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Susceptibility of chicken Kupffer cells to Chinese virulent infectious bursal disease virus
Veterinary Microbiology 164 (2013) 270–280
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Susceptibility of chicken Kupffer cells to Chinese virulent infectious bursal disease virus Haiyan Ma, Sufen Zhao, Yunfei Ma, Xin Guo, Deping Han, Yuanyuan Jia, Weiwei Zhang, Kedao Teng * College of Veterinary Medicine, China Agricultural University, Beijing, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 November 2012 Received in revised form 25 February 2013 Accepted 1 March 2013
Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although the effects of IBDV on bursa of Fabricius in chickens have been well reported, the impacts of IBDV on liver after IBDV infection are still unclear. In the present study, specific pathogen free (SPF) chickens were experimentally inoculated with IBDV Chinese virulent strain BC6/85, and the cells in liver and bursa were examined by immunohistochemisrty and transmission electron microscopy (TEM). The congestion of liver tissue and fatty degeneration of hepatocytes were characteristics of microscopical changes in chicken liver at 3 days post infection (d.p.i.), whereas there were follicular lymphoid necrosis, apoptosis, depletion, as well as edema and congestion in bursa. In addition, the number of IBDV-positive cells peaked at 4 d.p.i. in bursa and at 3 d.p.i. in liver, respectively. With respect to ultrastructural pathological changes of hepatocytes, mitochondria swelled and nucleus deformed into an irregular shape or its chromatin peripherally condensed which indicated that the hepatocyte was at the early stage of apoptosis, and the electron-lucent lipid droplets in a variety of sizes were observed within cytoplasm. Kupffer cells became ‘‘swollen-like’’ and the electron-density of their cytoplasm was lower than that of cells in uninfected group. Liver glycogen deposits significantly declined from 2 to 5 d.p.i. and recovered strongly at 6 d.p.i. More importantly, KLU01 (macrophage marker) positive (KUL01+) cells were infiltrated in bursa and liver in IBDV-exposed chickens by immunoperoxidase staining. To demonstrate the correlation between IBDV and macrophages in bursa and liver, we further investigated the colocalization of viral antigens and macrophages by double immunofluorescence labeling. At 4 d.p.i., the percentage of double positive cells (IBDV positive and KUL01+ cells) accounted for 26.5 percent of the total IBDV positive cells or 57 percent of the total KUL01+ cells in bursa. In comparison, the percentage of double positive cells in liver constituted 97 percent of the total IBDV positive cells or 99 percent of the total KUL01+ cells. These results suggest that IBDV was susceptible to KUL01+ cells in liver (mainly Kupffer cells) and replicated in the KUL01+ cells. By comparison with the influence of IBDV on bursa, our findings were the first to elucidate the pathological changes in liver after IBDV infection on a microscopical and ultrastructural scale, and, especially, to gain the initial insight into the susceptibility of Kupffer cells to IBDV. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Infectious bursal disease virus (IBDV) Histopathology Liver Kupffer cells Immunohistochemisrty Transmission electron microscopy(TEM)
1. Introduction * Corresponding author at: College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, People’s Republic of China. Tel.: +86 01 62733013. E-mail addresses: [email protected], [email protected] (K. Teng). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.03.002
Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is a highly contagious and immunosuppressive viral disease in young chickens during
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3 to 6 weeks of age. IBDV infection may aggravate infection with other pathogenic agents, and IBDV-infected chickens have the low ability of response to subsequent vaccinations (Mu¨ller et al., 2003). IBDV can be classified into two serotypes (serotype 1 and 2) by virus neutralization test (McFerran et al., 1980). Serotype 1 strains, differentiated as classical strains (cIBDV), very virulent strains (vvIBDV) and variant strains (vIBDV), can cause pathogenicity and mortality in young chickens, while serotype 2 strains are avirulent (Liu et al., 2010; Rauf et al., 2011). The causative agent, IBDV, belongs to the family Birnaviridae, and is non-enveloped, double-stranded (ds) RNA virus consisting of two segments (segment A and segment B) (Dobos et al., 1979). Segment A encodes the VP5, VP4 (28 kDa), VP3 (32 kDa), and VP2 (41 kDa), and segment B encodes VP1 (98 kDa) (Mundt et al., 1995; Birghan et al., 2000; Von Einem et al., 2004). IBDV infects and destroys actively dividing IgM-bearing B cells in the bursa of Fabricius (Hirai et al., 1981; Rodenberg et al., 1994). Additionally, the extensive viral replication is present in bursa (Dobos et al., 1979). So viral infection causes severe depletion of lymphocytes and destruction of bursa tissues of chickens infected with IBDV (Ka¨ufer and Weiss, 1980). Although bursa is the principal target organ for IBDV, recent studies show that the virus also infects and possibly replicates in other organs, such as spleen, thymus, bone marrow, cecal tonsil, liver and kidney, at early stage of infection (Tanimura et al., 1995; Oladele et al., 2009). It has been shown that B cells may not be the sole target for the virus. Macrophages may serve as host cells for IBDV (Khatri et al., 2005; Ka¨ufer and Weiss, 1976; Savova and Bozhkov, 1985; Palmquist et al., 2006). Macrophages, on the one hand, can phagocytize and clear pathogens by recognizing pathogen-associated molecular patterns (PAMPs) via their pattern-recognition receptors (PRRs) in innate immune system (Akira et al., 2006). On the other hand, macrophages secrete a variety of functional cytokines which can act as immunomodulatory, antiviral or proinflammatory factors, such as typeI interferon (IFN-a and IFN-b), IL-6, TNF-a, etc (Zhou, 2007). However, the role of macrophages in the pathogenesis of IBDV has not been extensively examined so far. It has been reported that gut-associated macrophages are hypothesized to be the initial transporters of IBDV from the digestive tract to the bursa and other peripheral organs (Ka¨ufer and Weiss, 1976). There are reports that infection with IBDV causes the production of proinflammatory mediators and cytokines by macrophages. The levels of induced proinflammatory mediators and cytokines reached the peak during active virus replication and correlated with extensive inflammatory response in both chicken bursa and spleen (Kim et al., 1998; Khatri et al., 2005; Palmquist et al., 2006). These macrophages were activated by viral infection and upregulated mRNA expression of IL-1b, IL-6, IL-18, and iNOS (Palmquist et al., 2006). Kupffer cells (liver macrophages) represent one of the largest reservoirs of resident tissue macrophages, which are located in the liver sinusoids in close contact with endothelial cells. As for mammals, in addition to Kuffer cells, there are endothelial cells in the hepatic sinusoid. The
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Disse’s space of liver harbors fat-storing cells and pit cells (Wisse et al., 1996). There are some distinctive characteristics of bile ductules and sinusoidal cells between mammals and chickens, although the ultrastructure of chicken liver is principally similar to that of rat or human liver. Investigation of ultrastructural chicken liver revealed that intercalated cells (extra-sinusoidal macrophages of chicken liver), a unique kind of chicken hepatic parenchyma cells, mostly present in interhepatocytic or Disse’s spaces (Ohata et al., 1982; Ohata and Ito, 1986; Ghoddusi and Kelly, 2004). So far, little is known about the pathological damage of virus replication to chicken livers and the susceptibility of Kupffer cells to virus during the acute phase of the IBDV infection. Thus, in the present study we examined the impacts of IBDV infection on chicken liver tissues and, in particular, on Kupffer cells by immunohistochemisrty and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Animals, viruses and rabbit anti-IBDV serum The specific-pathogen-free (SPF) male white Leghorn chickens were purchased from Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China) and reared in CC. JH-1 type positive pressure isolation units which were purchased from Jinhang Purifying Air Conditioning Equipment Co., Ltd. (Tianjin, China) under the supervision of the Animal Facility Management of China Agricultural University. Chickens for different experimental groups were housed in the separate isolation units. Water and feed were provided ad libitum during the entire experimental period. Virulent strain of IBDV (BC6/85, CVCC AV7) was obtained from China Institute of Veterinary Drug Control (Beijing, China). At 3 weeks of age, chickens were inoculated with 1000 50% egg infective doses (EID50) of IBDV strain BC6/85 or phosphate-buffered saline (PBS) by eye drop. Rabbit anti-IBDV serum was produced as previously described (Tanimura et al., 1995; Oladele et al., 2009). And the titer of rabbit antiserum was 1: 16 by agar gel precipitation (AGP) assay. Both IBDV (BC6/85) antigen and chicken anti-IBDV serum as a positive control in AGP assay were obtained from China Institute of Veterinary Drug Control (Beijing, China). 2.2. Experimental design A total of seventy 3-week-old SPF chickens were randomly distributed into two groups (the IBDV-exposed group and the control group) of 35 birds each, and were separately weighed before their sacrifice. IBDV-exposed birds were inoculated with IBDV strain BC6/85 and were euthanized (5 birds once) at 12, 24 h post infection (h.p.i.) and 2, 3, 4, 5, 6 days post infection (d.p.i.), and control birds were inoculated with PBS and were euthanized (5 birds once) at the same intervals. Of these chickens, the organs of bursa of Fabricius from both infected and uninfected chickens were sampled and weighed. The bursal weight index was calculated by dividing the value of bursal weight (g) by the value of chicken body weight (kg).
