Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus

Veterinary Immunology and Immunopathology 131 (2009) 229–237

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Research paper

Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus Decheng Wang a,b, Yufeng Liu a, Ruiping She a,*, Jingjing Xu c, Liqiang Liu a, Jinmao Xiong a, Yurong Yang a, Quan Sun a, Kaisong Peng d a

College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai 200032, China State Key Laboratory of AgroBiotechnology, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China d College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 November 2008 Received in revised form 13 April 2009 Accepted 17 April 2009

Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs (p < 0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased (p < 0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Mast cell SPF chickens Very virulent infectious bursal disease virus Tryptase Histamine

1. Introduction Infectious bursal disease viruses (IBDV), of the family Birnaviridae, are significant, widespread pathogens in the

Abbreviations: IBD, infectious bursal disease; IBDV, infectious bursal disease virus; vvIBDV, very virulent infectious bursal disease virus. * Corresponding author at: Department of Veterinary Pathology, College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China. E-mail addresses: [email protected], [email protected] (R. She). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.04.014

poultry industry. IBDV destroys B lymphocytes in the bursa of young chickens and causes infectious bursal disease (IBD), an acute, highly contagious immuno-suppressive disease among young chickens (Becht and Mu¨ller, 1991; van den Berg, 2000). First isolated in the town of Gumboro, DE, USA in 1957 (Cosgrove, 1962), IBDV has caused enormous worldwide economic loss in the poultry industry. Very virulent (vv) IBDV strains emerged in Europe in the late 1980s and have caused about 70% mortality in chickens in several European and Asian countries (Nunoya et al., 1992; van den Berg et al., 1991; Zhou et al., 1982). In spite of industrial vaccination

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programs, IBD continues to be a serious economic problem largely due to the spread and subsequent outbreak of vvIBDV (Saif, 1998; van den Berg, 2000), causing a higher incidence of acute disease and higher mortality (Nunoya et al., 1992; Snyder et al., 1992; van den Berg, 2000). In addition the virus-induced immune suppression results in secondary infections, growth retardation, and condemned carcasses at slaughter. It is generally accepted that the antigenicity and pathogenic signs of vvIBDV are similar to classical serotype I IBDV, but antibodies, including maternal antibodies, induced by attenuated serotype I vaccine do not provide satisfactory protection against vvIBDV (van den Berg, 2000). Currently, the depletion of B-lymphocytes in the bursa is known to be a crucial element in the early phase of vvIBDV infection, but it is not clear whether this effect is due to apoptosis or necrosis (Clouda et al., 1992; Jungmann et al., 2001; Ka¨ufer and Weiss, 1980; Mu¨ller et al., 2003). Although severe inflammation damage is a well documented consequence of IBDV infection, the exact mechanism of lymphocyte depletion is still not well characterized. Recently, the role of some immune cells, such as macrophages and mast cell, has attracted much attention from veterinary researchers (Khatri et al., 2005; Wang et al., 2008). Results from Khatri have demonstrated that IBDV can target macrophages and induce the release of mediators, ultimately resulting in inflammatory lesions in infected animals (Khatri et al., 2005). A previous study here revealed that mast cell populations and tryptase activity increased significantly in vvIBDV-infected birds (Wang et al., 2008). However, the exact process involved in mast cell switching to cause inflammatory injury of the bursa of Fabricius (BFs) in chickens infected with vvIBDV is still under debate. Mast cells, the key ‘‘effector cells’’ in inflammation, are resident in tissues throughout the body and particularly in tissues associated with structures such as skin, blood vessels and nerves, and in the tissues that interface with the external environment (Galli et al., 1999; Metcalfe et al., 1997). The critical role of mast cells in the immune response and in inflammation and physiological processes became clear after evidence indicated that mast cells are responsible for the production of many inflammatory molecules (Gordon and Galli, 1990; Metcalfe et al., 1997). As long-lived cells, mast cells play various roles in viral invasion, possibly contributing to the initiation of inflammatory responses (Kaliner, 1987; Kobayashi et al., 2000), or act as reservoirs, aiding viruses, such as macrophagetropic HIV-1 (Bannert et al., 2001), Dengue virus (King et al., 2000), cytomegalovirus (Gibbons et al., 1990) and adenovirus (King et al., 2002), by eluding immunosurveillance and hastening the inflammatory response. A preliminary study conducted by Sun et al. demonstrated that increased mast cell density in specific-pathogen-free (SPF) chickens after infection with New Castle Disease virus (NDV) was consistent with the infiltration of inflammatory cells and histopathological changes (Sun et al., 2008). Recently, it was found that mast cell populations were significantly increased in vvIBDVinfected SPF chickens. More interesting, mast cell populations and tryptase activity were markedly increased in the BFs, also showing the positive relevance of vvIBDV

