Differences in genetic background influence the induction of innate and acquired immune responses in chickens depending on the virulence of the infecting infectious bursal disease virus (IBDV) strain

Veterinary Immunology and Immunopathology 135 (2010) 79–92

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Research paper

Differences in genetic background influence the induction of innate and acquired immune responses in chickens depending on the virulence of the infecting infectious bursal disease virus (IBDV) strain Merve Aricibasi a, Arne Jung a, E. Dan Heller b, Silke Rautenschlein a,* a b

Clinic for Poultry, University of Veterinary Medicine Hannover, Hannover, Germany The Hebrew University, Robert H. Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel

A R T I C L E I N F O

A B S T R A C T

Article history: Received 25 March 2009 Received in revised form 4 November 2009 Accepted 11 November 2009

Previous studies and field observations have suggested that genetic background influences infectious bursal disease virus (IBDV) pathogenesis. However, the influence of the virulence of the infecting IBDV strain and the mechanisms underlying the differences in susceptibility are not known. In the present study IBDV pathogenesis was compared between specificpathogen-free layer-type (LT) chickens, which are the most susceptible chicken for IBDV and have been used as the model for pathogenesis studies, and broiler-type (BT) chickens, which are known to be less susceptible to clinical infectious bursal disease (IBD). The innate and acquired immune responses were investigated after inoculation of an intermediate (i), virulent (v) or very virulent (vv) strain of IBDV. IBDV pathogenesis was comparable among genetic backgrounds after infection with iIBDV. After infection with vIBDV and vvIBDV, LT birds showed severe clinical disease and mortality, higher bursal lesion scores and IBDVantigen load relative to BT birds. Circulating cytokine induction varied significantly in both timing and quantity between LT and BT birds and among virus strains (P < 0.05). Evaluation of different immune cell populations by flow-cytometric analysis in the bursa of Fabricius provided circumstantial evidence of a stronger local T cell response in BT birds vs. LT birds after infection with the virulent strain. On the other hand, LT birds showed a more significant increase in circulating macrophage-derived immune mediators such as total interferon (IFN) and serum nitrite than BT birds on days 2 and 3 post-vIBDV infection (P < 0.05). Stronger stimulation of innate immune reactions especially after vIBDV infection in the early phase may lead to faster and more severe lesion development accompanied by clinical disease and death in LT chickens relative to BT chickens. Interestingly, no significant differences were seen between genetic backgrounds in induction of the IBDV-specific humoral response: timing of IBDV-antibody induction and antibody levels were comparable between BT and LT birds. This study clearly demonstrates a significant influence of chickens’ genetic background on disease outcome. The difference between backgrounds in IBDV susceptibility is further influenced by the virulence of the infecting virus strain. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Infectious bursal disease virus Genetic background Bioactive cytokines

Abbreviations: BrL, brown Leghorn; BF, bursa of Fabricius; BSA, bovine serum albumin; BT, broiler-type chicken; CEF, chicken embryo fibroblasts; CPE, cytopathic effect; ELD, egg lethal dose; FBS, fetal bovine serum; HE, hematoxylin and eosin; IBD, infectious bursal disease; IBDV, infectious bursal disease virus; i, intermediate; Ig, immunoglobulin; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; IM, Irwin Moulthroph; LT, layer-type chicken; MHC, major histocompatibility complex; NO, nitric oxide; OD, optical density; PBS, phosphate-buffered saline; pi, post-inoculation; SPF, specificpathogen-free; VN, virus neutralization; VSV, vesicular stomatitis virus; v, virulent; vv, very virulent. * Corresponding author at: Clinic for Poultry, University of Veterinary Medicine Hannover, Bu¨nteweg 17, 30559 Hannover, Lower Saxony, Germany. Tel.: +49 511 953 8763; fax: +49 511 953 8580. E-mail address: [email protected] (S. Rautenschlein). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.11.005

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1. Introduction Infectious bursal disease virus (IBDV) causes an acute, highly contagious, immunosuppressive disease in chickens (Eterradossi and Saif, 2008). The causative agent belongs to the Birnaviridae family. Different pathotypes of IBDV have been classified in increasing order of virulence as mild, intermediate (i), classical virulent (v) and very virulent (vv). Classical virulent IBDV strains isolated from the United States in the early 1960s, such as the Irwin Moulthroph (IM) strain, induce hemorrhagic lesions accompanied by depletion of B cell follicles and mortality rates of 30–60% in layer-type chickens. In the mid 1990s, ‘‘very virulent’’ strains of IBDV, causing >70% mortality in chickens, emerged in several European and Asian countries. van den Berg (2000) suggested that vvIBDV causes clinical signs similar to those of the classical virulent strains with the same incubation time of 4 days but with an exacerbated acute phase. Chickens are highly susceptible to IBDV between 3 and 6 weeks after hatching. Experiments in which bursectomized chickens survived IBDV infection demonstrated that the bursa of Fabricius (BF) is the main target organ for IBDV. The acute phase usually lasts about 1 week, and peak clinical signs and mortality are recorded between 3 and 4 days after IBDV infection. The most common mode of IBDV infection is via the oral route. The virus is taken up from the gut and transported to other tissues by phagocytic cells, most likely resident macrophages. IBDV infects and destroys actively dividing IgM-bearing B cells in the BF (Rodenberg et al., 1994). Although B cells are the principal targets for IBDV, recent data show that the virus also infects and replicates in macrophages. Infection with IBDV causes the production of proinflammatory mediators and cytokines in macrophages, which peaks during the early phase of active virus replication (Khatri et al., 2005; Palmquist et al., 2006). IBDV induces expression of the following cytokines and cytokine genes: interleukin (IL)12, interferon (IFN)-g, IL-1b, IL-6 and CXCLi2 in bursal cells (Eldaghayes et al., 2006; Rauw et al., 2007), and expression of IL-1b, IL-6, IL-18 and inducible nitric oxide synthase (iNOS) in spleen cells (Palmquist et al., 2006). Nitric oxide (NO), which is produced by activated macrophages, may promote cellular destruction of both virus-infected and virus-free cells (Yeh et al., 2002). T cells, which are not infected by IBDV, may modulate the pathogenesis by limiting viral replication in the BF during the early phase of the disease at 5 days pi, by promoting bursal tissue damage and delaying tissue recovery, possibly through the release of cytokines and their concomitant cytotoxic effects (Rautenschlein et al., 2002a). Almost all these basic studies on IBDV pathogenesis have been done in specific-pathogen-free (SPF) layer-type chickens. Not much is known about the genetic influence on immune cell reactions in other types of chickens, which may show different susceptibility to infectious bursal disease (IBD) compared to SPF-layer-type chickens. It is difficult to compare the influence of genetic background on IBD with different commercially available chicken lines due to the maternal antibody levels, which may vary between birds of different background and parent flock in titer and half life time (de