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In addition, bursa of Fabricius and liver were examined throughout the experimental period for the observation of histopathological lesions by hematoxylin and eosin (HE) staining and for the detection of viral antigen by immunoperoxidase staining. To observe the ultrastructural lesions of chicken livers infected with IBDV, chicken livers at 2, 3, 6 d.p.i., as well as control groups were collected for transmission electron microscopy (TEM) examination. The livers at 12, 24 h.p.i. and 2, 3, 4, 5, 6 d.p.i. and these in control groups were harvested for examination of hepatic glycogens by Periodic Acid-Schiff (PAS) staining. The bursa and liver at 3 d.p.i. and 4 d.p.i., as well as these in control groups were collected for the production of cryostat sections to detect chicken macrophages in bursa and liver by immunoperoxidase staining. The cryostat sections of bursa and liver at 4 d.p.i. were prepared to colocalize viral antigens with macrophages by immunofluorescence double staining.
2.2.1. Histopathological examination The bursa and liver tissues of five IBDV-infected and five uninfected control chickens were collected at 12, 24 h.p.i. and 2, 3, 4, 5, 6 d.p.i., and then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 days at 4 8C, then were dehydrated in ascending grades of ethyl alcohol, cleared in benzene and embedded in melted paraffin wax (P3808, Sigma–Aldrich, St. Louis, MO, USA). Paraffin sections were cut into 4 mm thick, deparaffinized in xylene, rehydrated by passing through descending grades of ethyl alcohol to distilled water, and stained by hematoxylin and esosin, mounted with neutral balsam. The stained sections were observed under microscope for assessing histopathological changes. 2.2.2. Transmission electron microscopy (TEM) The tissue samples of liver (1 mm-thick) at 2, 3, 6 d.p.i. and in IBDV-uninfected chickens were fixed overnight at
Fig. 1. Microscopical lesions in bursa of Fabricious and liver following IBDV infection. SPF chickens were inoculated with IBDV strain BC6/85. Paraffin sections of bursa (left control, A and B; 20) and liver (right control, C and D; 40) were prepared to examine the histopathological changes by HE staining. At 3 d.p.i., the follicular lymphoid apoptosis and necrosis, as well as the bursal edema and congestion were observed in bursa (A). The detectable changes in liver were congestion in hepatic sinusoid and fatty degeneration in hepatocytes (C). At 6 d.p.i., there were lymphoid depletion in lymphoid follicles and fibrillation in interfollicular areas in bursa (B). The liver tissue was severely damaged (D). Control represents the uninfected control groups.
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48C in 2.5% glutaraldehyde-2%paraformaldehyde in 5 mM sodium phosphate (pH 7.4)-buffered 0.9% (w/v) saline (PBS), gently rinsed in the 0.1 M sodium phosphate buffer (PB; pH 7.2), postfixed with 1% osmium tetroxide in 0.1 M PB and rinsed thoroughly with the same buffer again. The tissues were then dehydrated through a series of ascending grades of acetone at room temperature, embedded in SUPRR resin, and polymerized at 608C for 2 days. Subsequently, they were cut into 70 nm-thick sections on a Leica ultramicrotome (Austria), and stained with uranyl acetate for 8 min, followed by lead citrate for 4 min.
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The ultrathin sections were examined under transmission electron microscope (JEM-1230, Japan). 2.2.3. Periodic Acid Schiff (PAS) staining The prepared paraffin sections of liver were deparaffinized and hydrated to water and then oxidized in 0.5% periodic acid solution for 10 min. After being rinsed 3 times in distilled water, the sections were stained in Schiff reagent for 30 min at 37 8C in darkness, washed in the running tap water, subsequently counterstained with hematoxylin, dehydrated and mounted with neutral balsam.
Fig. 2. Detection of viral antigens in bursa of Fabricious and liver at different intervals. SPF chickens were inoculated with IBDV strain BC6/85. Bursa of Fabricious and liver were harvested at 12, 24 h post infection (h.p.i.) and 2, 3, 4, 5, 6 days post infection (d.p.i.). Paraffin sections of bursa and liver were prepared to detect the distribution of viral antigens by immunoperoxidase staining using rabbit anti-IBDV antiserum. Representative sections of bursa (upper control and A; 40) and liver (lower control and B; 40) at 3 d.p.i. were shown. Brown color (arrow) indicates the positive staining. IBDV-positive cells in bursa (C) and liver (D) were counted (40) in five fields/bursa or liver/chicken at each designated time point. The values represent the mean SE of five bursa or liver. Control represents virus-free control groups.
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Fig. 3. Ultrastructural lesions of chicken liver after IBDV infection. There were large electron-lucent glycogen deposits (arrow in 1) and numerous mitochondria and rough endoplasmic reticulum (RER) in the hepatocyte of the virus-free control group. And the nucleus of a normal hepatocyte was round and large (1). At 2 d.p.i., Numerous swollen mitochondria (arrows in 2 and 3) scattered in the cytoplasm of hepatocytes (H), and deformed nucleus (3) and enlarged smooth endoplasmic reticulum were observed (arrowheads in 2). At 3 d.p.i., A lot of lipid droplets with electron-dense ring around them (arrow in 4) were found within the cytoplasm of hepatocytes (H) (4). The chromatin of a hepatocyte (H) peripherally condensed and so the hepatocyte (H) was at the early stage of apoptosis (5). At 6 d.p.i., Many patches of no electron-density were present throughout the hepatic cytoplasm and there was no clear
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2.2.4. Immunoperoxidase staining For immunolabeling of viral antigens and chicken monocyte/macrophage KUL01, we prepared separately paraffin sections and cryostat sections. The paraffin sections of liver and bursa (4 mm-thick) were deparaffinized and hydrated. For the preparation of cryostat sections, tissue samples were cut into 8 mm-thick sections on a freezing microtome after cryoprotection with 30% (w/v) sucrose in PBS at 48C overnight. The endogenous peroxidase activities were removed with 3% (v/v) hydrogen peroxide (H2O2) in PBS, and the non-specific binding sites were blocked by 10% normal goat serum. Without washing, Sections were incubated overnight in a humidified chamber at 48C with rabbit anti-IBDV antibody (1:20000) for paraffin sections or mouse anti-chicken monocyte/macrophage KUL01 (8420-01; Southern Biotech) (1:400) for cryostat sections, respectively. After being rinsed three times for 10 min each with PBS containing 0.05% (v/v) Tween 20 (PBST), the sections were incubated for 20 min with biotinylated goat antirabbit IgG or biotinylated goat anti-mouse IgG at 37 8C and then with HRP conjugated streptavidin for 20 min. After a rinse with PBST, the sections were reacted for 5–10 min with 0.02% (w/v) 3,3-diaminobenzidine (DAB)-4HCl (Zhongshan Golden Bridge Biotechnology Co. Ltd, China) and 0.001% (v/v) H2O2 in 50 mM Tris-HCl (pH 7.6). Subsequently, the sections were counterstained with hematoxylin, dehydrated and mounted. The procedure for negative control sections was the same as described above except the primary antibodies were substituted with PBS. In the experiment of the detection of viral antigens, IBDV positive cells were statistically determined at 40 magnification after counting five fields/bursa or liver/ chicken. 2.2.5. Immunofluorescence double staining Cryostat sections of liver and bursa (8 mm-thick) at 4 days post infection (d.p.i.) restored from 20 8C to room temperature, rinsed three times in PBS. The non-specific binding sites were blocked by normal goat serum. Without washing, sections were incubated overnight in a humidified chamber at 4 8C with rabbit anti-IBDV antibody (1:20000). After incubation with FITC-conjugated goat anti-rabbit IgG (whole molecule; F0382; Sigma–Aldrich) at 37 8C for 30 min, the sections were incubated with mouse anti-chicken monocyte/macrophage KUL01 (8420-01; Southern Biotech) (1:400) in the presence of 10% (v/v) normal rabbit. Subsequently, the sections were incubated with biotinylated goat anti-mouse IgG at 37 8C for 30 min and then with 5 mg/ml AlexaFluor594-conjugated streptavidin (S11223; Invitrogen) at 37 8Cfor 30 min. The sections were finally mounted with glycerin buffer, and examined under fluorescence microscope (SM-33TCI) immediately. The populations of IBDV positive cells,