distribution to vvIBDV antigen and tryptase content (Wang et al., 2008). These findings was found that mast cells degranulation might be a possible mechanism by which pathogens induced an inflammatory response, and that it is important to identify the exact mechanisms by which they may provide protection. Some studies have demonstrated that mast cell stabilization prevents histological damage of gastric mucosa induced by agents such as ethanol (Kalia et al., 2000; Kalia et al., 1997; Oates and Hakkinen, 1988), but it is not known whether they also prevent damage induced by vvIBDV in SPF chickens. In summary, these microscopic observations of mast cells, taken together with histological and cytochemical staining results, demonstrate changes that are indicative of the degranulation of mast cells. Tryptase and histamine are regarded as the principal products of activated mast cells (Fox et al., 1982; He and Walls, 1997; Metcalfe et al., 1997; Miadonna et al., 1994) and the release of these two factors from degranulated mast cells is the best known agent for a diversity of roles in many processes. Thus, the goal of the current study was to evaluate the effects of mast cell stabilization and its mediator antagonism on vvIBDV-induced acute inflammation using histochemical and histologic methods and to study the mechanisms by which mast cell stabilization may protect against inflammatory damage. 2. Materials and methods 2.1. Animals and experimental treatments One hundred and five 30-day-old, healthy, SPF chickens (White Avian, Merial Co. Ltd., Beijing, China), negative to NDV and IBD virus, were divided randomly into three groups (45 birds each in the vvIBDV infected and ketotifen pretreated groups, 15 birds as the control), which were then separated in individual negative-pressure isolators. All chickens were provided feed and water ad libitum. The vvIBDV SNJ93 strain was obtained from the China Institute of Veterinary Drug Control (Beijing, China) and had an estimated LD50 (eLD50) of vvIBDV at 3.2  106/ 0.2 mL, and was diluted 1/100 in sterile saline solution. Chickens in the vvIBDV infected group were inoculated with 0.2 mL of the vvIBDV dilution by means of nasal and eye drops. In the ketotifen pretreated group, birds were pretreated with 1 mL ketotifen (Sigma, Beijing, 1 mg/kg body weight), dissolved in 0.9% saline and prepared fresh on the day of use, by a stainless steel orogastric tube approximately 1 h before infection. In the control group, chickens received equal volumes of saline by the same route. Animals were visually examined daily, euthanasia was performed by intravenous injection of sodium pentobarbital in a brachial vein, and necropsies performed immediately postmortem. This study was approved by the Institutional Animal Care and Use Committee of China Agricultural University. 2.2. Sampling On days 1, 2, and 3 postinfection, 15 chickens from each test group and 5 control birds were randomly selected,