Wit, 1998). Previous preliminary studies indicated that detectable maternal antibodies above the break-through titer of the infecting intermediate (i) IBDV strain may delay virus replication and the induction of lesions but not the replication rate and severity of lesions in comparison to birds with antibody levels below the break-through levels or without detectable antibodies (Block et al., 2007; Jung, 2006). Some chicken lines with different major histocompatibility complex (MHC) haplotypes have been investigated following IBDV infection. Although no relation between MHC haplotype and resistance to IBDV has been observed to date, major differences have been found between different chicken lines. Bumstead et al. (1993) reported various mortality rates after infection of 11 inbred and partly inbred chicken lines with vvIBDV, being highest in a brown Leghorn line and lowest in some white Leghorn lines. Hassan et al. (2002) also reported major differences in mortality rates among six genetically different chicken lines. A recent study by Ruby et al. (2006) revealed differences in the transcript levels of some inflammatoryand immune cell-related genes during the acute phase of IBD between brown layer and white layer lines known to differ in IBD susceptibility. A notable MHC haplotype effect was observed on the specific antibody response against an inactivated IBDV as measured by ELISA (Juul-Madsen et al., 2006). The goal of this study was to understand the immune mechanisms contributing to the development of clinical disease and mortality. Our objectives were to compare innate and acquired immune reactions in the acute phase of IBDV infection between commercial Ross-type broilers (BT), known to resist clinical IBD under experimental conditions, and highly susceptible SPF-layer-type chickens (LT), which have been used extensively in many IBDV pathogenesis studies. The immune responses were determined after infection of BT and LT chickens with IBDV strains of different virulence – iIBDV, vIBDV, and vvIBDV. The development of clinical disease, macroscopic and microscopic lesions, IBDV-antigen load in the BF, IBDVantibody development, and the induction of circulating cytokines were determined during the acute phase of IBD. Serum samples were tested for serum nitrite and the following cytokines: IL-6, total IFN, IFN-g, and IL-1b, which are upregulated after IBDV infection (Eldaghayes et al., 2006; Rauw et al., 2007). 2. Material and methods 2.1. Virus The classical vIBDV strain IM was inoculated by the eyedrop route (Kim et al., 1999) at 104 egg lethal dose (ELD)50/bird. The vvIBDV strain 89163/7.3 (provided by N. Eterradossi, AFSSA, Ploufragan, France) was also inoculated by the eyedrop route at a dose of 103 ELD50/bird. These infectious doses for vIBDV and vvIBDV were chosen based on preliminary studies to reproduce the clinical disease in SPF LT chickens within 48 h pi. vIBDV and vvIBDV strains were propagated in 3-week old specific-pathogen-free (SPF) chickens (Rautenschlein et al., 2005). At 5 days after

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virus-inoculation, bursae from infected birds were harvested, homogenized, and titrated in embryonated chicken eggs as previously described (Kim et al., 1999; Tanimura and Sharma, 1997). In addition, a commercially available iIBDV vaccine, which was derived from the attenuated virulent IBDV strain LC75 and had been utilized in previous studies (Block et al., 2007; Jung, 2006), was inoculated orally following the manufacturer’s recommendations at a dose of 103 egg infectious dose (EID)50/bird. As previous laboratory studies indicated, this vaccine induces bursa lesions around 3 days pi in SPF LT and antibody-free broiler chickens (Jung, 2006). Vesicular stomatitis virus (VSV; kindly provided by Dr. G. Zimmer, Institute for Virology, University of Veterinary Medicine Hannover, Germany) was propagated and titrated in chicken embryo fibroblasts (CEFs) and used in the IFN bioassay at a dose of 4  104 TCID (tissue culture infectious dose)50/well. Virus titers were calculated by the method of Reed and Muench (1938). The intermediate IBDV strain Bursine 2 (Kim et al., 2000), which is not identical to the commercial IBDV vaccine strain, was propagated and titrated in CEFs and used in the virus neutralization (VN) test. All IBDV strains used in these experiments were free of detectable chicken anemia virus DNA as investigated by PCR (Sommer and Cardona, 2003). 2.2. Hematoxylin and eosin (HE) staining For the detection of histopathological lesions, the BF and spleen were collected, fixed in 10% phosphatebuffered formalin and stained with HE. Lesions were observed microscopically. Bursa lesion scores were determined and compared between groups (Kim et al., 1999; Sharma et al., 1989) as follows: score 1 = 1–25%, score 2 = 26–50%, score 3 = 51–75% and score 4 = 76–100% of follicles showing cellular depletion. 2.3. Detection of IBDV by immunohistochemistry BF was collected, fixed in 10% phosphate-buffered formalin, sectioned and processed for immunohistochemical staining using the Universal Vectorstain Kit (Vector Laboratories, Burlingame, CA, USA). A polyclonal rabbit anti-IBDV serum was used for IBDV-antigen detection (Rautenschlein et al., 2007). The group means of the number of IBDV-infected cells per field at a magnification of 400 were calculated based on the average number of positive cells in five microscopic fields for each bird/group. 2.4. Flow-cytometric analysis Single-cell suspensions were prepared from the BF and spleens as described previously (Kim et al., 2000). Lymphocytes were separated from red blood cells by a discontinuous Ficoll-Hypaque density gradient (Kim et al., 2000). The cells were double-stained with mouse antichicken-CD4 antibodies conjugated to phycoerythrin and mouse anti-chicken-CD8a antibodies conjugated to fluorescein (Southern Biotech, Birmingham, AL, USA). Unlabeled KUL-01 and BU-1 antibodies were used (Southern Biotech) to stain macrophage-like cells and B cells. In a

81

second reaction step, these cells were detected by goat anti-mouse IgG-FITC-labeled antibodies (Sigma, Taufkirchen, Germany). Leukocytes (1  106) isolated from spleen and bursa were suspended in 30 ml of antibody solution, incubated for 30 min on ice, washed twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), fixed with paraformaldehyde solution (3% in PBS), and stored at 7 8C until further analysis. Specific staining of lymphocyte fractions was determined by the Beckman Coulter Epics XL1 flowcytometer (Beckman Coulter GmbH, Krefeld). Gates were designated to analyze total live leukocytes in 5000 cells per sample. Data are expressed as group means, and presented in percentage of total gated leukocytes or lymphocytes. 2.5. Serum nitrite test Serum nitrite levels were determined following Schmidt et al. (1989). Briefly, serum nitrate was first reduced enzymatically to nitrite with nitrate reductase (Sigma), followed by addition of an equal amount of Griess reagent (a 1:1 mixture of 1% sulfanylamide in 2.5% phosphoric acid and 0.1% naphthylenediamine dihydrochloride in deionized water). The amount of nitrite was determined by measuring the OD570 with an automated microplate reader. Sodium nitrite was used as a standard to determine serum nitrite concentrations. 2.6. Enzyme-linked immunosorbent assay (ELISA) and VN test Sera were tested for IBDV-specific antibodies by a commercial IBDV-ELISA kit (Synbiotics IBD ProFLOK1, Synbiotics Corporation, San Diego, CA, USA), which detects anti-IBDV-antibodies of the IgG-type. Based on the OD, log10-titers were calculated and are expressed as group mean titers. Serum samples were also tested for IBDV-specific neutralizing antibodies (Winterfield et al., 1972). VN titers were calculated as the reciprocal of the highest serum dilution resulting in 100% neutralization. Geometric mean titers (log2) per group and day are presented. 2.7. IFNg ELISA A commercially available ELISA system was used for the detection of chicken IFNg (CytoSetTM, Biosource, Camarillo, CA, USA). The amount of IFNg was determined by measuring the OD450 with an automated microplate reader. According to the standard curve provided in the kit, an optical density (OD) of 1 corresponds to 1.26 ng/ml chicken IFNg. 2.8. IFN bioassay The IFN bioassay was performed to detect total IFN (type I and type II) (Karaca et al., 1996). CEFs were plated overnight at a cell density of 7.5  105 cells/ml in Leibovitz’s L-15 and McCoy’s 5A media (1:1) supplemented with antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin) and 1% fetal bovine serum (FBS). CEFs were