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KUL01 positive cells and double positive cells were counted in five fields/bursa or liver/chicken at 20 magnification. 2.2.6. Statistical analysis All the data were analyzed by one way ANOVA followed by multiple comparisons (LSD test) using SPSS Statistics Base 17.0 software (SPSS Inc., Chicago, USA). The value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. Clinical signs and gross macroscopical changes In the first 24 h after infection, no clinical signs were observed in the chickens infected with IBDV Chinese virulent strain BC6/85. IBDV-infected chickens started to show depression and ruffled feathers at 2 d.p.i. Two out of thirty-five chickens died of infection at 3 d.p.i., whereas one dead chicken was found at 4 d.p.i. Chickens recovered gradually from viral infection after 4 d.p.i. Eventually, clinical signs were hardly seen at 6 d.p.i. During postmortem examination, gross macroscopical changes of edema, atrophy and hemorrhage could be found in the bursa, branched hemorrhage in liver, and ecchymotic hemorrhage in the thigh muscles, in accordance with the description of Wang et al. (2011). The bursal weight index of IBDV-infected chickens experienced a gradual decrease from 12 h.p.i. to 6 d.p.i., which also had the similar results with the investigation of Wang et al. (2011). The index between 12 h.p.i. and 3 d.p.i. was higher than that of the uninfected group, while it was lower than that of the uninfected group from 4 d.p.i. to 6 d.p.i. In particular, the index at 6 d.p.i. was statistically significantly different from that of the control group (p < 0.01). 3.2. Microscopical changes in bursa of Fabricious and liver In IBDV-infected chickens, microscopical changes were first observed in bursa and liver at 2 d.p.i. The bursa lesions from 2 to 4 d.p.i. were characterized by lymphocytic necrosis, apoptosis, pyknotic nucleus, depletion in lymphoid follicles, as well as heterophil accumulation, bursal edema and congestion and hemorrhage in bursa tissues (Fig. 1(A)), in agreement with the observation of Tanimura et al. (1995). In addition, they were featured by the lymphocytic depletion in lymphoid follicles and the fibrillation in interfollicular areas from 5 to 6 d.p.i. (Fig. 1(B)). A large number of heterophils were recruited into hepatic sinusoid at 2 d.p.i. The livers in IBDV-infected chickens at 3 d.p.i. were diffusely affected by the congestion in central veins and in hepatic sinusoid, as well as the fatty degeneration in hepatocytes (Fig. 1(C)). The microscopical
demarcation between neighboring hepatocytes (H) (6). In virus-free chickens, a Kupffer cell (K) with a nucleus of irregular shape and numerous rough endoplasmic reticulums (RER) harbored in the sinusoidal lumen, and there was a secondary lysosome in its medium electron-dense cytoplasm (7). And an intercalated cell (Int) with electron-lucent cytoplasm interposed between hepatocytes (H) and was adjacent to the bile canaliculi (Bi) (9). At 2 d.p.i., the Kupffer cell (K) had close contact with the endothelial cell (En), and the disintegration of its mitochondria and RER, as well as the denudation of its ribosomes, gave rise to the swollen-like and low electron-dense cytoplasm (8). And the intercalated cell (Int) phagocytosed more nucleus debris and apoptosis bodies (arrow in 10).
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structure of liver was so severely damaged and disordered that hepatic sinusoid could not be easily distinguishable (Fig. 1(D)). 3.3. Detection of viral antigens in bursa of Fabricious and liver by immunoperoxidase staining To determine whether the pathological changes of livers in IBDV-infected chickens were related to IBDV proliferation, we detected viral proteins in bursa and liver by immunoperoxidase staining, and meanwhile, made a comparison of viral infectious characteristics between bursa, the target organ of IBDV infection, and liver. As a result, abundant viral proteins could be detected in the bursa tissues from 2 to 6 d.p.i., and in the liver from 2 to 4 d.p.i., respectively. Viral antigens in bursa at 4 d.p.i. reached a peak, while the peak population of IBDV positive cells in liver was at 3 d.p.i. The number of IBDV positive cells in bursa of Fabricious was about one and half times at 3 d.p.i. and three times at 4 d.p.i., respectively, more than that of IBDV positive cells in liver at corresponding time (Fig. 2(C and D)). Viral antigens scattered in the cortex and medulla of bursa, as well as in the interfollicular interstitium (Fig. 2(A)). The positive signals in chicken liver were largely seen in sinusoidal cells, or even in the monocytes of liver venule (Fig. 2(B)). No IBDV antigens were detected in the bursa and liver from virus-free control chickens. 3.4. Ultrastructural pathological changes in the chicken liver Based on above research, we have learned that the second day after infection was the early stage, and the third day was the peak stage, and the sixth day was the late stage in terms of IBDV replication and its microscopically damaged effects. Therefore, we selected the liver samples at 2, 3 and 6 d.p.i. to examine their ultrastructual changes after IBDV infection. There were a variety of pathological changes in chicken liver on the sub-cellular scale after infection with IBDV strain BC6/85. To be specific, the mitochondria in hepatocytes swelled and mitochondrial cristae fractured at 2 d.p.i. (Fig. 3(2 and 3)). The nucleus of
hepatocytes deformed into an irregular shape instead of a round one (Fig. 3(3 and 4)). Additionally, the flattened cisternaes of smooth endoplasmic reticulum (SER) were enlarged into vesicles in various sizes at 2 d.p.i. (Fig. 3(2)). Few lipid droplets were seen in the hepatocytes of the control group (Fig. 3(1)). In comparison, up to 3 d.p.i., the electron-lucent lipid droplets in various sizes were observed within the cytoplasm of hepatocytes, and an electron-dense ring tended to form around lipid globules (Fig. 3(4)). Some nucleus of hepatocytes revealed the typical feature of apoptosis, such as peripheral condensation of chromatin (Fig. 3(5)). Although hepatic glycogens appeared large electron-lucent deposits with amorphous appearance throughout the cytoplasm in the control group (Fig. 3(1)), there were no detectable glycogen deposits in hepatocytes at 2 and 3 d.p.i. However, interestingly, hepatic glycogens were dispersedly detected at 6 d.p.i. again. Kupffer cells and monocytes appeared ‘‘swollenlike’’ and took the form of lower electron-density cytoplasm by comparison with the control group as most organelles were damaged by infection with IBDV, for example, membrane-bound ribosomes denuded from rough endoplasmic reticulum (RER) into free ribosomes, and lysosomes, phagolysomes, and mitochondria were less found in the cytoplasm of Kupffer cells (Fig. 3(7 and 8)). Only few swollen mitochondria distributed throughout the cytoplasm of monocytes. Intercalated cells scattered in the Disse’ s spaces or between hepatocytes and there were much more engulfment of cell debris in the cytoplasm of intercalated cells as compared to the control group (Fig. 3(9 and 10)). In addition, at 2 d.p.i., Numerous heterophils, containing high electron-dense granules in the ovoid or rodshaped shape throughout the cytoplasm, were observed in the sinusoidal lumen, and a small amount of lymphocytes were recruited into hepatic sinusoids. 3.5. The significant decrease of hepatic glycogen in the liver of IBDV-infected chickens After observing the changes of glycogen granules in the liver of IBDV-infected chickens under sub-cellular level by TEM, PAS staining was used to further verify these findings.
Fig. 4. Changes of hepatic glycogen deposits after IBDV infection. SPF chickens were infected with IBDV strain BC6/85 and the livers were collected at 12, 24 h post infection (h.p.i.) and 2, 3, 4, 5, 6 days post infection (d.p.i.). Paraffin sections of liver were prepared to detect hepatic glycogen deposits by Periodic Acid-Schiff (PAS) staining. Numerous PAS positive cells (arrow) were seen in normal liver (Control; 40) and in liver at 6d.p.i. (B; 40). There were no PAS positive cells in liver at 3 d.p.i. (A; 40 ).
