Downregulation in cytokines profiles and immunopathological changes in chicks infected with chicken infectious anaemia virus

1 Immunology Section, Indian Veterinary Research Institute (IVRI), Izatnagar (U.P.)–243 122, India. 2 Avian Diseases Section, Division of Pathology, Indian Veterinary Research Institute (IVRI), Izatnagar (U.P.)-243 122, India. 3 Department of Microbiology, College of Veterinary Sciences, UP Pandit Deen Dayal Upadhyaya Veterinary University (DUVASU), Mathura (U.P.)-281001, India. 4 Division of Veterinary Biotechnology, Indian Veterinary Research Institute (IVRI), Izatnagar (U.P.)-243 122, India. 5 Division of Pathology, Indian Veterinary Research Institute (IVRI), Izatnagar (U.P.)-243 122, India.

The disease is caused by chicken infectious anemia virus (CIAV) that belongs to genus Gyrovirus of family Circoviridae (Pringle, 1999).CIAV is a non-enveloped, icosahedral DNA virus and is one of the smallest known avian pathogen with 23 to 25 nm diameter (Pringle, 1999;Schat, 2009).The virus has a 2.3 kb single stranded circular genome which encodes VP1 (the major capsid protein), VP2 (scaffold protein) and VP3 (apoptin) which causes p53 independent apoptosis in hematopoietic and thymic precursor cells (Schat, 2003(Schat, , 2009)).The virus has gained much importance due to unique features like highly contagious and hardy nature, vertical transmission and immunosuppressive potential.Due to these features, the virus has been placed in the list of emerging viruses that can cause severe threat to the Indian poultry industry (Bhatt et al., 2011).
CIAV being a potent immunosuppressive agent affects both cellular and humoral immune functions of the central as well as peripheral immune organs leading to decreased immunoprotective efficacies (Adair, 2000;Dhama, 2002;Hoerr, 2010).The virus has tropism for haematopoietic and thymic precursors (Noteborn and Koch, 1995;Scott et al., 1999).Earlier reports indicated that CIAV infection specifically causes severe defects in splenic T-lymphocyte functions leading to decreased responsiveness to non-specific mitogens like phytohemagglutinin, concanavalin A, and fall in interleukin production (Adair et al., 1991).Macrophage concentration and functions are also severely reduced after in vivo or in vitro exposure to the virus (Cloud et al., 1992;McConnell et al., 1993aMcConnell et al., , 1993b)).The virus has been found to be associated with the impaired generation of concurrent pathogen-specific cytotoxic T lymphocytes (Markowski and Schat, 2003).However, the cytokine responses and their role in the pathogenesis of CIAV have not been extensively studied yet.Therefore, the present study was designed with the aim to determine the effect of CIAV infection on cell mediated immune responses like blastogenic responses and cytokines viz., interlukin (IL)-1β, IL-10, IL-12β and granulocyte macrophage-colony stimulating factor (GM-CSF), and apoptosis in chicks infected with the virus at various intervals of their clinically susceptible period.

Virus and experimental chicks
Indian field isolate of CIAV (strain A; accession No. AY583755) maintained in Avian Diseases Section, Indian Veterinary Research Institute, Izatnagar, was used for experimental infection purposes.The SPF chickens obtained from M/S Venkateshwara Hatcheries Group Limited (VHL), Pune, Maharashta, were reared in the infection and control sheds of Avian Disease Section, Division of Pathology, under strict isolated conditions.Feed and water were provided ad libitum.All experimental procedures on animals were carried out according to the recommendations and approval of the Institute Animal Ethics Committee (IAEC) under the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Experimental design
The SPF chickens (n = 90) were randomly divided into five groups (A to E) with 15 chicks each in Groups A to D and 30 in control Group E. All the chicks of Groups A to D were inoculated intramuscularly (i/m) with 0.5 ml of 10 4.5 TCID 50 Marek's disease virus transformed -MSB1 cell culture passaged CIAV isolate as described previously (Natesan et al., 2006) at 1, 14, 21 and 28-dayold, respectively (Table 1).Chicks of the group E were kept as uninfected control, administered with 0.5 ml PBS i/m.Chicks of all the groups were regularly monitored for clinical signs of the disease and mortality, and cell mediated immune (CMI) responses at different intervals post infection by studying cytokine expression profile, lymphocyte transformation test (LTT), along with confirmation of virus infection by observation of haematological, gross, microscopic and apoptotic changes and polymerase chain reaction (PCR) testing.