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incubated with serially diluted serum samples at 37 8C for 16–24 h. Test-culture fluids were replaced with medium containing 4  104 TCID50/well of VSV, and incubated for 72 h. Virus-induced cytopathic effects were determined after staining the cells with crystal violet. The IFN activity of the test samples is expressed in U/ml. One unit is defined as the highest dilution of sample that caused 100% protection against VSV-induced cytopathic effects. 2.9. IL-6 assay The murine hybridoma cell line 7TD1 (kindly provided by Bernd Kaspers, Institute of Physiology, LMU Mu¨nchen) was used to detect chicken IL-6 in serum samples of infected and virus-free chickens as previously described (Lynagh et al., 2000). Diluted serum samples were tested in duplicates. Proliferation responses were calculated as the mean of duplicate wells. The amount of IL-6 was determined by measuring the OD450 with an automated microplate reader. According to the standard curve with recombinant chicken IL-6 (kindly provided by B. Kaspers, LMU Mu¨nchen, Germany) an optical density (OD) of 1 corresponds to 1.39 ng/ml IL-6. 2.10. IL-1b bioassay Chicken IL-1b was detected with a reporter assay as previously described (Gyorfy et al., 2003). The data are presented in relative light units of luminescence. 2.11. Chickens Embryonated eggs from SPF chickens (VALO1, Lohmann LSL-LITE) were purchased from Lohmann Tierzucht (Cuxhaven, Germany) and hatched at the Clinic for Poultry. One-day old commercial Ross-type broilers were provided by the BWE Hatchery Weser-Ems GmbH and Co. (VisbekRechterfeld, Germany). The chicks were transferred to the isolation facility at day old and no vaccination was administered. The BT birds were tested for maternally derived IBDV-antibodies at 10 days of age by a commercial IBDV-ELISA (Synbiotics IBD ProFLOK1, Synbiotics Corporation, San Diego, CA, USA). The virus antibody breakthrough levels were determined based on the Deventer formula (de Wit, 1998). Both LT and BT chickens were mixed sex. Chickens were reared in pressurized isolation units (Montaim Van Stratum, Kronsberg, Netherlands) for the duration of the study following German animal welfare guidelines. The birds were given food and water ad libitum. Chickens were distributed randomly based on SRS (simple random sample) to the different experimental groups and were housed in separate isolation units after infection. 2.12. Experimental procedure LT and BT chickens with no or low IBDV-maternal antibody levels, which were below the break-through titers of the used viruses (Eterradossi and Saif, 2008), were inoculated at 3 weeks of age with different IBDV strains or virus-free diluent. In Experiment 1 and 2, 61 LT and 60 BT birds each, and in Experiment 3 92 SPF LT and 65 BT

chicken were randomly divided into two groups of noninoculated control and IBDV-inoculated experimental groups. Groups of 36 (Exp. 1, 2) or 67 (Exp. 3) SPF LT and 35 (Exp. 1, 2) and 40 (Exp. 3) BT birds were IBDVinfected. They received an iIBDV strain (103 EID50/bird, Experiment 1) orally, as recommended by the manufacturer. In Experiments 2 and 3 chickens received a classical vIBDV (104 ELD50/bird) or vvIBDV (103 ELD50/bird) by the eyedrop route, respectively. Route of inoculation was based on previous studies (Rautenschlein et al., 2005, 2007). Chickens were observed daily for clinical signs, morbidity and mortality for up to 7 days post-inoculation (pi). In each experiment, five birds of each virus-free and infected group were randomly chosen based on SRS and sacrificed on days 1, 2, 3, 5 and 7 pi. Pathological lesions, and bursa- and spleen-to-body weight ratios were determined, and the following samples were collected for further investigation: serum for the detection of IBDVantibodies and circulating cytokines, spleen and BF samples for histology, and in Experiments 2 and 3 for flow-cytometric analysis of different immune cell populations. Furthermore, BF was also snap-frozen for immunohistochemical detection of viral antigen at 1, 2, 3, 5 and 7 days pi. Remaining surviving birds in the vIBDV and vvIBDV LT groups were sacrificed humanely at the end of the experiment. 2.13. Statistical analysis Group responses within experiments were analyzed and compared by Student’s t-test. P < 0.05 was considered significantly different from the virus-free control group or between groups with different genetic backgrounds within the same experiment. 3. Results 3.1. Clinical signs and pathological and histopathological changes induced by IBDV strains Clinical signs were monitored daily after IBDV infection of BT and LT chickens in all experiments. BT chickens showed no clinical signs or mortality during the course of the three experiments. Inoculation with iIBDV did not induce clinical signs or mortality in either infected group, independent of genetic background. Inoculation with vIBDV and vvIBDV resulted in 100% morbidity with clinical signs, such as depression, ruffled feathers and hunched posture, within the group of infected LT chickens (Table 1). The severity of clinical signs was comparable between vvIBDV- and vIBDV-inoculated LT chickens. The mortality rate was not evaluated because chickens were continuously removed from the groups for necropsy. One of 36 and 8 of 67 vIBDV- and vvIBDV-inoculated LT chickens were found dead within the first 3 days pi, respectively. None of the virus-free control groups showed clinical signs attributable to the disease. Mild pathological lesions such as marbled spleens and bursal edema were observed at necropsy in iIBDVinoculated LT and BT chickens between 2 and 7 days pi. vIBDV caused severe hemorrhages in pectoral muscles as

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Table 1 Induction of clinical signs, and pathological and histopathological lesions after inoculation of LT and BT chickens with different IBDV strains. Chicken

Virus

Morbidity (%)

LT BT

iIBDV

0 0

LT BT

vIBDV

100 0

LT BT

vvIBDV

100 0

Gross lesion-positive birds/totala on days pi

Group average of the histological bursa lesion score on days pi

1

2

3

5

7

1

2

3

5

7

0/5 0/5

0/5 1/5

1/5 0/5

3/5 5/5

3/5 2/5

0.0 0.0

0.0 0.0

1.0 1.0

3.0 2.0

2.5 3.0

0/5 0/5

5/5 0/5

5/5 2/5

3/5 5/5

3/5 1/5

1.0 0.0

4.0* 1.0

4.0 4.0

4.0 4.0

4.0 4.0

0/5 0/5

4/5 3/5

5/5 3/5

5/5 5/5

5/5 2/5

1.0 1.0

4.0* 1.0

4.0 4.0

4.0 4.0

4.0 4.0

a Gross lesions observed at necropsy included pathological changes observed in spleen and bursa samples such as mottling, gelatination and hemorrhages, respectively. Given as number of positive birds per group. * Significantly different from infected BT chickens in the same experiment (P < 0.05). Virus-negative birds did not show any clinical signs, pathological or histopathological lesions during the experiments.

well as marbled spleens and bursal hemorrhages in LT chickens. BT chickens inoculated with vIBDV also showed swollen bursae, a thin layer of gelatinous bursal edema covering the serous surface and mottling of the spleen, especially on the 3rd and 5th days pi. Inoculation with vvIBDV-induced macroscopic lesions on the spleen and BF that were comparable to the vIBDV-induced lesions (Table 1). The extent of histological bursa lesions differed among the virus-inoculated groups. iIBDV-inoculated chickens had only minor lesions scored between 1 and 3 in both genetic backgrounds. vIBDV-induced severe lesions in LT chickens of score 4 beginning on the 2nd day pi, which lasted throughout the experimental period. vIBDVinfected BT birds showed severe bursal lesions beginning on day 3 pi (Table 1). The differences in lesion scores between vIBDV-inoculated LT and BT chickens were significant (P < 0.05) on day 2 pi. Massive infiltration of heterophiles was observed in 100% of the vIBDV-inoculated birds on days 2 and 3 pi. vvIBDV-induced 100% follicle destruction in the BF of all infected birds 3 days pi. However, LT chickens showed a significantly higher bursal lesion score than BT chickens on day 2 pi (Table 1). Hyperplasia of the white pulp of the spleen was observed in vIBDV- as well as vvIBDV-infected birds of both genetic backgrounds on days 3 and 5 pi (data not shown). No pathological or histopathological lesions were seen in spleens or BF of virus-free control birds.