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The PAS staining of chicken livers suggested that a large number of hepatic glycogens were stored in the cytoplasm of hepatocytes in IBDV-uninfected chickens, whereas they decreased dramatically from 2 to 5 d.p.i. in IBDV-infected groups. However, glycogens started to reappear within the hepatic cytoplasm at 6 d.p.i. (Fig. 4). The results were consistent with the above observation by TEM in chicken livers. 3.6. Exposure to IBDV resulted in infiltration of macrophages in bursa and in liver As the above experiments investigated, viral antigens in bursa and liver primarily replicated at 3 d.p.i. and 4 d.p.i. Previous studies have showed that the number of macrophages changed in the bursa of IBDV-infected chickens (Rauf et al., 2011; Khatri et al., 2005). We therefore speculated that viral replication correlated with macrophage changes in bursa and in liver. So in the present study, to determine whether macrophages are infiltrated in bursa and liver of IBDV-infected chickens, Bursa and liver at 3 d.p.i. and 4 d.p.i. were examined by immunohistochemistry (IHC) using the antibody against chicken monocytes/macrophages (KLU01). The results showed that the infiltration of KLU01 positive (KUL01+) cells in bursa and in liver was significantly apparent at 3 d.p.i. and 4 d.p.i. Especially, KUL01+ cells mainly distributed in the interfollicular interstitium of bursa. Few KUL01+ cells were detected in the bursa and liver of uninfected chickens by
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immunoperoxidase staining. Representative sections of bursa and liver at 3 d.p.i. and the uninfected group were shown in Fig. 5. 3.7. IBDV replicated in the Kupffer cells of liver Previously published data demonstrated that macrophages in bursa were infected with IBDV (Ka¨ufer and Weiss, 1976; Savova and Bozhkov, 1985). Meanwhile, present studies indicated that IBDV caused severe ultrastructural pathological lesions in the Kupffer cells of chicken liver. Viral antigens could be detected in liver. In view of these findings, we next investigated whether IBDV replicated in Kupffer cells or other cells in liver. Accordingly, we co-localized viral antigens with Kupffer cells in liver at 4 d.p.i. by immunofluorescence double staining using the rabbit anti-IBDV polyclonal antibodies and KUL01 antibodies, because the KUL01 antibody primarily marks Kupffer cells in chicken liver (Mast et al., 1998). Furthermore, we compared the different features of the colocalization of macrophages and viral antigens between in bursa and in liver. The results showed that the percentage of double positive cells (IBDV positive and KUL01 positive cells) accounted for 26.5 percent of the total IBDV positive cells or 57 percent of the total KUL01 positive (KUL01+) cells in bursa at 4 d.p.i. Numerous KUL01+ cells in the interfollicular area of bursa were negative for IBDV, which suggested that these subpopulations of macrophages are insusceptible to viral infection. Additionally, a lot of IBDV
Fig. 5. Infiltration of macrophages in bursa and liver. SPF chickens were inoculated with IBDV strain BC6/85 and bursa of Fabricious and liver were harvested at 3 and 4 days post infection (d.p.i.). Cryostat sections of bursa and liver were prepared to detect macrophages by immunoperoxidase staining using KUL01 antibody. Representative sections of bursa (upper control and A; 20) and liver (lower control and B; 40) at 3 d.p.i. were shown. Brown color (arrow) indicates the positive staining. KUL01 positive (KUL01+) cells predominantly distributed between lymphoid follicles in bursa (A).
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Fig. 6. Colocalization of IBDV and macrophages in bursa and liver. SPF chickens were inoculated with IBDV strain BC6/85. Bursa and liver were harvested at 4 days post infection (d.p.i.). Cryostat sections of bursa (A, B and upper Merge; 20 ) and liver (C, D and lower Merge; 20) were prepared to colocalize macrophages with IBDV by immunofluorescence double staining. The first primary antibody was rabbit anti-IBDV antibody, detecting IBDV proteins, and was visualized with FITC conjugate-goat anti-rabbit IgG (A and C). The second primary antibody was mouse anti-chicken monocyte/macrophage KUL01 and was visualized with AlexaFluor594- conjugated streptavidin (B and D). An overlay of immunofluorescent pictures was shown (Merge). IBDV positive cells, KUL01 positive cells and double positive cells were counted in five fields/bursa or liver/chicken at 20 magnification. The values represent the mean SE of five bursa or liver (E).
positive cells were negative for KUL01 marker, since IBDV mainly infected B lymphocytes in bursa. By contrast, the proportion of double positive cells in liver at 4 d.p.i. comprised 97 percent of the total IBDV positive cells or 99 percent of the total KUL01+ cells (Fig. 6). Data indicated that IBDV mainly proliferated in the Kupffer cells rather than hepatocytes or endothelial cells of liver.
4. Discussion Previous studies have clarified that IBDV mainly caused an acute infection in the dividing immunoglobulin Mbearing (IgM+) B lymphocytes and targeted the bursa tissues in its susceptible period (Ka¨ufer et al., 1980; Hirai et al., 1981). Researchers have attached importance to the
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lymphoid organs, such as, bursa, spleen, as well as thymus, rather than liver. Therefore, in the present study, we investigated the effects of IBDV on the chicken liver by comparing with its impacts on the bursa from the pathological perspective. Specifically, our data suggested that IBDV infection resulted in the severe lesions of chicken liver microscopically and ultrastructurally. Moreover, our results indicated that Kupffer cells were susceptible to IBDV due to the complete colocalization between viral antigens and macrophages examined by double immunofluorescence labeling, and provided further evidences for elucidation of IBDV pathogenesis. The liver glycogen is the storage form of carbohydrates and is a crucial source of energy. So the glycogen synthesis and degradation are tightly regulated. In the present study, the glycogen deposits in hepatocytes significantly dropped after viral infection, suggesting that IBDV infection inhibited the glycogen synthesis or accelerated the process of glycogen degradation. Admittedly, the possibility that the decrease of hepatic glycogen was due to the decreasing food intakes during infectious periods could not be excluded in this experiment. However, hepatic glycogen was detected again at 6 d.p.i. by transmission electron microscopy (TEM), and the result was subsequently confirmed by PAS staining (Fig. 4). Our data thereby demonstrated that the hepatic glycogen might play an important part in the restoration of damaged liver cells caused by viral infection. Ohata et al. (1982) pointed out that extrasinusoidal macrophages in chicken liver showed phagocytic activity, and they speculated that these macrophages might be the precursors of Kupffer cells. Ghoddusi and Kelly (2004) named these macrophages intercalated cells, because they mostly interposed between hepatocytes or in Disse’s spaces. In the same research work, authors proposed that intercalated cells were capable of self-proliferation in situ and mainly took two different forms, a poorly differentiated form, as well as a relatively highly differentiated form which was associated with the phagocytic activity. In this study, we noted that intercalated cells possessed numerous phagosomes containing cell debris after chicken infection with IBDV. In addition, at 6 d.p.i., intercalated cells could be frequently found between destroyed hepatocytes. In contrast, we just occasionally saw intercalated cells in the control group. Thus, our data indicated that these intercalated cells might take part in inhibiting viral infection and promoting recovery of liver tissue from infection. Hume (2006) once reviewed that some macrophages played a tropic role for other nearby cells in development and homeostasis. So we infer that the function of these tropic macrophages in mice is probably comparable to that of intercalated cells in birds. Nevertheless, the genuine roles and characteristics of intercalated cells are still unclear and need to be proven further. IBDV induced the degeneration, necrosis and apoptosis of hepatic cells that, however, are not susceptible to IBDV. These results might be attributable to damage effects of the inflammatory response resulting from viral infection, which is supported by evidences that numerous heterophils, a kind of well-known inflammatory cells, were seen at 2 d.p.i. by HE staining and TEM. Furthermore, the
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inflammatory response recruited activated macrophages into bursa and liver, which may explain the reason why IBDV infection resulted in infiltration of macrophages into bursa and that of Kupffer cells into liver in the present study. Similar to such our findings, Rauf et al. (2011) reported the infiltration of macrophages in IBDV-infected bursa by immnohistochemistry. Immunofluorescence double staining was used to colocalize IBDV with the macrophages in bursa and liver. As shown in Fig. 6, a large number of KUL01+ cells (accounting for 99 percent of the total KUL01+ cells) colocalized with viral proteins in liver. In addition, we saw severe ultrastructural lesion changes of Kupffer cells in liver by SEM after IBDV infection. Considering these evidences, we could rule out the possibility that viral antigens were phagocytosed by Kupffer cells, and therefore come to a conclusion that IBDV targeted Kupffer cells and replicated in them. By contrast, in bursa, there were a relatively small number of KUL01+ cells (accounting for roughly 57 percent of the total KUL01+ cells) to be colocalized with IBDV, suggesting that a minority of macrophages in bursa were infected with IBDV. Ka¨ufer and Weiss (1976) and Savova and Bozhkov (1985) observed viral particles in macrophages of bursa using transmission electron microscopy (TEM), which demonstrated that IBDV replicated in the macrophages of bursa. Similarly, IBDV was also detected in splenic macrophages (Palmquist et al., 2006). In the present study, our studies showed that the colocalization of macrophages and IBDV took place in bursa and liver in IBDV-exposed chickens. As these evidences demonstrated, we may therefore draw the conclusion that macrophages in various tissues infected with IBDV in vivo, although there were different characteristics of infection in these tissues, for example, Kupffer cells in liver completely colocalized with IBDV positive cells, whereas macrophages in bursa partially colocalized with IBDV. As a consequence, in addition to destroyed B lymphocytes, our study may be suggested that these IBDV-infected macrophages are a crucial factor leading to the immunosuppressive effects after viral infection, since macrophages are one of the most important immune cells both in the innate immunity and acquired immunity. Nonetheless, there are no specific data regarding the pathway of macrophages infection with IBDV. Therefore, further studies should be done to demonstrate whether there are specific receptors in macrophages to facilitate viral infection. Conflict of interest statement The authors have no conflicts of interest. Acknowledgements This study was supported by the ‘‘Twelfth Five YearPlan’’ of the National Science and Technology Support Project (2011BAD34B01) and Chinese Universities Scientific Fund (2011JS008) We thank Junzhen Jia and Haihong Liu for their technical assistance in transmission electron microscopy.
