals between 3 and 168 hours pi. Sampling for turkeys and ducks commenced at 6and 12 hours pi respectively.

Production of rabbit IBD hyperimmune serum Two adult rabbits (4 months old) wereinoculated with IBD bursal homogenate supernate diluted to 50% with complete Freund’sadjuvant (CFA). Rabbit A was injected with 0.5 ml of the diluted inoculum (equivalent to6.25×104.5LD50) intramuscularly in the thigh muscle while Rabbit B was injected withsame volume of inoculum intradermally into the foot-pad. Two booster doses wereadministered at two weeks intervals. Both rabbits tested strongly positive (titer 6 log2) forprecipitating IBD virus antibody by agar gel precipitation test, 6 weeks after the firstinoculation. They were terminally bled for IBD virus hyperimmune serum which wasstored at −200C.

Tissue harvesting and preparation for immunoperoxidase staining Thymus, bursa ofFabricius, spleen, ceacal tonsils, liver, proventriculus and kidney were harvested fromeuthanased birds and fixed in Bouin’s solution for 6 hours. The tissues were latertransferred through several changes of 70% ethanol to completely remove the Bouin’ssolution and embedded in low-melting point (56oC) paraffin wax. Sections (3 – 4 μm inthickness) were cut and mounted on microscope slides that were pre-coated with 2%gelatin. The mounts were placed on hotplate for 5 minutes and in oven at 60oC for 10minutes to ensure tissues were well adhered and properly dried. The tissue mounts weredeparaffinized in 2 changes of xylene (3 minutes each) and hydrated serially throughabsolute ethanol, 80%, 40%, 20% ethanol (2 changes each) and finally in water. Afterrinsing for 5 minutes in distilled water and 10 minutes in wash buffer, tissue endogenousperoxidase activity was blocked with either KPL’s (Kirkegard & Perry Laboratories)blocking solution for 10 minutes or 0.3% H2O2 in absolute methanol for 30 minutes. Thesections were then washed in distilled water and transferred into wash buffer for 5 minutes.

Immunoperoxidase staining HistoMark® Biotin Streptavidin-HRP System and HistoMark®Orange (Kirkegard & Perry Laboratories, Gaithersburg, MD, USA) were used forimmunoperoxidase staining. The tissue sections were covered, first with normal goatserum in a humidity chamber for 15 minutes and then with 1:1600 dilution of rabbit IBDvirus hyperimmune serum (determined after standardization) for 30 minutes. Primaryantibody was rinsed off and sections were covered with biotinylated goat anti-rabbit for 30minutes at room temperature, rinsed in wash buffer and treated with streptavidin peroxidasefor another 30 minutes at room temperature. Washing of sections was again done in washbuffer, treated with substrate (DAB-C) for 5 minutes and rinsed with distilled water.

Counter staining was done in contrast green for 3 minutes after which sections were rinsedin 2-propanol, air-dried and mounted with cover-slip in Depex. Stained sections were finallyobserved and scored based on the amount of antigen containing cells, under the lightmicroscope. Experimental controls consisted of tissue sections from infected birds stainedwithout anti-IBDV serum i.e. primary antibody and tissue sections from uninfected controls.

The presence of antigen-containing cells was scored on a scale of 0 to 3, with 0 for lessthan 5 stained cells per x40 objective lens of the light microscope, 1 for 5 to 50 stainedcells, 2 for 50 to 150 cells and 3 for over 150 stained cells. Three sites in each tissue sectionwere observed under the microscope and scored. The average score was regarded as thescore for the organ. The scores of all tissue sections (organs) for each species were thensummed up for each sampling time.