3.2. Detection of IBDV-antigen in lymphoid tissues None of the virus-free birds was positive for IBDVantigen in the BF at any investigated time point. None of the iIBDV- or vvIBDV-inoculated BT chickens and one out of five vvIBDV-inoculated LT chickens were positive by immunohistochemistry for IBDV-antigen on day 1 pi (Table 2). However, 100% of the vIBDV-inoculated LT birds and 40% of the BT birds were already positive for IBDVantigen on day 1 pi. On day 3 pi, all virus-infected groups showed IBDV-antigen in the bursa. The number of positive cells was significantly higher in the vIBDV-inoculated LT chickens than in the BT birds in the same experiment at 3 days pi (P < 0.05). At 5 and 7 days pi, significant differences in IBDV-antigen load were observed between vvIBDVinoculated LT and BT chickens (P < 0.05). 3.3. IBDV-induced B cell depletion and accumulation of intrabursal T cells No flow-cytometric analysis was performed after infection with the iIBDV strain. Significant bursal B cell depletion was detected after vIBDV infection of BT and LT chickens on day 7 pi (P < 0.001; Fig. 1). Surprisingly, infection with vvIBDV resulted in only a moderate change in the percentage of intrabursal B cells in both type of chickens at all investigated time points (Fig. 1a). Moreover, in the spleen, infection with vIBDV resulted in a reduction

Table 2 Detection of IBDV-antigen by immunohistochemistry following IBDV inoculation of LT and BT chickens. Average number of IBDV-positive cells in the bursa of Fabricius of infected chickens/microscopic field  SD (number of IBDV-positive birds per group) on days pi

Chicken

Virus

LT BT

iIBDV

0.0  0.0 (0/7) 0.0  0.0 (0/7)

4.9  8.3 (2/7) 0.0  0.0 (0/7)

11.0  7.6 (5/6) 30.7  58.9 (3/7)

45.6  45.3 (6/7) 29.5  41.5 (4/6)

47.8  43.3 (6/7) 26.0  34.6 (3/6)

LT BT

vIBDV

21.2  12.2 (5/5) 27.0  7.0 (2/5)

304.8  284.3 (5/5) 13.0  16.0 (3/5)

428.3  100.4* (5/5) 145.0  72.4 (5/5)

225.5  146.3 (5/5) 108.7  28.7 (5/5)

220.1  87.3 (5/5) 93.2  38.2 (5/5)

LT BT

vvIBDV

2.0 (1/5) 0.0  0.0 (0/5)

208.4  193.6 (5/5) 9.0  8.5 (2/5)

136.8  39.4 (5/5) 121.2  29.8 (5/5)

187.8  54.4* (5/5) 65.6  53.6 (5/5)

143.4  36.6* (5/5) 55.2  25.4 (5/5)

1

2

3

5

7

On different days post-IBDV inoculation, birds were sacrificed, and bursa samples investigated by immunohistochemistry for IBDV-antigen. The number of IBDV-positive cells/field at 400 was counted for five fields and the group average calculated. * Significantly different from infected BT chickens in the same experiment (Student’s t-test, P < 0.05).

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Fig. 1. Percentage of B cells per total number of lymphoid cells in the bursa of Fabricius (a) and spleen (b), as measured by flow-cytometric analysis. Presented is the fold change  SD in the infected group relative to the corresponding control group of the same experiment. BT and LT chickens were infected with vIBDV (Experiment 1) or vvIBDV (Experiment 2) at 3 weeks post-hatch. On different days pi, bursae and spleens were collected, and single-cell suspensions were stained with the B cell marker BU-1, and analyzed by flow-cytometry. *Significantly different from the virus-free control group of the same genetic background (P < 0.001), n = 3–5.

of B cells at 7 days pi, while vvIBDV-infected birds showed an increase in the percentage of splenic B cells in BT and LT chickens at 1 and 7 days pi, respectively, in comparison to virus-free birds of the same genetic background (Fig. 1b). No significant differences were observed in the percentage of bursal B cells between virus-free control birds and IBDVinfected ones on days 2, 3 and 5 pi (data not shown). The percentage of CD4+ and CD8+ T cells increased significantly in the BF of vIBDV- as well as vvIBDV-infected chickens of both genetic backgrounds. A significant increase of intrabursal CD4+ and CD8+ T cells was detected particularly on days 3, 5 and 7 (P < 0.05) and days 5 and 7 pi (P < 0.005) post-vIBDV infection of BT chickens, respectively (Fig. 2a and b). LT chickens showed a clear increase in CD4+ T cells in the BF on days 2, 5 and 7 (P < 0.05) and in CD8+ T cells on days 2, 3, and 7 (P < 0.05) post-vIBDV inoculation (Fig. 2a and b). Intrabursal CD4+ and CD8+ T cell accumulation was significant in BT chickens on day 3 after vvIBDV inoculation (P < 0.05), whereas the differences between virus-free and vvIBDVinoculated LT birds were not significant at the other days due to individual variation (Fig. 2a and b). The percentage of splenic CD4+ T cells increased significantly at 2 and 5 days, and on day 5 pi in vIBDV- and vvIBDV-infected LT birds, respectively (Fig. 2c; P < 0.05). Increase of CD4+ T cell percentages in spleen showed no significance in BT birds (Fig. 2c). The percentage of CD8+ spleen cells was significantly higher in vIBDV- and vvIBDV-infected LT birds on days 3 and 5 pi compared to virus-free birds (Fig. 2d; P < 0.05). Slight increases in splenic CD4+ T cells at the experimental days, and decreases in splenic CD8+ T cells at 3 and 5 days pi were detected in vIBDV-infected BT birds.

Although some T cell increase was observed in vvIBDVinfected BT birds on most of these experimental days, the differences were not significant due to high individual variation between animals. 3.4. Detection of circulating cytokines Inoculation of chickens with IBDV strains of different virulence induced systemic release of IFNs and the proinflammatory cytokines IL-6 and IL-1b. Significant differences in total circulating IFN levels were detected by the IFN bioassay on days 2 and 3 pi. The IFN levels differed between genetic backgrounds as well as among infecting viruses. The highest levels were induced on day 3 postvIBDV inoculation in the LT birds (Table 3). Minor differences were seen in IFN-g-levels between infected and non-infected groups, which varied a lot between individual birds of one group and did not correlate with the total IFN values (Table 4). Increased serum IL-1b levels were only detected in birds exposed to vIBDV (Fig. 3b) and vvIBDV (Fig. 3c) but not after iIBDV (Fig. 3a) inoculation, relative to virus-free controls at 5 days pi. None of the differences were statistically significant compared to controls or between the virus-inoculated groups. LT birds showed an average 1.91- and 2.38-fold increase in IL-1b after vIBDV and vvIBDV inoculation, respectively, on day 5 pi relative to virus-free controls (Fig. 3b and c). BT birds showed only a slight, less than 1-fold increase for most of the tested broilers in IL-1b level after inoculation of vIBDV relative to virus-free controls on the experimental days examined (Fig. 3b).