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Susceptibility of chicken Kupffer cells to Chinese virulent infectious bursal disease virus Haiyan Ma, Sufen Zhao, Yunfei Ma, Xin Guo, Deping Han, Yuanyuan Jia, Weiwei Zhang, Kedao Teng * College of Veterinary Medicine, China Agricultural University, Beijing, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 November 2012 Received in revised form 25 February 2013 Accepted 1 March 2013
Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although the effects of IBDV on bursa of Fabricius in chickens have been well reported, the impacts of IBDV on liver after IBDV infection are still unclear. In the present study, specific pathogen free (SPF) chickens were experimentally inoculated with IBDV Chinese virulent strain BC6/85, and the cells in liver and bursa were examined by immunohistochemisrty and transmission electron microscopy (TEM). The congestion of liver tissue and fatty degeneration of hepatocytes were characteristics of microscopical changes in chicken liver at 3 days post infection (d.p.i.), whereas there were follicular lymphoid necrosis, apoptosis, depletion, as well as edema and congestion in bursa. In addition, the number of IBDV-positive cells peaked at 4 d.p.i. in bursa and at 3 d.p.i. in liver, respectively. With respect to ultrastructural pathological changes of hepatocytes, mitochondria swelled and nucleus deformed into an irregular shape or its chromatin peripherally condensed which indicated that the hepatocyte was at the early stage of apoptosis, and the electron-lucent lipid droplets in a variety of sizes were observed within cytoplasm. Kupffer cells became ‘‘swollen-like’’ and the electron-density of their cytoplasm was lower than that of cells in uninfected group. Liver glycogen deposits significantly declined from 2 to 5 d.p.i. and recovered strongly at 6 d.p.i. More importantly, KLU01 (macrophage marker) positive (KUL01+) cells were infiltrated in bursa and liver in IBDV-exposed chickens by immunoperoxidase staining. To demonstrate the correlation between IBDV and macrophages in bursa and liver, we further investigated the colocalization of viral antigens and macrophages by double immunofluorescence labeling. At 4 d.p.i., the percentage of double positive cells (IBDV positive and KUL01+ cells) accounted for 26.5 percent of the total IBDV positive cells or 57 percent of the total KUL01+ cells in bursa. In comparison, the percentage of double positive cells in liver constituted 97 percent of the total IBDV positive cells or 99 percent of the total KUL01+ cells. These results suggest that IBDV was susceptible to KUL01+ cells in liver (mainly Kupffer cells) and replicated in the KUL01+ cells. By comparison with the influence of IBDV on bursa, our findings were the first to elucidate the pathological changes in liver after IBDV infection on a microscopical and ultrastructural scale, and, especially, to gain the initial insight into the susceptibility of Kupffer cells to IBDV. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Infectious bursal disease virus (IBDV) Histopathology Liver Kupffer cells Immunohistochemisrty Transmission electron microscopy(TEM)
1. Introduction * Corresponding author at: College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, People’s Republic of China. Tel.: +86 01 62733013. E-mail addresses: [email protected], [email protected] (K. Teng). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.03.002
Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is a highly contagious and immunosuppressive viral disease in young chickens during
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3 to 6 weeks of age. IBDV infection may aggravate infection with other pathogenic agents, and IBDV-infected chickens have the low ability of response to subsequent vaccinations (Mu¨ller et al., 2003). IBDV can be classified into two serotypes (serotype 1 and 2) by virus neutralization test (McFerran et al., 1980). Serotype 1 strains, differentiated as classical strains (cIBDV), very virulent strains (vvIBDV) and variant strains (vIBDV), can cause pathogenicity and mortality in young chickens, while serotype 2 strains are avirulent (Liu et al., 2010; Rauf et al., 2011). The causative agent, IBDV, belongs to the family Birnaviridae, and is non-enveloped, double-stranded (ds) RNA virus consisting of two segments (segment A and segment B) (Dobos et al., 1979). Segment A encodes the VP5, VP4 (28 kDa), VP3 (32 kDa), and VP2 (41 kDa), and segment B encodes VP1 (98 kDa) (Mundt et al., 1995; Birghan et al., 2000; Von Einem et al., 2004). IBDV infects and destroys actively dividing IgM-bearing B cells in the bursa of Fabricius (Hirai et al., 1981; Rodenberg et al., 1994). Additionally, the extensive viral replication is present in bursa (Dobos et al., 1979). So viral infection causes severe depletion of lymphocytes and destruction of bursa tissues of chickens infected with IBDV (Ka¨ufer and Weiss, 1980). Although bursa is the principal target organ for IBDV, recent studies show that the virus also infects and possibly replicates in other organs, such as spleen, thymus, bone marrow, cecal tonsil, liver and kidney, at early stage of infection (Tanimura et al., 1995; Oladele et al., 2009). It has been shown that B cells may not be the sole target for the virus. Macrophages may serve as host cells for IBDV (Khatri et al., 2005; Ka¨ufer and Weiss, 1976; Savova and Bozhkov, 1985; Palmquist et al., 2006). Macrophages, on the one hand, can phagocytize and clear pathogens by recognizing pathogen-associated molecular patterns (PAMPs) via their pattern-recognition receptors (PRRs) in innate immune system (Akira et al., 2006). On the other hand, macrophages secrete a variety of functional cytokines which can act as immunomodulatory, antiviral or proinflammatory factors, such as typeI interferon (IFN-a and IFN-b), IL-6, TNF-a, etc (Zhou, 2007). However, the role of macrophages in the pathogenesis of IBDV has not been extensively examined so far. It has been reported that gut-associated macrophages are hypothesized to be the initial transporters of IBDV from the digestive tract to the bursa and other peripheral organs (Ka¨ufer and Weiss, 1976). There are reports that infection with IBDV causes the production of proinflammatory mediators and cytokines by macrophages. The levels of induced proinflammatory mediators and cytokines reached the peak during active virus replication and correlated with extensive inflammatory response in both chicken bursa and spleen (Kim et al., 1998; Khatri et al., 2005; Palmquist et al., 2006). These macrophages were activated by viral infection and upregulated mRNA expression of IL-1b, IL-6, IL-18, and iNOS (Palmquist et al., 2006). Kupffer cells (liver macrophages) represent one of the largest reservoirs of resident tissue macrophages, which are located in the liver sinusoids in close contact with endothelial cells. As for mammals, in addition to Kuffer cells, there are endothelial cells in the hepatic sinusoid. The
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Disse’s space of liver harbors fat-storing cells and pit cells (Wisse et al., 1996). There are some distinctive characteristics of bile ductules and sinusoidal cells between mammals and chickens, although the ultrastructure of chicken liver is principally similar to that of rat or human liver. Investigation of ultrastructural chicken liver revealed that intercalated cells (extra-sinusoidal macrophages of chicken liver), a unique kind of chicken hepatic parenchyma cells, mostly present in interhepatocytic or Disse’s spaces (Ohata et al., 1982; Ohata and Ito, 1986; Ghoddusi and Kelly, 2004). So far, little is known about the pathological damage of virus replication to chicken livers and the susceptibility of Kupffer cells to virus during the acute phase of the IBDV infection. Thus, in the present study we examined the impacts of IBDV infection on chicken liver tissues and, in particular, on Kupffer cells by immunohistochemisrty and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Animals, viruses and rabbit anti-IBDV serum The specific-pathogen-free (SPF) male white Leghorn chickens were purchased from Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China) and reared in CC. JH-1 type positive pressure isolation units which were purchased from Jinhang Purifying Air Conditioning Equipment Co., Ltd. (Tianjin, China) under the supervision of the Animal Facility Management of China Agricultural University. Chickens for different experimental groups were housed in the separate isolation units. Water and feed were provided ad libitum during the entire experimental period. Virulent strain of IBDV (BC6/85, CVCC AV7) was obtained from China Institute of Veterinary Drug Control (Beijing, China). At 3 weeks of age, chickens were inoculated with 1000 50% egg infective doses (EID50) of IBDV strain BC6/85 or phosphate-buffered saline (PBS) by eye drop. Rabbit anti-IBDV serum was produced as previously described (Tanimura et al., 1995; Oladele et al., 2009). And the titer of rabbit antiserum was 1: 16 by agar gel precipitation (AGP) assay. Both IBDV (BC6/85) antigen and chicken anti-IBDV serum as a positive control in AGP assay were obtained from China Institute of Veterinary Drug Control (Beijing, China). 2.2. Experimental design A total of seventy 3-week-old SPF chickens were randomly distributed into two groups (the IBDV-exposed group and the control group) of 35 birds each, and were separately weighed before their sacrifice. IBDV-exposed birds were inoculated with IBDV strain BC6/85 and were euthanized (5 birds once) at 12, 24 h post infection (h.p.i.) and 2, 3, 4, 5, 6 days post infection (d.p.i.), and control birds were inoculated with PBS and were euthanized (5 birds once) at the same intervals. Of these chickens, the organs of bursa of Fabricius from both infected and uninfected chickens were sampled and weighed. The bursal weight index was calculated by dividing the value of bursal weight (g) by the value of chicken body weight (kg).