PBMC separation and RNA extraction
For cytokine studies in CIAV infected chicks, peripheral blood mononuclear cells (PBMCs) were collected from three chicks of each group on 4 and 7 days post infection (dpi) as described previously (Kaiser et al., 2006) with slight modification.PBMCs *Corresponding author.E-mail: kdhama@rediffmail.com.Tel: +91-581-2310074 or +919837654996.Fax: 0091-581-2303284 or 2302179.were also collected for CMI studies at 1st, 2nd and 3rd week post infection (wpi) from five chicks of each group.Briefly, about 3 ml blood samples were collected, diluted with equal volume of sterile PBS (pH 7.2), over-layered on 3 ml of histopaque (1.077 g/ml, Sigma Co., USA) and centrifuged at 400 ×g for 30 min.The gradient interface of PBMCs were collected and washed twice with PBS by centrifugation at 100 ×g for 10 min and finally resuspended in 1 ml of Trizol (Invitrogen, Carlsbad, CA).The resulting Trizol-PBMC mixture was incubated at room temperature for complete cell lysis.RNA was extracted using chloroform for aqueous phase separation, precipitated with isopropanol, washed with 75% ethanol, dried at 37°C and finally resuspended in RNase-free water.

Complementary DNA (cDNA) synthesis
The total RNA of the PBMCs obtained from the experimental chicks was reverse transcribed for cDNA synthesis using M-MLV RT enzyme (M1705, Promega, Madison, WI) according to the manufacturer's recommendations.Briefly, 2 µg of total RNA and 0.5 µg of random hexamer primer were taken in 15 µl nuclease free water, incubated for 5 min at 70°C then chilled on ice.Resulting reaction mixture was combined with 5 µl of M-MLV 5X Reaction Buffer, 10 mM dNTP mixture, 40 U/µl of RNasin®, 200 IU of M-MLV RT and RNase-free water to final volume of 25 µl.The reaction mixture was incubated at 37°C for 1 h, and the reaction was finally stopped by heating at 72°C for 10 min in the thermal cycler (MJ Research, San Gabriel, USA).The prepared cDNAs were checked by PCR using housekeeping gene, chicken β-actin specific primers (Table 2) and stored at -20°C until used.

Quantification of cytokines by real time PCR
For quantifying of the expression levels of cytokines (IL-1β, IL-10, IL-12β and GM-CSF), the cDNA samples of the respective PBMCs were subjected to real time PCR analysis using gene specific primers.The cDNAs prepared were run in duplicate sets on STRATAGENE Q-CYCLER with MX 3000P software using SYBR green master mix (QuantiFast SYBR Green PCR Master Mix, Qiagen, USA).The reaction mixture consisted of 12.5 μl of SYBR green master mix, 3 pmole each of gene specific forward and reverse primers, 100 ng of c-DNA and nuclease free water to 25 μl along with at least one non-template control.The quantitative SYBR green master mix contained hot start Taq DNA polymerase, hence the real-time PCR reaction started with initial incubation at 95°C for 5 min followed by 35 cycles of amplification with denaturation at 95°C for 10 s, combined annealing and extension at 60°C for 30 s for all genes.The specificity of the target gene specific product was confirmed by dissociation curve generated by the software at annealing temperature through 95°C.Primers sets used in this study were either previously published primer sequences or were self designed (Table 2).Data analysis was performed by 2 - Ct method of Livak and Schmittgen (2001), and presented as the mean relative fold change in gene expression normalized to an endogenous reference gene and relative to uninfected control.