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weighed, euthanized, and a portion of the BFs collected immediately and fixed in 2.5% (v/v) glutaraldehyde– polyoxymethylene solution. A second portion (0.5 g) of the same tissues was concurrently preserved at 70 8C in liquid nitrogen for later histamine analysis. 2.3. Histopathology examination The fixed cloacal bursa (bursa of Fabricius) samples were dehydrated, embedded in paraffin, serial sectioned (5 mm thickness), and kept at 37 8C for more than 12 h. The sections were washed three times in xylol for 5 min to remove paraffin, hydrated by five washings successively in solutions of 100, 100, 95, 80, and 70% alcohol. Sections were then stained with haematoxylin–eosin solution according to route protocol and the slides coded to prevent observer bias during examination and evaluation under an Olympus light microscope (Olympus Optical Co, Beijing, China). 2.4. Immunological assays 2.4.1. Measurement and distribution of vvIBDV viral antigen A second set of serial paraffin sections were immersed in three consecutive washings in xylol for 5 min to remove paraffin, hydrated with five consecutive washings with alcohol 100, 95, 80, 70%, and PBS, respectively, and treated with 3% peroxide at room temperature for 30 min to produce endogenous peroxidase ablation. All steps were carried out in a moist chamber, according to previous reports from this laboratory (Wang et al., 2008), sections for the negative controls were prepared using the same steps as described above except vvIBDV anti-sera was replaced with PBS. The number of positives (vvIBDV viral antigens) present in the bursa was counted under a microscope and Motic Images 2000 (Motic China Group Co., Guangzhou, China) used for characterization of vvIBDV positives changes, with results were expressed as the number of cells per mm2. 2.4.2. Improved toluidine blue staining procedure for mast cells Mast cells were examined by an improved toluidine blue staining method performed according to previous reports (Ha˚kanson et al., 1972; Humanson, 1979). Briefly, tissue samples were dehydrated, embedded in paraffin, deparaffinaged, rehydrated, and immersed in 0.8% toluidine blue (Sigma Co.) for 15 s. Slides were washed with distilled water for 30 s, immediately placed into 95% alcohol until the mast cells appeared deep reddish purple under the microscope, immersed for 3 min successively in alcohol 100%, alcohol–xylol (1:1, v/v), and xylol, and then mounted with neutral gums. The number of mast cells was counted in five fields of view under the microscope (40 magnification) and the means calculated. Sampling of the sections was unbiased, with the samples coded and examinations performed by one investigator. Each section was scanned for general qualitative observations, but detailed examination focused on mast cells.

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2.4.3. Measurement of tryptase with immunohistochemical staining Tryptase is synthesized almost exclusively by mast cells, being released after mast cell degranulation (Schwartz et al., 1981; Walls et al., 1990) and, because it is stored almost exclusively in mast cells, this proteinase has attracted particular attention as a marker for mast cells and these cells’ activation. Examination of tryptase in tissue samples were performed by immunohistochemical analyses, based on a recent report (Wang et al., 2008), and tryptase positive cells in bursal samples counted by microscopy and recorded as cells per mm2. 2.5. Assay of histamine content with fluorospectrophotometry Histamine, another important secreting factor accompanying mast cell degranulation (Glenner and Cohen, 1960). In the present study, the role of histamine was investigated using a fluorospectrophotometry technique, described previously for the histamine determinations (Ha˚kanson et al., 1972). BF tissues were homogenized in 2 mL of 25% (w/v) trichloroacetic acid, settled for 10 min, and 1.0 mL of the supernatant diluted to 2.0 mL by the addition of double-distilled water. Then, 0.5 mL each of 0.4N NaOH and 0.1% OPT methanol (Sigma, Beijing) was added, the solution mixed gently, reacted at 22 8C in darkness for 10 min, and stopped by the addition of 0.5 mL of 0.5N HCl. A blank was created by combining, in order, 0.5 mL each of 0.5N HCl, 0.1% OPT methanol, and 0.4N NaOH. The fluorescence intensities of the samples were determined by fluorescence spectrophotometry (Cary Eclipse, Varian Co., USA) using an activation wavelength of 360 nm and fluorescent monitoring at 450 nm. 2.6. Statistical analysis Experimental data were analyzed by one-way ANOVA using SAS statistical program (SAS Institute Inc., Cary, NC, USA) and the results were expressed as means and standard errors. And differences were considered significant at p < 0.05 or 0.01. 3. Results 3.1. Clinical and gross pathologic observations The health and behavior of the control group chickens were consistently normal throughout the experiment, but chickens infected with vvIBDV showed slightly altered gait, inactivity, ruffled plumes, diarrhea, decreased appetite, dehydration, and behavioral changes beginning on day 1 postinfection. By day 2 postinfection, most of the chickens abruptly presented clinical signs of a multifaceted syndrome, including visual signs of labored respiration, respiratory distress, increased inappetence, emaciation, and diarrhea. Most of the infected chickens (40 of 45, 89%) died during days 1–3 postinfection and gross observation showed severe hemorrhaging and swelling in the BFs, which had a purple grape-like appearance (Fig. 1). In contrast, on day 1 postinfection, the behavior of the ketotifen pretreated birds was normal, on day 2, a few