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In addition, bursa of Fabricius and liver were examined throughout the experimental period for the observation of histopathological lesions by hematoxylin and eosin (HE) staining and for the detection of viral antigen by immunoperoxidase staining. To observe the ultrastructural lesions of chicken livers infected with IBDV, chicken livers at 2, 3, 6 d.p.i., as well as control groups were collected for transmission electron microscopy (TEM) examination. The livers at 12, 24 h.p.i. and 2, 3, 4, 5, 6 d.p.i. and these in control groups were harvested for examination of hepatic glycogens by Periodic Acid-Schiff (PAS) staining. The bursa and liver at 3 d.p.i. and 4 d.p.i., as well as these in control groups were collected for the production of cryostat sections to detect chicken macrophages in bursa and liver by immunoperoxidase staining. The cryostat sections of bursa and liver at 4 d.p.i. were prepared to colocalize viral antigens with macrophages by immunofluorescence double staining.
2.2.1. Histopathological examination The bursa and liver tissues of five IBDV-infected and five uninfected control chickens were collected at 12, 24 h.p.i. and 2, 3, 4, 5, 6 d.p.i., and then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 days at 4 8C, then were dehydrated in ascending grades of ethyl alcohol, cleared in benzene and embedded in melted paraffin wax (P3808, Sigma–Aldrich, St. Louis, MO, USA). Paraffin sections were cut into 4 mm thick, deparaffinized in xylene, rehydrated by passing through descending grades of ethyl alcohol to distilled water, and stained by hematoxylin and esosin, mounted with neutral balsam. The stained sections were observed under microscope for assessing histopathological changes. 2.2.2. Transmission electron microscopy (TEM) The tissue samples of liver (1 mm-thick) at 2, 3, 6 d.p.i. and in IBDV-uninfected chickens were fixed overnight at
Fig. 1. Microscopical lesions in bursa of Fabricious and liver following IBDV infection. SPF chickens were inoculated with IBDV strain BC6/85. Paraffin sections of bursa (left control, A and B; 20) and liver (right control, C and D; 40) were prepared to examine the histopathological changes by HE staining. At 3 d.p.i., the follicular lymphoid apoptosis and necrosis, as well as the bursal edema and congestion were observed in bursa (A). The detectable changes in liver were congestion in hepatic sinusoid and fatty degeneration in hepatocytes (C). At 6 d.p.i., there were lymphoid depletion in lymphoid follicles and fibrillation in interfollicular areas in bursa (B). The liver tissue was severely damaged (D). Control represents the uninfected control groups.
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48C in 2.5% glutaraldehyde-2%paraformaldehyde in 5 mM sodium phosphate (pH 7.4)-buffered 0.9% (w/v) saline (PBS), gently rinsed in the 0.1 M sodium phosphate buffer (PB; pH 7.2), postfixed with 1% osmium tetroxide in 0.1 M PB and rinsed thoroughly with the same buffer again. The tissues were then dehydrated through a series of ascending grades of acetone at room temperature, embedded in SUPRR resin, and polymerized at 608C for 2 days. Subsequently, they were cut into 70 nm-thick sections on a Leica ultramicrotome (Austria), and stained with uranyl acetate for 8 min, followed by lead citrate for 4 min.
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The ultrathin sections were examined under transmission electron microscope (JEM-1230, Japan). 2.2.3. Periodic Acid Schiff (PAS) staining The prepared paraffin sections of liver were deparaffinized and hydrated to water and then oxidized in 0.5% periodic acid solution for 10 min. After being rinsed 3 times in distilled water, the sections were stained in Schiff reagent for 30 min at 37 8C in darkness, washed in the running tap water, subsequently counterstained with hematoxylin, dehydrated and mounted with neutral balsam.
Fig. 2. Detection of viral antigens in bursa of Fabricious and liver at different intervals. SPF chickens were inoculated with IBDV strain BC6/85. Bursa of Fabricious and liver were harvested at 12, 24 h post infection (h.p.i.) and 2, 3, 4, 5, 6 days post infection (d.p.i.). Paraffin sections of bursa and liver were prepared to detect the distribution of viral antigens by immunoperoxidase staining using rabbit anti-IBDV antiserum. Representative sections of bursa (upper control and A; 40) and liver (lower control and B; 40) at 3 d.p.i. were shown. Brown color (arrow) indicates the positive staining. IBDV-positive cells in bursa (C) and liver (D) were counted (40) in five fields/bursa or liver/chicken at each designated time point. The values represent the mean SE of five bursa or liver. Control represents virus-free control groups.
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Fig. 3. Ultrastructural lesions of chicken liver after IBDV infection. There were large electron-lucent glycogen deposits (arrow in 1) and numerous mitochondria and rough endoplasmic reticulum (RER) in the hepatocyte of the virus-free control group. And the nucleus of a normal hepatocyte was round and large (1). At 2 d.p.i., Numerous swollen mitochondria (arrows in 2 and 3) scattered in the cytoplasm of hepatocytes (H), and deformed nucleus (3) and enlarged smooth endoplasmic reticulum were observed (arrowheads in 2). At 3 d.p.i., A lot of lipid droplets with electron-dense ring around them (arrow in 4) were found within the cytoplasm of hepatocytes (H) (4). The chromatin of a hepatocyte (H) peripherally condensed and so the hepatocyte (H) was at the early stage of apoptosis (5). At 6 d.p.i., Many patches of no electron-density were present throughout the hepatic cytoplasm and there was no clear
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2.2.4. Immunoperoxidase staining For immunolabeling of viral antigens and chicken monocyte/macrophage KUL01, we prepared separately paraffin sections and cryostat sections. The paraffin sections of liver and bursa (4 mm-thick) were deparaffinized and hydrated. For the preparation of cryostat sections, tissue samples were cut into 8 mm-thick sections on a freezing microtome after cryoprotection with 30% (w/v) sucrose in PBS at 48C overnight. The endogenous peroxidase activities were removed with 3% (v/v) hydrogen peroxide (H2O2) in PBS, and the non-specific binding sites were blocked by 10% normal goat serum. Without washing, Sections were incubated overnight in a humidified chamber at 48C with rabbit anti-IBDV antibody (1:20000) for paraffin sections or mouse anti-chicken monocyte/macrophage KUL01 (8420-01; Southern Biotech) (1:400) for cryostat sections, respectively. After being rinsed three times for 10 min each with PBS containing 0.05% (v/v) Tween 20 (PBST), the sections were incubated for 20 min with biotinylated goat antirabbit IgG or biotinylated goat anti-mouse IgG at 37 8C and then with HRP conjugated streptavidin for 20 min. After a rinse with PBST, the sections were reacted for 5–10 min with 0.02% (w/v) 3,3-diaminobenzidine (DAB)-4HCl (Zhongshan Golden Bridge Biotechnology Co. Ltd, China) and 0.001% (v/v) H2O2 in 50 mM Tris-HCl (pH 7.6). Subsequently, the sections were counterstained with hematoxylin, dehydrated and mounted. The procedure for negative control sections was the same as described above except the primary antibodies were substituted with PBS. In the experiment of the detection of viral antigens, IBDV positive cells were statistically determined at 40 magnification after counting five fields/bursa or liver/ chicken. 2.2.5. Immunofluorescence double staining Cryostat sections of liver and bursa (8 mm-thick) at 4 days post infection (d.p.i.) restored from 20 8C to room temperature, rinsed three times in PBS. The non-specific binding sites were blocked by normal goat serum. Without washing, sections were incubated overnight in a humidified chamber at 4 8C with rabbit anti-IBDV antibody (1:20000). After incubation with FITC-conjugated goat anti-rabbit IgG (whole molecule; F0382; Sigma–Aldrich) at 37 8C for 30 min, the sections were incubated with mouse anti-chicken monocyte/macrophage KUL01 (8420-01; Southern Biotech) (1:400) in the presence of 10% (v/v) normal rabbit. Subsequently, the sections were incubated with biotinylated goat anti-mouse IgG at 37 8C for 30 min and then with 5 mg/ml AlexaFluor594-conjugated streptavidin (S11223; Invitrogen) at 37 8Cfor 30 min. The sections were finally mounted with glycerin buffer, and examined under fluorescence microscope (SM-33TCI) immediately. The populations of IBDV positive cells,