Lymphocytic transformation test (LTT)
Lymphocytic transformation test (LTT) was conducted as described previously (Bounous et al., 1992) with slight modification.Briefly, PBMCs were separated from five chicks of each group at 1st, 2nd and 3rd wpi as mentioned above, and collected in RPMI-1640 growth media (without phenol red, with NaHCO 3 , Sigma, USA) to which 0.25 g/100 ml HEPES, 100 IU/ml of benzyl penicillin and 100 μg/ml of streptomycin sulphate were added.Viability of the cells was ascertained by trypan blue dye exclusion method and the cells were adjusted to give 5 x 10 6 viable cells/ml and platted in 96-well flat-bottomed tissue culture plate (Nunclon, Denmark).Cells were stimulated (10 µg ml) either with Con-A (Sigma-Aldrich, St. Louis, Missouri, USA) or CIAV antigen (whole purified CIAV-A inactivated virus) dissolved in RPMI in triplicate wells along with unstimulated control wells.The plates were incubated at 37°C in a humidified chamber at 5% CO 2 tension.After 72 h, 20 µl (5 mg/ml) of MTT (3-[4, 5-Dimethyl thiazolyl]-2, 5-diphenyl tetrazolium bromide) solution was added to all the wells and incubated further for 4 h, following which the plate was centrifuged at 1200 rpm for 15 min.The supernatant was removed carefully from all the wells.The MTT formazan was extracted from the cells, using DMSO (150 µl/well).The optical densities (OD) of wells were measured at a test wavelength of 510 nm and a reference wavelength of 650 nm.Stimulation index (SI) was calculated by dividing the OD value of stimulated with the OD of unstimulated.

Clinical, haematological, gross and histopathological changes
Chicks of all the groups (A to D) were observed for clinical signs and mortality, if any along with similar examination in the control (Group E) chicks.At 12 dpi, 1 ml of blood was collected from three chicks of each group for estimation of packed cell volume (PCV) using Wintrobe's macrohaematocrit method (Wintrobe, 1967).Wintrobe tubes were filled with blood with the help of long needled syringe to avoid air bubbles and centrifuged at 900 xg for 45 min to determine the PCV (%) values.For observation of gross and histopathological lesions, three chicks from each groups were sacrificed at 12th dpi and lesions recorded in various organs.Tissue samples (about 10 × 5 mm thick pieces) of thymus, liver, spleen and bursa were collected in 10% neutral buffered formalin and processed for histopathological examination following standard protocol and Haematoxylin and Eosin (H&E) staining.

Detection of CIAV using PCR
PCR was employed for confirmation of the establishment of CIAV infection in experimentally inoculated chicks.Total DNA was isolated from the pooled tissues of liver, thymus, spleen and bone marrow of at least three birds from each group sacrificed at 12 dpi, using DNeasy® Blood and Tissue Kit (QIAGEN, Germany) following manufacturer's instructions.The eluted DNA samples were stored at -20°C until used.PCR amplification of VP2 was performed using gene specific primers (F:ATGCACGGGAACGGCGGAC and R: TCACACTATACGTACCGGGG).Briefly to 12.5 μl of 2×PCR master mix, 10 pmol/µl of each primer and 1µg of DNA was added using nuclease free water to the final volume of 25 μl in an automatic thermal cycler (PTC 200 MJ Research, USA).A known CIAV DNA and DNA isolated from control birds were used as positive control and experimental negative control, respectively.

DNA fragmentation assay for apoptotic changes
For determining CIAV induced apoptosis in infected chicks, DNA fragmentation assay (laddering assay) was performed using the DNA extracted from the thymus of chicks of all five groups (A-E) at an interval of 7, 10 and 14 days post infection (DPI).10 µl of each DNA sample was analyzed in 2% agarose gel containing ethidium bromide (0.5 μg/ml) as described previously (Jeurissen et al., 1992).

Statistical analysis
All the data are given as mean ± SD.Different experimental groups were compared by ANOVA followed by a post hoc Tukey's test using SPSS v.16.0 statistical software, and values with p ≤ 0.05 were considered statistically significant.

Quantitation of cytokine gene expression by real time PCR
The cytokine gene expression levels in CIAV infected chicks of all the groups were determined at 4 and 7 dpi.CIAV was found to down regulate the mRNA expression levels of IL-1β, IL-10, IL-12β and GM-CSF, significantly (p<0.05) in all infective groups at both intervals as compared to control group.The highest fold change was observed in IL-1β m-RNA expression level in infected chicks of group A (4924.8 fold) as compared to Group B (1060), Group C (2369) and Group D (596) at 4 dpi.
At 7 dpi, all CIAV infected groups showed similar decrease in IL-1β levels (~5000).Downregulatory change in IL-10 expression levels was greater at 4 dpi which was decreased by 33.11, 17.63, 13.90 and 6.75 folds in groups A to D, respectively, as compared to 7 dpi levels decreasing only by 16.31, 3.53, 2.98 and 2.73 folds, respectively.IL-12β expression profile was also downregulated like IL-10 and was more pronounced at 4 dpi, while GM-CSF followed the pattern of IL-1β.However, the effect of CIAV infection on IL-12β and GM-CSF as compared to IL-1β was very low (Figure 1).