M. Aricibasi et al. / Veterinary Immunology and Immunopathology 135 (2010) 79–92 Fig. 2. Percentage of CD4+ (a and c) and CD8+ (b and d) T cells in the bursa of Fabricius (a and b) and spleen (c and d) of IBDV-infected birds. Presented is the fold change in the infected group relative to the corresponding control group of this experiment. Error bars indicate the SD per group. *Significant difference between virus-free and infected groups of the same experiment (P < 0.05), n = 3–5.

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Table 3 Induction of circulating IFN after inoculation of LT and BT chickens with different IBDV strains. Groups

Group average of total IFN (U/ml) on days pi

Chickens

Virus

2

LT

iIBDV

0 18* 0 0

0 5 0 35

0 35 0 8

0 98* 0 2

0 384* 0 0

0 6 0 0

0 0 0 60

0 5 0 0

0 50 0 18

+ BT + LT

vIBDV +

BT + LT

vvIBDV +

BT +

3

5

The group average of circulating IFNs was calculated on the basis of the IFN units determined in the IFN bioassay. * Significantly different from infected BT chickens in the same experiment (P < 0.05), n = 3–5.

A significant 4.9-fold increase in circulating IL-6 levels was detected in LT birds on day 2 pi with iIBDV relative to virus-free controls based on the calculated IL-6 concentration in the serum (P < 0.05). BT birds showed only a slight, in average 1.7-fold increase in IL-6 levels after iIBDV inoculation. After inoculation with the other IBDV strains infected LT birds showed a slight but non-significant increase in IL-6 levels while vIBDV- and vvIBDV-inoculated BT birds showed a slight decrease relative to virus-free controls at 2 days pi (Table 4). Overall, the IL-6 background levels were high as it has been shown previously also by Rauw et al. (2007). 3.5. Stimulation of macrophages after IBDV inoculation No flow-cytometric analysis was conducted in Experiment 1. Infection with vIBDV and vvIBDV led to an increase in the number of macrophage-like cells in the BF of LT

chickens beginning at day 1 pi. At day 1 pi the average percentage of intrabursal KUL-01+ cells was increased 1.5and 1.6-fold in vIBDV- and vvIBDV-inoculated LT birds, respectively, as compared to virus-free birds. Significant changes in macrophage-like cells in the BF of vIBDVinoculated LT- and BT birds were observed at 5 and 7 days pi (P < 0.05). vvIBDV-inoculated BT birds showed an increase in the percentage of intrabursal KUL-01+ cells at 5 days pi, whereas vvIBDV-inoculated LT birds showed an increase in the macrophage-like cells population only at 1 day pi, but these differences are not significant (Fig. 4a). Changes in the percentage of macrophages were also observed in spleens of vIBDV- and vvIBDV-infected birds. While vIBDV-infected BT birds showed in average a significant 3.6-fold decrease in the relative number of splenic macrophages at 1 day pi (P < 0.05), vIBDV-infected LT chickens had increased macrophage numbers of 2–3fold vs. virus-free controls at days 1, 2, and 5 pi, but these differences were not significant due to high individual variation (Fig. 4b). The numbers of splenic macrophages in vvIBDV-inoculated LT and BT birds increased 2–3-fold at days 5 and 7 pi, respectively, as compared to virus-free controls, but these differences were only significant for vvIBDV-inoculated BT birds (P < 0.05; Fig. 4b). Circulating serum nitrite, an indicator of macrophage stimulation, was detected mainly in vIBDV-inoculated LT birds at 2, 3 (Fig. 5) and 4 days pi (data not shown). In all other groups, this increase was less clear and not significant. vIBDV-inoculated BT chickens showed increased circulating nitrate levels on days 2 and 3 pi, but due to high individual variation, these values were not significantly different from those in virus-free control BT birds. 3.6. Induction of serum IBDV-antibodies The VN and ELISA IBDV-antibody titers were not statistically different (P > 0.05) between genetic backgrounds 7 days pi with the different IBDV strains (Table 5). BT birds still had low residual maternal antibody levels between 2 and 5 days pi. These antibody levels were

Table 4 Induction of circulating IL-6 and IFNg after inoculation of LT and BT chickens with different IBDV strains. Chicken

LT

Virus

iIBDV +

BT + LT

vIBDV +

BT + LT

vvIBDV +

BT + a b *

Group average OD value  SD for IL-6a at days pi

Group average OD value  SD for IFN-gb at days pi

2

3

5

7

2

3

5

7

0.8  0.2 2.0  0.6* 1.0  0.6 1.3  0.5

0.4  0.0 0.4  0.0 0.4  0.0 0.4  0.0

0.4  0.1 0.4  0.0 0.4  0.0 0.4  0.1

0.4  0.1 0.4  0.0 0.4  0.1 0.4  0.1

0.052  0.004 0.055  0.006 0.043  0.002 0.048  0.006

0.044  0.002 0.045  0.002 0.047  0.002 0.045  0.003

0.047  0.002 0.055  0.013 0.044  0.003 0.049  0.006

0.046  0.004 0.048  0.005 0.048  0.003 0.045  0.002

1.6  0.3 2.0  0.4 1.8  0.3 1.6  0.1

1.6  0.0 1.3  0.2 1.3  0.1 1.6  0.1

1.5  0.2 1.3  0.0 1.5  0.3 1.4  0.2

1.9  0.4 2.0  0.1 1.5  0.6 2.0  0.1

0.073  0.004 0.129  0.038 0.082  0.002 0.139  0.098

0.075  0.010 0.063  0.002* 0.067  0.004 0.070  0.011

0.067  0.004 0.079  0.017 0.075  0.019 0.064  0.003

0.062  0.003 0.068  0.005 0.061  0.001 0.064  0.002*

1.6  0.4 2.2  0.1 2.0  0.1 1.7  0.1

2.0  0.1 1.9  0.5 1.8  0.0 1.7  0.1

1.6  0.0 1.5  0.2 1.3  0.8 1.5  0.7

1.4  0.0 1.7  0.0* 2.1  0.1 1.8  1.0

0.057  0.002 0.083  0.029 0.062  0.003 0.062  0.003

0.064  0.005 0.052  0.004 0.061  0.004 0.094  0.043

0.051  0.006 0.071  0.041 0.053  0.015 0.057  0.015

0.050  0.008 0.104  0.112 0.049  0.007 0.062  0.022

Data represent the mean  SD of absorbance values by the IL-6 bioassay on the 7TD1 cell line. Data represent the mean  SD of absorbance values in the chIFNg-specific ELISA. Serum samples were diluted 1:10. Significantly different from the infected corresponding control group of the same experiment (P < 0.05). n = 3–5.

M. Aricibasi et al. / Veterinary Immunology and Immunopathology 135 (2010) 79–92 Fig. 3. Detection of circulating IL-1b after inoculation with different IBDV strains. The amount of detected IL-1b in serum samples is presented as group average in relative light units correlating with the activity of the luciferase reagent: (a) iIBDV-inoculated birds, (b) vIBDV-inoculated birds, and (c) vvIBDV-inoculated birds. Error bars indicate the SD per group.