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Fig. 1. Gross observation and histopathological examination of BFs. (A) Bursa from the control group, no petechia shown in mucosal surface. (B) Bursa from ketotifen pretreated group, slight swelling, and a few hemorrhages in mucosa. (C) Bursa from vvIBDV infected group, severe swelling and hemorrhages, and purple grape-like appearance. (D) BF texture in control is normal, boundary among lymphoid nodules obvious, integrated lymphaticus folliculus and mucosa of BF, and orderly lymphocytes spread in lymphoid nodules. (E) Connective tissues among lymphoid nodules in ketotifen pretreated birds remain complete, few lymphocytes gone, and lymphoid follicles intact. (F) Degeneration and necrosis of lymphocytes in medullary area of bursal follicles in vvIBDV infected chickens, lymphocytes missing and replaced by segmented neutrocytes, pyknotic debris, and hyperplastic reticuloendothelial cells, and connective tissues among lymphoid nodules dissolved and vanished. Bar = 150 mm.

chickens presented with diarrhea, depressed appetite, or inappetence; and, on day 3, some chickens still showed diarrhea and depressed appetite and about 31% (14 of 45) died. Gross pathological findings showed relatively light hemorrhaging and swelling in the bursa (Fig. 1A–C).

scopically, were mainly located in the cortex and stroma of the lymphoid folliculus, with fewer present in the medullary zone (Fig. 2). Compared with day 1 postinfection, the dispersion of positives in the bursa was increased significantly (p < 0.01) by days 2 and 3 (Fig. 3).

3.2. Histopathologic lesions in chickens

3.4. The distribution and number of mast cells

The most severe pathological changes were observed in the BFs of the infected birds. By day 1 postinfection, degeneration and necrosis of lymphocytes in the medullary area of bursal follicles were observed, with the lymphocytes replaced by segmented neutrocytes, pyknotic debris, and hyperplastic reticuloendothelial cells. By day 2 postinfection, red cells had flooded the lymphoid nodes, the interstitial spaces of lymphoid folliculi had markedly increased, and there was obvious inflammatory cells infiltration en masse into the bursal follicles. All lymphoid follicles were affected by day 3, with typical ‘‘satellite-like vacuity’’ changes seen in the follicular medullary areas. During the course of the inflammatory response, there was necrosis and phagocytosis of heterophils, and fibroplasia in interfollicular connective tissues (Fig. 1D–F).

Mast cells were recognized as round or elongated cells with metachromatic granular staining produced by toluidine blue staining used to identify mast cells in tissues (Carlson and Hacking, 1972). Mast cells were not detected in the tissues taken from the control group throughout the study. In the infected group, mast cells presented typical deep reddish purple coloring on day 1 postinfection and exhibited rounded outlines filled with granules. On days 2 and 3, some mast cells had extruded particles dispersed around the cell bodies. Significant differences in the distribution of mast cells were found between the control group and vvIBDV infected group (p < 0.01, Fig. 4), while ketotifen pretreatment appeared to have a profound effect on the BF mast cell numbers in all 3 days postinfection (Fig. 5). Overall, the number of mast cells was dramatically greater in the vvIBDV infected group compared with the control (p < 0.01), and significantly decreased in the ketotifen inhibited infected group relative to the infected group (p < 0.01, Fig. 5).

3.3. The distribution of the vvIBDV viral antigen The positive indications of the presence of vvIBDV viral antigen, a brownish colored bursa when viewed micro-

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Fig. 2. Distribution of vvIBDV viral antigen in BFs. (A) No vvIBDV viral antigen positive signals in control group. (B) and (C) vvIBDV viral antigen positives generally distributed in BFs in ketotifen pretreated and vvIBDV infected chickens, respectively (250).