Lymphocytic proliferation assay
The blastogenic responses of lymphocytes stimulated with non-specific (Con A) and specific (CIAV antigen) antigen were significantly (p<0.05)low in chicks of CIAV infected groups as compared to control group at all the three intervals of 1st, 2nd and 3rd wpi (Figure 2).The SI   values ranged from 0.459 ± 0.062 to 0.563 ± 0.023 and 0.601 ± 0.076 to 0.702 ± 0.046 in infected groups as compared to 0.653 ± 0.027 to 0.710 ± 0.025 and 0.792 ± 0.045 to 0.917 ± 0.056 in control (Group E) on stimulation with CIAV antigen and Con A antigens, respectively.Higher SI values were observed on Con A stimulation as compared to stimulation CIAV antigen both in infected and control groups at all post infection intervals.

Clinical, hematological, gross and histopathological changes
Chicks of the CIAV infected groups (A to D) showed clinical signs of CIA viz., weakness, anorexia with ruffled feathers, anemic (pallor), and stunted growth, which were more pronounced in chicks infected at 1 and 14-day-old as compared to chicks infected at 21-day-old, and were lower in chicks infected at 28-day-old.The PCV (%) values in all the CIAV infected chicks were significantly low as compared to that of uninfected healthy control (26.67 ± 0.88) at 12 dpi.Among the infected groups, the mean PCV value was lowest (8 ± 0.58) in Group A chicks inoculated on 1 st day of age and it showed increasing trends in Group B (11 ± 0.57), Group C (16.33 ± 0.88) and Group D (20 ± 0.58) inoculated at 14th, 21st and 28th day, respectively (Figure S1).
The gross lesions observed in various organs like thymus, liver, spleen, bone marrow and bursa of CIAV infected chicks (Group A to D) at 12 dpi included thymus atrophy, whitish pale bone marrow, mild to moderately atrophied spleen and bursa, and pale liver (Figure S2).The lesions were more intense and characteristic in chicks inoculated at 1st and 14th days of age (Group A and B) as compared to those inoculated at 21st and 28th day (Group C and D).Histopathological changes were also similarly reflecting the gross lesions in both intensity and severity in chicks infected at various age intervals.Histopathology of liver indicated mild degeneration of hepatocytes, dilated blood capillaries and focal lymphoid aggregation.In thymus and bursa, mild to moderate atrophy and depletion of lymphocytes in cortex and medulla was observed (Figure 6).The microscopic lesions were less severe in chicks of Group C than that of Groups A and B, and were least in Group D. All the chicks of uninfected control Group E remained healthy throughout the experimental period and did not show any signs and lesions of CIA.

CIAV detection by PCR
PCR testing of the pooled tissues (liver, thymus, spleen and bone marrow) from chicks of CIAV inoculated groups (A to D) were found positive for viral DNA at 12 dpi.The CIAV VP2 specific PCR showed a distinct amplicon of 651 bp in 1% agarose gel electrophoresis.No such amplification from samples of uninfected control chicks (Group E) was observed (Figure 3).

Apoptotic changes
The cell death due to VP3 induced apoptosis caused by CIAV infection was investigated by testing thymic tissues for apoptotic changes in chicks of Groups A to D. The thymic DNAs showed nucleosomal laddering at all the three time intervals of 7, 10, 14 dpi only in chicks of Groups A to D. This laddering pattern was more marked and highest at 7 and 10 dpi and was lower at 14 dpi (Figures 4 and 5).This laddering pattern was lower in the chicks inoculated at 28 days of age (Group D).