Vet Res Commun (2009) 33:111–121 113

Results

Presence of bound and stained IBD virus antigen was observed as red to brownish, fine orcoarse granules or globules in cytoplasm of infected cells, while nuclei stained bluish.Positive reactions (stained cells) were absent in tissue sections from uninfected controls(Fig. 1) and also in control tissue sections from infected birds stained without rabbit anti-IBDV serum i.e. primary antibody (Fig. 2). The summary of scoring of tissue sections forchickens, turkeys and ducks are presented in Tables 1, 2 and 3 respectively. Figure 3represents the total antigens scores for infected chickens, turkeys and ducks at differentsampling times.

Stained IBD virus antigen cells were present in all seven organs examined (i.e. bursa ofFabricius, thymus, spleen, ceacal tonsil, proventriculus, liver and kidney) as from 3 hours to168 hours pi in chickens and 6 hours to 168 hours pi in turkeys, which were the lengths ofthe observation periods. In ducks however, antigen-bearing cells were absent in ceacal

Fig. 1 Liver of uninfected chick(a) stained antigen absent. Com-pare (b), liver of infected chickshowing stained antigen in tissue(x100). Immunoperoxidase stain-ing with contrast green counterstain

Fig. 2 Liver of infected chicken48 hours pi. (a) Primary antibodywas omitted during staining;stained antigen absent. Compare(b), liver of infected chicken 48hours pi showing stained antigenin tissue (×100). Immunoperox-idase staining with contrast greencounter stain

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tonsil at 72 hours and 120 hours pi. In chickens, antigen was detected from 3 hours pi whena total antigen score of 7 was recorded from all tissues. The scores totaled 11.5 at 6 hours piand peaked with 17.5/17 at 24/48 hours pi. The scores declined to 9 at 96 hours pi, butattained a second peak of 13.5 at 144 hours.

In turkeys, antigen stained cells were detected and scored 13 at 6 hours pi. The scoresdeclined slightly and stabilized between 10 and 11 till 168 hours pi. In ducks, stained cellswere detected in all tissues and scored 10 at 12 hours pi. This also declined to stabilize atabout 7.5 up to 168 hours pi.

The bursa of Fabricius of chickens showed IBD virus antigen in cortical tissues by 3–6hours pi, increased interfollicular spaces due to oedema were observed by 12 hours pi whileantigen bearing cells were present in the medulla by 48 hours pi. Antigen positiveinflammatory cells, which were mostly macrophages, were observed in interfollicularspaces by 96 hours pi (Fig. 4). Some epithelial cells were also positively stained at thisstage. Fibroplasia and cystic follicles were observed from 72 hours pi and 120 hours pirespectively and up till 168 hours pi (Fig. 5). In bursae of Fabricius of turkeys and duckshowever, antigen was detected only in lymphoid cells and few macrophages dispersed inthe cortex and medulla of bursal follicles from 6 hours pi in turkeys and 12 hours pi inducks up till 168 hours pi (Figs. 6 and 7). The accompanying histopathological changes

Table 1 Antigen scores from sections of various organs of IBD virus infected chickens

Sampling timepi

Bursa ofFabricius

Thymus Spleen CeacalTonsil

Proven-triculus

Liver Kidney TotalScore

3 hrs 1 1 1 1 1 1 1 76 hrs 1.5 2 2 1.5 2 1 1.5 11.512 hrs 3 2.5 1 2 2 1 1.5 1324 hrs 3 3 3 2.5 2 2 2 17.548 hrs 3 3 2.5 3 3 1 1.5 1772 hrs 2 2.5 2 2 2 1.5 1.5 13.596 hrs 1.5 1.5 1 1 1.5 1.5 1 9120 hrs 1 1 1.5 1 1 1 1 7.5144 hrs 1.5 2.5 2.5 1 1.5 2 2.5 13.5168 hrs 1 2 1.5 1.5 2 1 1.5 10.5Total score/organ

18.5 21 18 16.5 18 13 15

Table 2 Antigen scores from sections of various organs of IBD virus infected turkeys

Sampling timepi

Bursa ofFabricius

Thymus Spleen CeacalTonsil

Preven-triculus

Liver Kidney Totalscore

6 hrs 2 2 2 2.5 1.5 1 2 1312 hrs 1.5 2 2 1.5 1 1 1.5 10.524 hrs 2 2 2 1 1 1 1.5 10.572 hrs 3 2 1 1 1 1 1 10120 hrs 2 2 1 1 1 1 2 10168 hrs 2 2 2 1 1 1 2 11Total score/organ

12.5 12 10 8 6.5 6 10

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namely; oedema, fibroplasia and cyst formation observed in the bursae of Fabricius ofchickens were absent in turkeys and ducks.