87

88

M. Aricibasi et al. / Veterinary Immunology and Immunopathology 135 (2010) 79–92

Fig. 4. Percentage of KUL-01+ cells in the bursa of Fabricius (a) and spleen (b) of IBDV inoculated and virus-free control groups. Presented is the average (as xfold change from virus-negative groups) percentages of macrophage-like cells per total counted live cells in the organ cell suspension/per group  SD, n = 3–5.

considered to be below the virus-neutralizing breakthrough levels of 250–500 of the infecting IBDV strains (Eterradossi and Saif, 2008). vvIBDV- and vIBDV-induced VN and ELISA antibodies more rapidly in infected birds than iIBDV (Table 5). 4. Discussion Previous studies and field observations have indicated that IBDV pathogenesis may vary with the genetic background of the chicken but also with the IBDV strain under investigation (Bumstead et al., 1993; Ruby et al., 2006). However, no studies have ever been performed combining a comparison of genetically different chickens and IBDV strains of different virulence. A retrospective comparison of different studies is not possible due to the variation in experimental conditions applied by different researchers (Ahmed et al., 2007; Hudson et al., 2002). To better understand virus–host interactions, we compared the innate and acquired immune responses of LT and BT chickens after inoculation with intermediate, virulent and very virulent strains of IBDV. As expected from previous observations, there were significant differences between the genetic backgrounds in susceptibility to IBD. The

extent of this difference depended significantly on the infecting IBDV strain. While the pathogenesis of the intermediate strain was comparable between BT and LT chickens, the vIBDV and vvIBDV strains induced different clinical disease and innate and acquired immune reactions in the different genetic backgrounds. LT birds showed severe clinical disease and mortality, higher bursal lesion scores and IBDV-antigen load, especially during the first 3 days pi, than BT birds. Moreover, the induction of circulating cytokines varied significantly in terms of both timing and quantity between LT and BT birds. Interestingly, no significant differences were seen between genetic backgrounds in the induction of the humoral response. The overall timing of antibody increase and antibody levels were comparable between BT and LT birds. On the other hand, variations were seen between the different virus strains with respect to onset and antibody titers: the classical vIBDV and vvIBDV strains induced the most rapid onset and the vvIBDV strain had the highest titers at 7 days pi relative to the intermediate strain (P < 0.05). Although broilers had low maternal antibodies at the time of virusinoculation, these detected titers were below the breakthrough titers of the infecting viruses (Eterradossi and Saif, 2008). In this experiment the day 7-pi data clearly indicate

M. Aricibasi et al. / Veterinary Immunology and Immunopathology 135 (2010) 79–92

89

Fig. 5. Circulating nitrite levels in serum samples of LT and BT chickens after inoculation with vIBDV (a) and vvIBDV (b). The amount of detected nitrite is presented in mM  SD. *Significant difference as compared to virus-free controls (P < 0.05), n = 3–5. No significant differences between infected and non-infected chickens were seen at 5 and 7 days pi.

that the remaining maternal antibody levels at the time of infection did not affect the beginning of virus clearance, which was comparable between LT and BT chickens for all the viruses. Furthermore, the induced antibody levels in BT and LT chickens were comparable also indicating that the low maternal antibody levels did not affect IBDV infection significantly. Previous preliminary vvIBDV infection studies in IBDV-maternal antibody negative commercial broilers further confirm this speculation. After inoculation of IBDV-antibody negative broilers with the same vvIBDV strain and dose used in the presented study broilers did not develop clinical signs or mortality with similar IBDV-ELISA antibody levels at 7 days pi as seen in this study (Aricibasi et al., unpublished data). Surprisingly, vIBDV showed the highest virus replication rate of the three virus strains at all investigated time points. Possibly the higher virus dose at inoculation may have affected the number of detected IBDV-antigen positive cells in vIBDV-infected birds in comparison to the iIBDV and vvIBDV, where a 10-fold lower dose was used. Moreover, LT chickens showed significantly faster virus replication than BT chickens in the BF (P < 0.05). This massive virus replication appeared to be correlated with

the onset of bursa lesions, the initiation of proinflammatory cytokine release, and the rapid manifestation of the disease accompanied by mortality in LT chickens. Although the clinical disease induced by vvIBDV is more severe than that induced by vIBDV, this was not reflected in viral antigen load, depletion of intrabursal B cells or onset of bursa lesions. Thus other factors, such as circulating cytokines, may have a more significant influence on the clinical disease than viral load or lesions. Major differences between vIBDV- and vvIBDV-infected LT birds were found in the induction of total IFN. We speculate that type I IFNs dominate the antiviral activity detected in the IFN bioassay. Type I IFNs have been previously shown to play an important role in the regulation of virus replication and the anti-inflammatory response (Kochs et al., 2007). Furthermore, the capacity for a strong type I IFN response has been shown to be closely connected to genetic background as well as to the properties of the infecting virus strain in other infection models (Abdul-Careem et al., 2008; Watanabe, 2007; Zhou et al., 2008). In vivo and in vitro effects of chicken IFNa have been investigated in IBDV-infected SPF Leghorn-type chickens and in a Chinese strain of commercial broiler-

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90

Table 5 Serum ELISA antibody development and induction of VN antibodies in serum after IBDV inoculation of LT and BT chickens. Groups

Mean ELISA titer (log10)/group on days pi

Mean VN titer (log2)/group on days pi

Chickens

Virus

3

5

7

3

5

7

LT

iIBDV

nd nd nd nd

0.0 0.0 0.6 0.8

0.0 1.1 0.2 2.0

0.0 0.0 2.8 3.0

0.0 0.8 3.0 3.8

0.0 7.6 3.5 6.5

0.0 0.0 0.0 0.0

0.0 3.2* 0.0 1.7

0.0 3.2 0.0 2.9

0.0 6.4 4.1 2.1

0.0 9.3 0.0 7.5

0.0 8.8 5.3 8.8

0.0 0.0 0.9 1.0

0.0 2.1 2.2 2.0

0.0 3.3 0.0 3.2

0.0 8.1 5.2 7.1

0.0 7.1 5.0 5.6

0.0 9.7 3.5 9.1

+ BT + LT

vIBDV +

BT + LT

vvIBDV +

BT +

nd, not done. * Significantly different as compared to the BT-infected birds in the same experiment (P < 0.05), n = 5–10.

type chickens (Mo et al., 2001). In that study, recombinant IFNa suppressed IBDV plaque formation in a dosedependent manner, and the antiviral effect of IFNa was more significant in commercial chickens than in their SPF counterparts (Mo et al., 2001). Although B cells are the main targets for IBDV, recent data show that the virus also infects and replicates in macrophages (Khatri et al., 2005; Palmquist et al., 2006). Qureshi’s studies (Qureshi, 2003) drew attention to genetic differences in embryonic hemopoietic progenitors’ ability to differentiate into macrophages: starting with the same number of bone-marrow stem cells, broiler-type chickens produced a significantly lower number of macrophagetype colonies than White Leghorn chickens (Hussain and Qureshi, 1997; Qureshi, 2003). These observations of genetic differences in macrophages between layer- and broiler-type chickens may explain the observations made in the present study. vvIBDV-inoculated LT chickens showed higher numbers of accumulating macrophagelike cells in the BF than BT chickens at 1 day pi. We can speculate that these macrophages are activated and release enhanced amounts of cytokines, adhesion molecules, and enzymes that mediate and modulate various immune responses. iNOS is one of the enzymes induced in macrophages, catalyzing the biosynthesis of free radical NO (MacMiking et al., 1997). NO release mediated by iNOS is an important host-defense mechanism against microbial pathogens in mononuclear phagocytes (Crippen et al., 2003; Hibbs et al., 1988) and may limit IBDV replication as it has been shown for other viruses (Djeraba et al., 2002). Low-pathogenic or intermediate IBDV strains were found not to induce NO or circulating nitrites (Rautenschlein et al., 2003), as also found in this study. Here, the more virulent strains induced comparable levels of circulating nitrite up to 3 days pi. vIBDV-infected LT chickens had higher circulating nitrite levels than BT chickens, which may explain the development of clinical signs and mortality in LT chickens as compared to the latter. In