DISCUSSION
CIAV is an important pathogen that poses threat to the poultry industry worldwide.It causes immunosuppression, directly by severe depletion of lymphocytes from primary and secondary lymphoid organs (Taniguchi et al., 1982), or indirectly by participating with other immunosuppressive viruses (Bülow et al., 1986;Cloud et al., 1992).For the control of viral infections, cell mediated response is regarded as an integral and important component of immune system and it mainly involves various cytokines in comparison with the humoral immune response which acts through  antibodies.However, the effect of CIAV infection on various cytokines has not been well studied in detail.The data presented in this work showed that CIAV has marked effect in down regulation of IL-1β, IL-10, IL-12 and GM-CSF in virus infected chicks.The down regulation of cytokines was more pronounced in chicks infected at 1st and 14th days of age as compared to chicks inoculated at 21st and 28th days of age, supporting the well established fact that chicks are more susceptible to disease during their first two weeks of age.Previously, it was reported that natural exposure of chickens to CIAV resulted in impaired functional activity and reduction of IFN-γ, IL-1β and IL-2 expression (McConnell et al., 1993a;Ragland et al., 2002).The present experimental data indicated not only IL-1β decreases but also IL-10, IL-12 and GM-CSF at various post infection intervals of the clinically susceptible age, and the effects were more pronounced in young chicks.The immuno-regulatory cytokine, IL-10, decrease was more pronounced during earlier stages of infection (4 dpi) than at the latter stages (7 dpi), indicating higher immunosuppressive stage at 7 dpi.A change in cytokine expression levels may be closely related to replication of CIAV, but the mechanism is still not fully understood.However, it could be due to depletion of lymphocytes in lymphoid organs and there by decreased count in the peripheral blood.
Pathogenesis of CIAV indicates that primary target cells are haematopoietic precursor (haematocytoblasts) and thymic precursor (lymphoblasts) cells in the bone marrow and thymic cortex (Jeurissen et al., 1992;Noteborn and Koch, 1995).In this study, it was found that thymus is indeed primary target organ for the viral replication as confirmed by nucleosomal fragmentation, an indicator of apoptosis.Nucleosomal fragmentation was higher at 7, 10 dpi than at 14 dpi in the CIAV infected groups; also, the apoptotic pattern was least in chicks of Group D, infected at 28 days of age.This may be due to refractoriness of thymocytes to CIAV infection or depletion of the thymocytes in the thymus at latter stages and as age progresses (Goryo et al., 1985;Jeurissen et al., 1992;Noteborn and Koch, 1995).This type of apoptosis is an important phenomenon during the pathogenesis of CIAV and is associated with VP3 protein, apoptin (Noteborn and Koch, 1995).Due to this apoptosis in thymic and haematopoietic precursor cells, possibly lower circulating lymphocyte were present in peripheral blood of infected groups as certain correlation was found between apoptotic pattern in the thymus and cytokine down regulation in the peripheral blood.
The blastogenic responses, as an indicator of cell mediated immunity (CMI) indicated lower stimulation indices in chicks of CIAV infected groups than the control chicks.The depression in CMI response in groups infected at younger age was more pronounced than those groups infected at later ages.Also, it was found that blastogenic responses were lower at 1 and higher at 3 wpi in young chicks infected at 1st and 14th day of age, while in older chicks infected with the virus at 21 and 28 days of age, the stimulation indices were comparatively higher which infers that older birds are comparatively less vulnerable to virus infection.This may be due to lower T lymphocytes in the peripheral blood associated with higher apoptosis of thymic and haematopoietic precursor cells in younger chicks as compared to older birds in which the virus may have replicated slowly because of development of age resistance to CIAV and due to maturation of immune cells.
After adsorption and penetration, CIAV enters the haematopoietic and thymic precursors and multiplies in their nucleii by a rolling circle model (Noteborn and Koch, 1995;Scott et al., 1999).The virus then affects both cellular and humoral immune function directly by affecting helper (CD4 + ) and cytotoxic (CD8 + ) T-lymphocytes in the thymus and indirectly through cytokine responses, respectively (Hu et al., 1993;Todd, 2000).By inhibiting