Stained antigen was also observed in lymphoid cells of thymus, spleen, and ceacaltonsil; tubular epithelial cells and glomerulli in kidney (Fig. 8); glandular and mucosalepithelial cells in proventriculus, as well as Kupfer cells in the liver (Fig. 9) of the 3 species.The tissues in turkeys and ducks however, showed fewer stained cells by 168 hours pi thanat onset of sampling compared with chicken tissues. Ceacal tonsils of chickens werehyperplastic by 6 hours pi and it became marked by 48 hours pi.

Discussion

In this study the amount of positively stained cells was highest in chickens and least inducks as reflected by average of total scores/sampling time of 12.0, 10.8 and 8.0 inchickens, turkeys and ducks respectively. All tissue sections from chickens showedpresence of viral antigen from 3 hours to 168 hours pi. In a similar study by Muller et al.(1979), IBD virus antigen was detected 4 hours pi in macrophages and lymphoid cells of

Table 3 Antigen scores from sections of various organs of IBD virus infected ducks

Sampling timepi

Bursa ofFabricius

Thymus Spleen CeacalTonsil

Preven-triculus

Liver Kidney Totalscore

12 hrs 2 1 2 1 2 1 1 1024 hrs 1 1 2.5 0.5 1 1 1 872 hrs 1 1 2 0 1.5 1 1 7.5120 hrs 1 1 2 0 1 1 1 7168 hrs 1 1 1 1 1 1.5 1 7.5Total score/organ

6 5 9.5 2.5 6.5 5.5 5

0

2

4

6

8

10

12

14

16

18

20

3 6 12 24 48 72 96 120 144 168

Hours post-infection

To

tal A

nti

gen

Sco

res

Chickens

Turkeys

Ducks

Fig. 3 Total antigen scores/sam-pling time in tissues of IBD virusinfected chickens, turkeys andducks

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chicken ceacum. In another study by Tanimura et al.( 1994) on bursa of Fabricius, ceacaltonsil, thymus, spleen and bone marrow of chicken, IBD virus antigen was not detected intissue sections of these organs prior to 6 hours pi, but was detected for up to 10 days pi.Therefore, the detection of antigen 3 hours pi in this study provides new information on theearliest detection of IBD virus antigen by immunohistochemistry.

The antigen detection made at 3 hours pi which was simultaneous in all 7 organssampled compared to only the ceacum in the study by Muller et al.( 1979) could be due todifferences in the virulence of the strains of IBD virus used in the two studies. Earlierstudies (Nunoya et al. 1992; Tanimura et al. 1994; Lui and Vakharia 2004) have shown thatIBD virus strains differ in tissue tropism which had been observed to be dependent on

Fig. 5 Bursa of Fabricius ofinfected chicken, 120 hours pishowing fibroplasia (a) and cystformation (b) (×100). Immuno-peroxidase staining with contrastgreen counter stain

Fig. 4 Bursa of Fabricius ofinfected chicken, 96 hours pishowing stained inflammatorycells and interfollicular spaces.(a) Immunoperoxidase stainingwith contrast green counter stain

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virulence. Also, while the conjuctival route was used in this study, Muller et al. (1979) usedthe oral route. Expectedly, from the conjuctiva, the virus gets into the bloodstream and it isdistributed to all organs almost simultaneously. However with the oral route, the virusmoves through the length of the gastrointestinal tract, gets into the gut-associated lymphoid

Fig. 7 Bursa of Fabricius ofinfected duck, 168 hours pishowing fewer antigen bearingcells. Immunoperoxidase stainingwith contrast green counter stain

Fig. 6 Bursa of Fabricius of infected turkey, 24 hours pi showing stained lymphoid cells and macrophages(a). Immunoperoxidase staining with contrast green counter stain

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tissues before moving into the bloodstream for the first viraemia described by Weiss andKaufer-Weiss (1994).