previous studies, it was further demonstrated that bursal macrophages isolated from IBDV-infected chickens also show upregulated gene expression of IL-1b, IL-6, and IL-18 during the acute phase of infection (Khatri et al., 2005). In this study, we investigated the bioactive proteins that were actually expressed, because the detection of gene expression may not directly correlate with this parameter. We detected increased levels of circulating IL-1b in LT chickens with a peak 5 days pi with vIBDV and vvIBDV. IL-6 levels clearly peaked 2 days pi in iIBDV-inoculated LT birds, while the differences in the vIBDV- and vvIBDVinoculated groups were less clear due to high standard deviations. This is in contrast to the other investigated cytokines, which showed their highest upregulation in the groups infected with the more virulent strains. This observation suggests that IL-6 does possibly not contribute to clinical IBD or lesion development. Studies by Rauw et al. (2007) demonstrated upregulation of bioactive IFN-g and IL-6 the first 5 days pi and at 3 and 5 days pi, respectively, after infection of SPF LT chickens with a Belgium vvIBDV strain. The upregulation of the IL-6 and IFN-g levels in our study is less clear than in the study by Rauw et al. (2007). On one side this may be due to high individual variation, but also the differences suggest that strain variations may play a significant role in detectable IL-6 and IFNg levels after infection. Virus replication in the BF is known to be associated with the accumulation of CD4+ and CD8+ T lymphocytes (Kim and Sharma, 2000; Kim et al., 2000; Rautenschlein et al., 2002a,b; Tanimura and Sharma, 1997). In this study, T cell influx into the BF was observed in both genetic backgrounds. In addition to genetic background, viral strains also had an influence on time of onset of T cell accumulation. Interestingly, BT chickens showed a higher number of intrabursal T cells than LT chickens, especially after vIBDV infection. The ability to mount a strong local T cell-mediated immune response during the early phase of infection, between days 3 and 5 pi, may influence the clinical outcome of the disease and protect BT chickens from clinical disease. The T cells may contribute to the control of IBDV replication (Rautenschlein et al., 2002a), explaining the lower IBDV-antigen load in BT chickens as compared to LT chickens, but they may also release cytokines, which could contribute to bursal destruction (Rautenschlein et al., 2007; Ruby et al., 2006; Wong et al., 2007). The ability of IBDV to replicate in different extrabursal lymphoid tissues appears to correlate with the induction of a strong systemic humoral immune response: the more virulent the virus, the more efficient the induction of IBDVantibodies, as detected in the ELISA and VN test. Previous studies have suggested a correlation between the induction of high IBDV-antibody levels during the early phase of infection and disease protection in less susceptible birds (Pitcovski et al., 2001; Yunis et al., 2002). This speculation could not be supported in this study because the more susceptible LT chickens developed comparable IBDVantibody levels after infection as compared to the less susceptible BT birds. Overall, this study provides important information on IBDV pathogenesis and immune responses in susceptible

M. Aricibasi et al. / Veterinary Immunology and Immunopathology 135 (2010) 79–92

and less susceptible birds. Our data indicate that a higher proinflammatory response after infection may lead to more severe lesion development and clinical disease with associated mortality rates. The induction of a high mortality rate after IBDV infection of susceptible chickens with virulent strains correlated with the ability of the bird to mount a rapid systemic cytokine-mediated immune response, which may lead to a shock-like syndrome followed by death (Asif et al., 2007; Jarosinski et al., 2005; Schat and Xing, 2000). More bioactive cytokines have to be evaluated in repeat experiments to elaborate on this speculation. The immune response after IBDV infection in BT chickens may be better regulated, not allowing the massive proinflammatory cytokine expression observed in the LT chickens. Higher activity of macrophagelike cells in LT chickens during the early phase of infection may lead to more severe lesion development in these birds relative to BT chickens. This study emphasizes the importance of the influence of bird genotype and infectious agent strain on actual disease outcome. Acknowledgements We would like to thank Christine Haase, Sonja Bernhardt und Martina Koschorrek for their excellent technical support in this study. This work was funded by the German Israeli Foundation (GIF 818-65.11/2004). References Abdul-Careem, M.F., Read, L.R., Parvizi, P., Thanthrige-Don, N., Sharif, S., 2008. Marek’s disease virus-induced expression of cytokine genes in feathers of genetically defined chickens. Dev. Comp. Immunol. 33, 618–623. Ahmed, K.A., Saxena, V.K., Ara, A., Singh, K.B., Sundaresan, N.R., Saxena, M., Rasool, T.J., 2007. Immune response to Newcastle disease virus in chicken lines divergently selected for cutaneous hypersensitivity. Int. J. Immunogenet. 34, 445–455. Asif, M., Lowenthal, J.W., Ford, M.E., Schat, K.A., Kimpton, W.G., Bean, A.G., 2007. Interleukin-6 expression after infectious bronchitis virus infection in chickens. Viral Immunol. 20, 479–486. Block, H., Meyer-Block, K., Rebeski, D.E., Scharr, H., de Wit, S., Rohn, K., Rautenschlein, S., 2007. A field study on the significance of vaccination against infectious bursal disease virus (IBDV) at the optimal time point in broiler flocks with maternally derived IBDV antibodies. Avian Pathol. 36, 401–409. Bumstead, N., Reece, R.L., Cook, J.K., 1993. Genetic differences in susceptibility of chicken lines to infection with infectious bursal disease virus. Poult. Sci. 72, 403–410. Crippen, T.L., Sheffield, C.L., He, H., Lowry, V.K., Kogut, M.H., 2003. Differential nitric oxide production by chicken immune cells. Dev. Comp. Immunol. 27, 603–610. de Wit, S., 1998. Gumboro disease: estimation of optimal time of vaccination by the Deventer formula. Pol. Vet. J. 3, 19–22. Djeraba, A., Musset, E., van Rooijen, N., Quere, P., 2002. Resistance and susceptibility to Marek’s disease: nitric oxide synthase/arginase activity balance. Vet. Microbiol. 86, 229–244. Eldaghayes, I., Rothwell, L., Williams, A., Withers, D., Balu, S., Davison, F., Kaiser, P., 2006. Infectious bursal disease virus: strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa. Viral Immunol. 19, 83–91. Eterradossi, N., Saif, Y.M., 2008. In: Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K., Swayne, D.E. (Eds.), Infectious Bursal Disease. 12th edition. Blackwell Publishing, Ames, IA, USA, pp. 185–208. Gyorfy, Z., Ohnemus, A., Kaspers, B., Duda, E., Staehli, P., 2003. Truncated chicken interleukin-1beta with increased biologic activity. J. Interferon Cytokine Res. 23, 223–228. Hassan, M.K., Afify, M., Aly, M.M., 2002. Susceptibility of vaccinated and unvaccinated Egyptian chickens to very virulent infectious bursal disease virus. Avian Pathol. 31, 149–156.