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KUL01 positive cells and double positive cells were counted in five fields/bursa or liver/chicken at 20 magnification. 2.2.6. Statistical analysis All the data were analyzed by one way ANOVA followed by multiple comparisons (LSD test) using SPSS Statistics Base 17.0 software (SPSS Inc., Chicago, USA). The value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. Clinical signs and gross macroscopical changes In the first 24 h after infection, no clinical signs were observed in the chickens infected with IBDV Chinese virulent strain BC6/85. IBDV-infected chickens started to show depression and ruffled feathers at 2 d.p.i. Two out of thirty-five chickens died of infection at 3 d.p.i., whereas one dead chicken was found at 4 d.p.i. Chickens recovered gradually from viral infection after 4 d.p.i. Eventually, clinical signs were hardly seen at 6 d.p.i. During postmortem examination, gross macroscopical changes of edema, atrophy and hemorrhage could be found in the bursa, branched hemorrhage in liver, and ecchymotic hemorrhage in the thigh muscles, in accordance with the description of Wang et al. (2011). The bursal weight index of IBDV-infected chickens experienced a gradual decrease from 12 h.p.i. to 6 d.p.i., which also had the similar results with the investigation of Wang et al. (2011). The index between 12 h.p.i. and 3 d.p.i. was higher than that of the uninfected group, while it was lower than that of the uninfected group from 4 d.p.i. to 6 d.p.i. In particular, the index at 6 d.p.i. was statistically significantly different from that of the control group (p < 0.01). 3.2. Microscopical changes in bursa of Fabricious and liver In IBDV-infected chickens, microscopical changes were first observed in bursa and liver at 2 d.p.i. The bursa lesions from 2 to 4 d.p.i. were characterized by lymphocytic necrosis, apoptosis, pyknotic nucleus, depletion in lymphoid follicles, as well as heterophil accumulation, bursal edema and congestion and hemorrhage in bursa tissues (Fig. 1(A)), in agreement with the observation of Tanimura et al. (1995). In addition, they were featured by the lymphocytic depletion in lymphoid follicles and the fibrillation in interfollicular areas from 5 to 6 d.p.i. (Fig. 1(B)). A large number of heterophils were recruited into hepatic sinusoid at 2 d.p.i. The livers in IBDV-infected chickens at 3 d.p.i. were diffusely affected by the congestion in central veins and in hepatic sinusoid, as well as the fatty degeneration in hepatocytes (Fig. 1(C)). The microscopical
demarcation between neighboring hepatocytes (H) (6). In virus-free chickens, a Kupffer cell (K) with a nucleus of irregular shape and numerous rough endoplasmic reticulums (RER) harbored in the sinusoidal lumen, and there was a secondary lysosome in its medium electron-dense cytoplasm (7). And an intercalated cell (Int) with electron-lucent cytoplasm interposed between hepatocytes (H) and was adjacent to the bile canaliculi (Bi) (9). At 2 d.p.i., the Kupffer cell (K) had close contact with the endothelial cell (En), and the disintegration of its mitochondria and RER, as well as the denudation of its ribosomes, gave rise to the swollen-like and low electron-dense cytoplasm (8). And the intercalated cell (Int) phagocytosed more nucleus debris and apoptosis bodies (arrow in 10).
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structure of liver was so severely damaged and disordered that hepatic sinusoid could not be easily distinguishable (Fig. 1(D)). 3.3. Detection of viral antigens in bursa of Fabricious and liver by immunoperoxidase staining To determine whether the pathological changes of livers in IBDV-infected chickens were related to IBDV proliferation, we detected viral proteins in bursa and liver by immunoperoxidase staining, and meanwhile, made a comparison of viral infectious characteristics between bursa, the target organ of IBDV infection, and liver. As a result, abundant viral proteins could be detected in the bursa tissues from 2 to 6 d.p.i., and in the liver from 2 to 4 d.p.i., respectively. Viral antigens in bursa at 4 d.p.i. reached a peak, while the peak population of IBDV positive cells in liver was at 3 d.p.i. The number of IBDV positive cells in bursa of Fabricious was about one and half times at 3 d.p.i. and three times at 4 d.p.i., respectively, more than that of IBDV positive cells in liver at corresponding time (Fig. 2(C and D)). Viral antigens scattered in the cortex and medulla of bursa, as well as in the interfollicular interstitium (Fig. 2(A)). The positive signals in chicken liver were largely seen in sinusoidal cells, or even in the monocytes of liver venule (Fig. 2(B)). No IBDV antigens were detected in the bursa and liver from virus-free control chickens. 3.4. Ultrastructural pathological changes in the chicken liver Based on above research, we have learned that the second day after infection was the early stage, and the third day was the peak stage, and the sixth day was the late stage in terms of IBDV replication and its microscopically damaged effects. Therefore, we selected the liver samples at 2, 3 and 6 d.p.i. to examine their ultrastructual changes after IBDV infection. There were a variety of pathological changes in chicken liver on the sub-cellular scale after infection with IBDV strain BC6/85. To be specific, the mitochondria in hepatocytes swelled and mitochondrial cristae fractured at 2 d.p.i. (Fig. 3(2 and 3)). The nucleus of
hepatocytes deformed into an irregular shape instead of a round one (Fig. 3(3 and 4)). Additionally, the flattened cisternaes of smooth endoplasmic reticulum (SER) were enlarged into vesicles in various sizes at 2 d.p.i. (Fig. 3(2)). Few lipid droplets were seen in the hepatocytes of the control group (Fig. 3(1)). In comparison, up to 3 d.p.i., the electron-lucent lipid droplets in various sizes were observed within the cytoplasm of hepatocytes, and an electron-dense ring tended to form around lipid globules (Fig. 3(4)). Some nucleus of hepatocytes revealed the typical feature of apoptosis, such as peripheral condensation of chromatin (Fig. 3(5)). Although hepatic glycogens appeared large electron-lucent deposits with amorphous appearance throughout the cytoplasm in the control group (Fig. 3(1)), there were no detectable glycogen deposits in hepatocytes at 2 and 3 d.p.i. However, interestingly, hepatic glycogens were dispersedly detected at 6 d.p.i. again. Kupffer cells and monocytes appeared ‘‘swollenlike’’ and took the form of lower electron-density cytoplasm by comparison with the control group as most organelles were damaged by infection with IBDV, for example, membrane-bound ribosomes denuded from rough endoplasmic reticulum (RER) into free ribosomes, and lysosomes, phagolysomes, and mitochondria were less found in the cytoplasm of Kupffer cells (Fig. 3(7 and 8)). Only few swollen mitochondria distributed throughout the cytoplasm of monocytes. Intercalated cells scattered in the Disse’ s spaces or between hepatocytes and there were much more engulfment of cell debris in the cytoplasm of intercalated cells as compared to the control group (Fig. 3(9 and 10)). In addition, at 2 d.p.i., Numerous heterophils, containing high electron-dense granules in the ovoid or rodshaped shape throughout the cytoplasm, were observed in the sinusoidal lumen, and a small amount of lymphocytes were recruited into hepatic sinusoids. 3.5. The significant decrease of hepatic glycogen in the liver of IBDV-infected chickens After observing the changes of glycogen granules in the liver of IBDV-infected chickens under sub-cellular level by TEM, PAS staining was used to further verify these findings.
Fig. 4. Changes of hepatic glycogen deposits after IBDV infection. SPF chickens were infected with IBDV strain BC6/85 and the livers were collected at 12, 24 h post infection (h.p.i.) and 2, 3, 4, 5, 6 days post infection (d.p.i.). Paraffin sections of liver were prepared to detect hepatic glycogen deposits by Periodic Acid-Schiff (PAS) staining. Numerous PAS positive cells (arrow) were seen in normal liver (Control; 40) and in liver at 6d.p.i. (B; 40). There were no PAS positive cells in liver at 3 d.p.i. (A; 40 ).