3.5. The expression and distribution of tryptase Considered the most important protease released by degranulating mast cells, the expression and distribution of tryptase was examined using an established histochemical technique (He et al., 1998; Schwartz et al., 1981). Tryptase positive indications were scarcely observed in BFs from the control groups, but were observed extensively in infected birds, mostly distributed in the lymph nodule and stroma of BFs (Fig. 6) and being significantly increased from day 1 to 3 postinfection in infected animals compared with the controls (p < 0.01, Fig. 7). In contrast, the production of tryptase was clearly reduced from day 1 to 3 in ketotifen pretreated infected birds compared with the infected group (p < 0.01, Fig. 7). Furthermore, tryptase expression had a positive correlation with mast cell distribution as observed microscopically. 3.6. Histamine content in bursa Histamine contents in the bursa of all infected birds were significantly increased from day 1 to 3 postinfection compared with the control birds (p < 0.01), with the concentration peaking at day 2. The bursa of ketotifen pretreated infected birds showed significantly reduced histamine content relative to the infected birds (p < 0.01, Fig. 8). 4. Discussion This study evaluated the presence of mast cells and acute inflammatory injury of BFs induced by vvIBDV infection. Significantly increased mast cell populations as well as acute inflammatory injury were found in vvIBDV infected chickens. However, pretreatment with ketotifen

before infection decreased mast cell numbers markedly compared with the infected birds, and acute injury in the pretreated group was ameliorated significantly. These results implicated mast cells as part of the cause of the acute injury observed during vvIBDV infection. Mast cells are usually distributed in the skin, the entire gastrointestinal tract, and the airways, where they are in close contact with exterior spaces. Studies of mast cells have demonstrated that they play an important role in some viral infections (Bannert et al., 2001; Castleman et al., 1990; Gibbons et al., 1990; King et al., 2002; Sundstrom et al., 2004). Recent studies here have shown that the number and morphology of mast cells changed strikingly in SPF chickens after vvIBDV infection (Wang et al., 2008); however, the role of mast cells in the pathogenesis of acute inflammatory injury in BFs during vvIBDV infection remained a puzzle. The present results show that mast cells were increased greatly in infected tissues, with a large number of infiltrated inflammatory cells, while in the ketotifen pretreated group inflammatory cells were rarely observed. In explanation, mast cells could serve as a primary detector mechanism for responding to tissue infection or invasion and in the release of proinflammatory mediators for that recruitment of leukocytes to the tissues site at risk (Gaboury et al., 1995). With few studies addressing this area of research in birds, mast cells might play a crucial role in some avian diseases (Sun et al., 2008; Wang et al., 2008). Mast cells are known to release several mediators that have been suggested to induce gastric injury, resulting in severe hemorrhaging and tissue necrosis in rats (Ogle and Lau, 1980; Takeuchi et al., 1986). In addition, previous results have demonstrated that increased mast cell numbers result in an increased release of some mediators by mast cells, which may contribute to tissue damage after NDV

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Fig. 3. Dynamic changes of vvIBDV viral antigen in BFs. Area density of vvIBDV positive signals in control (C), vvIBDV infected (T), and ketotifen pretreated (K) groups at various times postinfection. No antigens present in the control. ‘‘**’’ indicates significant difference from the control at p < 0.01. ‘‘a’’ indicates no significant difference between vvIBDV infected and ketotifen pretreated groups at p > 0.05.

infection (Sun et al., 2008). More recently, it was found here that, during vvIBDV infection, mast cell populations and tryptase release were markedly increased in primary immune organs, such as the BFs, thymus and spleen, which also showed increased vvIBDV viral antigen expression and severe tissues injury (Wang et al., 2008). Mast cell are important participants in the response to infection due to their ability to release quantities of active mediators, including tryptase, histamine, 5-HT, and other cytokines (Galli et al., 2005a; Marshall, 2004; Metcalfe et al., 1997). Tryptase is produced almost exclusively by mast cells and plays a multifaceted role in many physiological and abnormal conditions during degranulation (Galli et al., 2005a,b). The present results revealed that large quantities of tryptase were produced in the bursa during vvIBDV infection, while no expression was observed in bursa of the control group. These results indicated that the release of tryptase may have correlated with the inflammatory response. Interestingly, observations here found that tryptase expression had a positive

Fig. 5. Dynamic changes of mast cells in BFs. Number of mast cell in the bursa of the control (C), vvIBDV infected (T) and ketotifen pretreated (K) groups at various times postinfection. No mast cells observed in the control. ‘‘**’’ indicates significant difference from the control at p < 0.01. ‘‘*’’ indicates significant difference from the vvIBDV-infected group at p < 0.01.