91

Hibbs Jr., L.B., Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1988. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157, 87–94. Hudson, J.C., Hoerr, E.J., Parker, S.H., Ewald, S.J., 2002. Quantitative measures of disease in broiler breeder chicks of different major histocompatibility complex genotypes after challenge with infectious bursal disease virus. Avian Dis. 46, 581–592. Hussain, I., Qureshi, M.A., 1997. Nitric oxide synthase activity and mRNA expression in chicken macrophages. Poult. Sci. 76, 1524–1530. Jarosinski, K.W., Njaa, B.L., O’Connell, P.H., Schat, K.A., 2005. Pro-inflammatory response in chicken spleen and brain tissues after infection with very virulent plus Marek’s disease virus. Viral Immunol. 18, 148– 161. Jung, A., 2006. Pathogenesestudie eines intermedia¨rvirulenten Gumborovirus in spezifiziert-pathogen-freien (SPF) Hu¨hnern und kommerziellen Broilern. Doctoral Thesis. University of Veterinary Medicine, Hannover, Germany. Juul-Madsen, H.R., Dalgaard, T.S., Rontved, C.M., Jensen, K.H., Bumstead, N., 2006. Immune response to a killed infectious bursal disease virus vaccine in inbred chicken lines with different major histocompatibility complex haplotypes. Poult. Sci. 85, 986–998. Karaca, K., Sharma, J.M., Tomai, M.A., Miller, R.L., 1996. In vivo and in vitro interferon induction in chickens by S-28828, an imidazoquinolinamine immunoenhancer. J. Interferon Cytokine Res. 16, 327–332. Khatri, M., Palmquist, J.M., Cha, R.M., Sharma, J.M., 2005. Infection and activation of bursal macrophages by virulent infectious bursal disease virus. Virus Res. 113, 44–50. Kim, I.J., Gagic, M., Sharma, J.M., 1999. Recovery of antibody-producing ability and lymphocyte repopulation of bursal follicles in chickens exposed to infectious bursal disease virus. Avian Dis. 43, 401–413. Kim, I.J., Sharma, J.M., 2000. IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes. Vet. Immunol. Immunopathol. 74, 47–57. Kim, I.J., You, S.K., Kim, H., Yeh, H.Y., Sharma, J.M., 2000. Characteristics of bursal T lymphocytes induced by infectious bursal disease virus. J. Virol. 74, 8884–8892. Kochs, G., Koerner, I., Thiel, L., Kothlow, S., Kaspers, B., Ruggli, N., Summerfield, A., Pavlovic, J., Stech, J., Staeheli, P., 2007. Properties of H7N7 influenza A virus strain SC35 M lacking interferon antagonist NS1 in mice and chickens. J. Gen. Virol. 88, 1403–1409. Lynagh, G.R., Bailey, M., Kaiser, P., 2000. Interleukin-6 is produced during both murine and avian Eimeria infections. Vet. Immunol. Immunopathol. 76, 89–102. MacMiking, J., Xie, Q.W., Nathan, C., 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15, 323–350. Mo, C.W., Cao, Y.C., Lim, B.L., 2001. The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and Newcastle disease virus infection. Avian Dis. 45, 389–399. Palmquist, J.M., Khatri, M., Cha, R.M., Goddeeris, B.M., Walcheck, B., Sharma, J.M., 2006. In vivo activation of chicken macrophages by infectious bursal disease virus. Viral Immunol. 19, 305–315. Pitcovski, J., Cahaner, A., Heller, E.D., Zouri, T., Gutter, B., Gotfried, Y., Leitner, G., 2001. Immune response and resistance to infectious bursal disease virus of chicken lines selected for high or low antibody response to Escherichia coli. Poult. Sci. 80, 879–884. Qureshi, M.A., 2003. Avian macrophage and immune response: an overview. Poult. Sci. 82, 691–698. Rautenschlein, S., Yeh, H.Y., Njenga, M.K., Sharma, J.M., 2002a. Role of intrabursal T cells in infectious bursal disease virus (IBDV) infection: T cells promote viral clearance but delay follicular recovery. Arch. Virol. 147, 285–304. Rautenschlein, S., Yeh, H.Y., Sharma, J.M., 2002b. The role of T cells in protection by an inactivated infectious bursal disease virus vaccine. Vet. Immunol. Immunopathol. 89, 159–167. Rautenschlein, S., Yeh, H.Y., Sharma, J.M., 2003. Comparative immunopathogenesis of mild, intermediate, and virulent strains of classic infectious bursal disease virus. Avian Dis. 47, 66–78. Rautenschlein, S., Kraemer, C., Vanmarcke, J., Montiel, E., 2005. Protective efficacy of intermediate and intermediate plus infectious bursal disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers. Avian Dis. 49, 231–237. Rautenschlein, S., von Samson-Himmelstjerna, G., Haase, C., 2007. A comparison of immune response to infection with virulent infectious bursal disease virus (IBDV) between specific-pathogen-free chickens infected at 12 and 28 days of age. Vet. Immunol. Immunopathol. 115, 251–260. Rauw, F., Lambrecht, B., van den Berg, T., 2007. Pivotal role of ChIFNgamma in the pathogenesis and immunosuppression of infectious bursal disease. Avian Pathol. 36, 367–374. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497.

92

M. Aricibasi et al. / Veterinary Immunology and Immunopathology 135 (2010) 79–92

Rodenberg, J., Sharma, J.M., Belzer, S.W., Nordgren, R.M., Naqi, S., 1994. Flow cytometric analysis of B cell and T cell subpopulations in specific-pathogen-free chickens infected with infectious bursal disease virus. Avian Dis. 38, 16–21. Ruby, T., Whittaker, C., Withers, D.R., Chelbi-Alix, M.K., Morin, V., Oudin, A., Young, J.R., Zoorob, R., 2006. Transcriptional profiling reveals a possible role of the timing of the inflammatory response in determining susceptibility to a viral infection. J. Virol. 80, 9207–9216. Schat, K.A., Xing, Z., 2000. Specific and nonspecific immune response to Marek’s disease virus. Dev. Comp. Immunol. 24, 201–221. Schmidt, H.H., Wilke, P., Evers, B., Bohme, E., 1989. Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol. Biochem. Biophys. Res. Commun. 165, 284–291. Sharma, J.M., Dohms, J.E., Metz, A.L., 1989. Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens. Avian Dis. 33, 112–124. Sommer, F., Cardona, C., 2003. Chicken anemia virus in broilers: dynamics of the infection in two commercial broiler flocks. Avian Dis. 47, 1466– 1473. Tanimura, N., Sharma, J.M., 1997. Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens. Avian Dis. 41, 638–645.

Watanabe, T., 2007. Polymorphism of the chicken antiviral MX gene. Cytogenet. Genome Res. 117, 370–375. Winterfield, R.W., Fadly, A.M., Bickford, A., 1972. Infectivity and distribution of infectious bursal disease virus in the chicken. Persistence of the virus and lesions. Avian Dis. 16, 622–632. Wong, R.T., Hon, C.C., Zeng, F., Leung, F.C., 2007. Screening of differentially expressed transcripts in infectious bursal disease virus-induced apoptotic chicken embryonic fibroblasts by using cDNA microarrays. J. Gen. Virol. 88, 1785–1796. van den Berg, T.P., 2000. Acute infectious bursal disease in poultry: a review. Avian Pathol. 29, 175–194. Yeh, H.Y., Rautenschlein, S., Sharma, J.M., 2002. Protective immunity against infectious bursal disease virus in chickens in the absence of virus-specific antibodies. Vet. Immunol. Immunopathol. 89, 149–158. Yunis, R., Ben-David, A., Heller, A.D., Cahaner, A., 2002. Genetic and phenotypic correlations between antibody responses to Escherichia coli, infectious bursal disease virus (IBDV), and Newcastle disease virus (NDV), in broiler lines selected on antibody response to Escherichia coli. Poult. Sci. 81, 302–308. Zhou, J., Smith, D.K., Lu, L., Poon, V.K., Ng, F., Chen, D.Q., Huang, J.D., Yuen, K.Y., Cao, K.Y., Zheng, B.J., 2008. A non-synonymous single nucleotide polymorphism in IFNAR1 affects susceptibility to chronic hepatitis B virus infection. J. Viral Hepat. 16, 45–52.