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The PAS staining of chicken livers suggested that a large number of hepatic glycogens were stored in the cytoplasm of hepatocytes in IBDV-uninfected chickens, whereas they decreased dramatically from 2 to 5 d.p.i. in IBDV-infected groups. However, glycogens started to reappear within the hepatic cytoplasm at 6 d.p.i. (Fig. 4). The results were consistent with the above observation by TEM in chicken livers. 3.6. Exposure to IBDV resulted in infiltration of macrophages in bursa and in liver As the above experiments investigated, viral antigens in bursa and liver primarily replicated at 3 d.p.i. and 4 d.p.i. Previous studies have showed that the number of macrophages changed in the bursa of IBDV-infected chickens (Rauf et al., 2011; Khatri et al., 2005). We therefore speculated that viral replication correlated with macrophage changes in bursa and in liver. So in the present study, to determine whether macrophages are infiltrated in bursa and liver of IBDV-infected chickens, Bursa and liver at 3 d.p.i. and 4 d.p.i. were examined by immunohistochemistry (IHC) using the antibody against chicken monocytes/macrophages (KLU01). The results showed that the infiltration of KLU01 positive (KUL01+) cells in bursa and in liver was significantly apparent at 3 d.p.i. and 4 d.p.i. Especially, KUL01+ cells mainly distributed in the interfollicular interstitium of bursa. Few KUL01+ cells were detected in the bursa and liver of uninfected chickens by
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immunoperoxidase staining. Representative sections of bursa and liver at 3 d.p.i. and the uninfected group were shown in Fig. 5. 3.7. IBDV replicated in the Kupffer cells of liver Previously published data demonstrated that macrophages in bursa were infected with IBDV (Ka¨ufer and Weiss, 1976; Savova and Bozhkov, 1985). Meanwhile, present studies indicated that IBDV caused severe ultrastructural pathological lesions in the Kupffer cells of chicken liver. Viral antigens could be detected in liver. In view of these findings, we next investigated whether IBDV replicated in Kupffer cells or other cells in liver. Accordingly, we co-localized viral antigens with Kupffer cells in liver at 4 d.p.i. by immunofluorescence double staining using the rabbit anti-IBDV polyclonal antibodies and KUL01 antibodies, because the KUL01 antibody primarily marks Kupffer cells in chicken liver (Mast et al., 1998). Furthermore, we compared the different features of the colocalization of macrophages and viral antigens between in bursa and in liver. The results showed that the percentage of double positive cells (IBDV positive and KUL01 positive cells) accounted for 26.5 percent of the total IBDV positive cells or 57 percent of the total KUL01 positive (KUL01+) cells in bursa at 4 d.p.i. Numerous KUL01+ cells in the interfollicular area of bursa were negative for IBDV, which suggested that these subpopulations of macrophages are insusceptible to viral infection. Additionally, a lot of IBDV
Fig. 5. Infiltration of macrophages in bursa and liver. SPF chickens were inoculated with IBDV strain BC6/85 and bursa of Fabricious and liver were harvested at 3 and 4 days post infection (d.p.i.). Cryostat sections of bursa and liver were prepared to detect macrophages by immunoperoxidase staining using KUL01 antibody. Representative sections of bursa (upper control and A; 20) and liver (lower control and B; 40) at 3 d.p.i. were shown. Brown color (arrow) indicates the positive staining. KUL01 positive (KUL01+) cells predominantly distributed between lymphoid follicles in bursa (A).
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Fig. 6. Colocalization of IBDV and macrophages in bursa and liver. SPF chickens were inoculated with IBDV strain BC6/85. Bursa and liver were harvested at 4 days post infection (d.p.i.). Cryostat sections of bursa (A, B and upper Merge; 20 ) and liver (C, D and lower Merge; 20) were prepared to colocalize macrophages with IBDV by immunofluorescence double staining. The first primary antibody was rabbit anti-IBDV antibody, detecting IBDV proteins, and was visualized with FITC conjugate-goat anti-rabbit IgG (A and C). The second primary antibody was mouse anti-chicken monocyte/macrophage KUL01 and was visualized with AlexaFluor594- conjugated streptavidin (B and D). An overlay of immunofluorescent pictures was shown (Merge). IBDV positive cells, KUL01 positive cells and double positive cells were counted in five fields/bursa or liver/chicken at 20 magnification. The values represent the mean SE of five bursa or liver (E).
positive cells were negative for KUL01 marker, since IBDV mainly infected B lymphocytes in bursa. By contrast, the proportion of double positive cells in liver at 4 d.p.i. comprised 97 percent of the total IBDV positive cells or 99 percent of the total KUL01+ cells (Fig. 6). Data indicated that IBDV mainly proliferated in the Kupffer cells rather than hepatocytes or endothelial cells of liver.
4. Discussion Previous studies have clarified that IBDV mainly caused an acute infection in the dividing immunoglobulin Mbearing (IgM+) B lymphocytes and targeted the bursa tissues in its susceptible period (Ka¨ufer et al., 1980; Hirai et al., 1981). Researchers have attached importance to the
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lymphoid organs, such as, bursa, spleen, as well as thymus, rather than liver. Therefore, in the present study, we investigated the effects of IBDV on the chicken liver by comparing with its impacts on the bursa from the pathological perspective. Specifically, our data suggested that IBDV infection resulted in the severe lesions of chicken liver microscopically and ultrastructurally. Moreover, our results indicated that Kupffer cells were susceptible to IBDV due to the complete colocalization between viral antigens and macrophages examined by double immunofluorescence labeling, and provided further evidences for elucidation of IBDV pathogenesis. The liver glycogen is the storage form of carbohydrates and is a crucial source of energy. So the glycogen synthesis and degradation are tightly regulated. In the present study, the glycogen deposits in hepatocytes significantly dropped after viral infection, suggesting that IBDV infection inhibited the glycogen synthesis or accelerated the process of glycogen degradation. Admittedly, the possibility that the decrease of hepatic glycogen was due to the decreasing food intakes during infectious periods could not be excluded in this experiment. However, hepatic glycogen was detected again at 6 d.p.i. by transmission electron microscopy (TEM), and the result was subsequently confirmed by PAS staining (Fig. 4). Our data thereby demonstrated that the hepatic glycogen might play an important part in the restoration of damaged liver cells caused by viral infection. Ohata et al. (1982) pointed out that extrasinusoidal macrophages in chicken liver showed phagocytic activity, and they speculated that these macrophages might be the precursors of Kupffer cells. Ghoddusi and Kelly (2004) named these macrophages intercalated cells, because they mostly interposed between hepatocytes or in Disse’s spaces. In the same research work, authors proposed that intercalated cells were capable of self-proliferation in situ and mainly took two different forms, a poorly differentiated form, as well as a relatively highly differentiated form which was associated with the phagocytic activity. In this study, we noted that intercalated cells possessed numerous phagosomes containing cell debris after chicken infection with IBDV. In addition, at 6 d.p.i., intercalated cells could be frequently found between destroyed hepatocytes. In contrast, we just occasionally saw intercalated cells in the control group. Thus, our data indicated that these intercalated cells might take part in inhibiting viral infection and promoting recovery of liver tissue from infection. Hume (2006) once reviewed that some macrophages played a tropic role for other nearby cells in development and homeostasis. So we infer that the function of these tropic macrophages in mice is probably comparable to that of intercalated cells in birds. Nevertheless, the genuine roles and characteristics of intercalated cells are still unclear and need to be proven further. IBDV induced the degeneration, necrosis and apoptosis of hepatic cells that, however, are not susceptible to IBDV. These results might be attributable to damage effects of the inflammatory response resulting from viral infection, which is supported by evidences that numerous heterophils, a kind of well-known inflammatory cells, were seen at 2 d.p.i. by HE staining and TEM. Furthermore, the
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inflammatory response recruited activated macrophages into bursa and liver, which may explain the reason why IBDV infection resulted in infiltration of macrophages into bursa and that of Kupffer cells into liver in the present study. Similar to such our findings, Rauf et al. (2011) reported the infiltration of macrophages in IBDV-infected bursa by immnohistochemistry. Immunofluorescence double staining was used to colocalize IBDV with the macrophages in bursa and liver. As shown in Fig. 6, a large number of KUL01+ cells (accounting for 99 percent of the total KUL01+ cells) colocalized with viral proteins in liver. In addition, we saw severe ultrastructural lesion changes of Kupffer cells in liver by SEM after IBDV infection. Considering these evidences, we could rule out the possibility that viral antigens were phagocytosed by Kupffer cells, and therefore come to a conclusion that IBDV targeted Kupffer cells and replicated in them. By contrast, in bursa, there were a relatively small number of KUL01+ cells (accounting for roughly 57 percent of the total KUL01+ cells) to be colocalized with IBDV, suggesting that a minority of macrophages in bursa were infected with IBDV. Ka¨ufer and Weiss (1976) and Savova and Bozhkov (1985) observed viral particles in macrophages of bursa using transmission electron microscopy (TEM), which demonstrated that IBDV replicated in the macrophages of bursa. Similarly, IBDV was also detected in splenic macrophages (Palmquist et al., 2006). In the present study, our studies showed that the colocalization of macrophages and IBDV took place in bursa and liver in IBDV-exposed chickens. As these evidences demonstrated, we may therefore draw the conclusion that macrophages in various tissues infected with IBDV in vivo, although there were different characteristics of infection in these tissues, for example, Kupffer cells in liver completely colocalized with IBDV positive cells, whereas macrophages in bursa partially colocalized with IBDV. As a consequence, in addition to destroyed B lymphocytes, our study may be suggested that these IBDV-infected macrophages are a crucial factor leading to the immunosuppressive effects after viral infection, since macrophages are one of the most important immune cells both in the innate immunity and acquired immunity. Nonetheless, there are no specific data regarding the pathway of macrophages infection with IBDV. Therefore, further studies should be done to demonstrate whether there are specific receptors in macrophages to facilitate viral infection. Conflict of interest statement The authors have no conflicts of interest. Acknowledgements This study was supported by the ‘‘Twelfth Five YearPlan’’ of the National Science and Technology Support Project (2011BAD34B01) and Chinese Universities Scientific Fund (2011JS008) We thank Junzhen Jia and Haihong Liu for their technical assistance in transmission electron microscopy.
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