correlation with mast cell populations. A previous study has demonstrated that tryptase released from degranulated mast cells had the capacity to activate certain mast cell populations and modulate some of the mediators released by mast cells. Moreover, the released tryptase from activated mast cells may stimulate secretions by neighboring mast cells and thus produce a feedback cycle as the disease progresses (He et al., 1998). Comparison of results from examination of vvIBDV viral antigen observed in the vvIBDV infected and ketotifen pretreated groups showed no significant difference, but the histopathology in these two groups showed significant difference, suggesting that ketotifen man play a positive role of mitigating injury by some unknown pathway. The present results were somewhat similar to previous reports that mast cell stabilization protects against ethanol-induced gastric mucosal injury (Kalia et al., 2000). Histamine is a multifunctional mediator stored within cytoplasmic granules of mast cells, and is known to play a central role in pathophysiologic processes (Black, 2002; Graham et al., 1955; Lagunoff and Benditt, 1960). A previous study has demonstrated that mast cell play a key role in gastric mucosal injury due to histamine release (Bechi et al., 1995), which can interact with endothelial

Fig. 4. Toluidine blue staining for mast cells in BF. (A) No mast cells in BFs of control group and bursal structure normal. (B) Mast cells reddish purple and distributed in stroma of BFs lymphoid follicles (arrows) in ketotifen pretreated birds. (C) Mast cells distributed in stroma of BFs lymphoid nodules with typical features of ‘‘metachromatic-granule’’ in vvIBDV infected group, some cell activation, and some degranulated during vvIBDV infection (arrow) (A, 200; B and C, 400).

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Fig. 6. Expression of tryptase positive signals in BFs. (A) Control group, tryptase positive signals rarely observed in bursa. (B) Ketotifen pretreated group, few positives (arrow) distributed in BFs. (C) vvIBDV infected group, tryptase positive stains dispersedly distributed in bursal folliculus lymphaticus (A and C, 200; B, 400).

Fig. 7. The dynamic expression of tryptase positive signals in BFs. Area density of tryptase in control (C), vvIBDV infected (T), and ketotifen pretreated (K) groups at various times postinfection. No tryptase detected in the control. ‘‘**’’ indicates significant difference from the control at p < 0.01. ‘‘*’’ indicates significant difference from the vvIBDV-infected group at p < 0.01.

cells (Geng et al., 1990), directly activate neutrophils (Benbarek et al., 1999), produce bronchoconstriction and vasodilation (Geng et al., 1990), induces exocytosis and inflammatory response by stimulating interleukin-6 production from human lung macrophages, and modulate the cytokine network (Sirois et al., 2000). In this study, histamine production was markedly increased in the vvIBDV infected chickens compared with the control and ketotifen pretreated groups and also, the histamine content in these tissues correlated with the number of mast cells. In contrast, the mast cell population and histamine content in ketotifen pretreated birds were significantly decreased and the injury of BFs was remitted as well, suggesting that histamine released from degranulated mast cells was involved in the injury process. This study provides further evidence associating mast cells with histamine release and tryptase production in chickens, but the possible contributing role of other mast cell mediators in the pathogenesis of vvIBDV induced disease could not be ruled out without further investigation.

Fig. 8. Dynamic changes of histamine contents in BFs (mg/mg). Changes in content of histamine (mg/mg) in BFs from control (C), vvIBDV infected (T), and ketotifen pretreated (K) groups at various times postinfection. ‘‘**’’, significantly different from the control at p < 0.01. ‘‘*’’ indicates significant difference from the vvIBDV-infected group at p < 0.01.

5. Conclusion In summary, the kinetics of mast cell populations, tryptase expression, and histamine release in the bursa of vvIBDV-infected birds showed acute inflammatory responses and tissue damage. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased, which correlated with the soft tissue damage observed in experiments. The overall results strongly supported the idea that the activation and degranulation of mast cells maybe an important means of injury to tissues with vvIBDV infection. In a word, this study provides interesting insights into the role of mast cell and their mediators in vvIBDV induced damage in chickens, showing that, with infection, increased mast cells correlated with tryptase and histamine production and that the inhibition of mast cell

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