In the bursa of Fabricius of IBD virus infected chickens, antigen was detected incytoplasm of lymphocytes, epithelial cells and inflammatory cells (mostly macrophages), aspreviously reported by Nunoya et al. (1992) and Tanimura et al. (1994). Jonsson andEngstrom (1986) however, observed antigen only in bursal lymphocytes. Histopathologicalfindings such as cellular infiltration, fibroplasia and formation of cystic follicles in thebursae of Fabricius of chickens are indications of bursal damage and the full susceptibilityof chickens in contrast to turkeys and ducks. The detection of antigen in tissues of all 3species indicates that they all became infected with the viral inoculum.

Fig. 9 Liver of infected turkey, 6hours pi showing stained antigenin Kupfer cells (×400). Immuno-peroxidase staining with contrastgreen counter stain

Fig. 8 Kidney of infected turkey,6 hours pi showing stained anti-gen in tubular epithelial cells (a)and glomerulli (b). Immunoper-oxidase staining with contrastgreen counter stain

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Two phases of increased concentration of IBD virus positive cells were observed inchickens; the first was between 6 hours and 48 hours pi and the second was between 144and 168 hours pi. On the contrary, high concentrations of positive cells were observed inturkeys and ducks only at the onset of sampling (6 and 12 hours pi, respectively) withminimal decrease throughout the period of the experiment. This study however, did notinvestigate tissue antigen concentration prior to 6 and 12 hours in turkeys and ducksrespectively. Kaufer and Weiss (1980) observed the highest tissue antigen concentration at48 hours pi in chickens, as was the case in this study.

With the presence of detectable antigen concentrations at the onset of sampling, it maybe inferred that the early events of IBD virus infection did occur in turkeys and ducks.While marked differences in tissue antigen concentration per sampling time were observedin chicks as reflected by a range of 7.0–17.5 in total antigen score, little differences wereobserved in turkeys and ducks with ranges of 10–13 and 7–10 per sampling time,respectively. It could therefore be inferred that, out of the 3 poultry species studied, viralreplication was marked in chickens but minimal in turkeys and ducks, in which the decisivesecond viraemic phase described by Weiss and Kaufer-Weiss (1994) appeared to have beenaborted.

As Kaufer and Weiss (1980) suggested, probably there was not enough amount of highlysusceptible cells in the bursa of Fabricius of turkeys and ducks. Thus, viral multiplicationwas moderate and was kept in check by the host defense mechanism. Nakai and Hirai(1981) reported that immunoglobulin M (IgM)-bearing B cells are the specific targets forIBD virus.

This study has demonstrated bursal damage and two phases of increased tissue antigenconcentration in chickens in agreement with bi-phasic viraemia described by Weiss andKaufer-Weiss (1994). On the contrary, there was no bursal damage but there was an earlyphase, high tissue antigen concentration in turkeys and ducks, which decreased gradually.Thus, these two species are susceptible to IBD virus infection, but not to the secondviraemia and clinical disease. Thus, emphasizing the full susceptibility of chickens andtolerance of turkeys and ducks. It appears that the chicken host has a facilitating inherent“factor” which permits maximal replication of the IBD virus compared with turkey andduck, thus, the virus is able to run its full course resulting in clinical disease.

Acknowledgements We would like to thank Dr. Femi Obateru for generously donating the immunoper-oxidase kits used for this work and the management of Nucryst Pharmaceutical Company, Wakefield, MA,USA for access to photomicrography facility.

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