Packed cell volume (%)
PCV values (%) Figure S1.Effect of CIAV infection on %PCV values in chicks inoculated at 1 st (Group A), 14 th (Group B), 21 st (Group C) and 28 th (Group D) days of age.IL-1, IL-2, IFN and other cytokine production, it adversely affects the inflammatory and immunoregulatory responses, the expression of surface receptors and the cytotoxic activities of pool of cells including macrophages and T lymphocyte (CTL) (McNulty, 1991;Todd, 2000;Schat, 2003).These detrimental effects on T-cell mediated functions adversely affects lymphocyte transformation response to mitogens, cytokine production; reduces macrophage functions namely, IL-1, FcR expression, phagocytosis and bactericidal activities (Cloud et al., 1992;McConnell et al., 1993aMcConnell et al., , 1993b)).All of such changes have substantial negative effects on immune response leading to enhancement of the concurrent infection with other pathogens and vaccination failure.
To confirm that the establishment of CIAV infection in experimentally infected chicks and the changes in the immune profile were indeed caused by CIAV, pathology, haematology and PCR testing of various tissues of the virus infected chicks was carried out.Generally normochromic anemia with watery blood, paler plasma and increased clotting time and associated symptoms are recorded in CIAV infections (Taniguchi et al., 1982;Ramadan et al., 1998).Accordingly, in the present study, PCV was decreased in all the infected groups at 12 DPI, but the decrease was more pronounced in groups infected at younger ages (group A and B), suggesting the more severity and susceptibility of young chicks to the infection as compared to that at later stages (Groups C and D).CIAV VP2 specific PCR assay yielded desired virus specific amplicon of 651 bp indicating that CIAV replicated in the tissues causing associated signs and lesions.The pooled tissue (thymus, bone marrow, spleen and bursa) samples were found positive on PCR assay at 12 dpi from all the infected groups.PCR has several advantages over conventional diagnostic tests and previous reports indicated that it is very useful for detecting CIAV infection (Dhama, 2002;Hussein et al., 2002;Yassir et al., 2011;Snoeck et al., 2012).Clinical signs and gross lesions were more pronounced in chicks infected at young age (1 and 14 day of age) as compared to that of chicks infected at later stages (21 and 28 day of age).Similarly, histopatholgical alterations in tissues of the CIAV infected chicks were more pronounced in chicks infected at younger ages.The histopathology of thymus and bursa showed mild to moderate atrophy and mild depletion of lymphocytes.In liver, mild degeneration of hepatocytes, dilated blood capillaries and focal lymphoid aggregation were observed.These observations were in agreement with the earlier reports (Goryo et al., 1989;McNulty, 1991;Sakr and Talaat, 1991;Smyth et al., 1993;Dhama, 2002).
Immunodepression caused by CIAV infection has dramatic effects on the poultry birds as it increases their susceptibility to secondary infections (viral, bacterial or fungal origin), depresses vaccinal immunity against other pathogens, complications like vaccinal reactions, aggravates residual pathogenicity of attenuated vaccine viruses and emergence of variant virus and most importantly leads to vaccination failure (Liu et al., 1997;Pascucci, 1997;Todd, 2000;Dhama et al., 2008).The important concurrent infections associated with CIAV infection include MD, ND, FP, ILT, IBD, etc.In the field, CIAV infection seems to cause few clinical signs of disease; however, the production losses both in terms of weight gain and due to concurrent infections are very high.Sub-clinical CIAV infections in older chickens have also been reported to cause substantial economic losses by affecting growth and health of birds and production of commercial SPF eggs.
In conclusion the present study provide supportive evidence that CIAV infection has profound effect on immunological profile of the young chicks, and reports the downregulatory changes in cytokines (IL-1, IL-12, IL-10 and GM-CSF) in chicks infected with CIAV at various intervals of clinically susceptible period, with younger ages of upto 2 weeks being more vulnerable.The cytokine down regulation correlated to some extent with apoptotic pattern in the thymus.CMI responses were also depressed in CIAV infected chicks as assayed by LTT, with a similar pattern as with cytokine downregulation.This has further provided evidence regarding the apoptotic involvement of lymphocytes in thymus due to which there was decreased peripheral pool and the decreased blastogenic responses in infected chicks.The observations of clinical signs and disease (CIA), anaemia, gross lesions and histopathological studies and PCR testing confirmed the establishment of the CIAV infection in experimental chicks.However, further studies are required regarding the role of other cytokines and the impact of viral load on the cytokine profile and immunopathogenesis both in young and adult birds.

Figure 1 .
Figure 1.Relative fold change in PBMCs mRNA expression of cytokine levels (IL-1β, IL-10, IL-12β and GM-CSF) in CIAV infected chicks (Groups A to D) at 4 and 7 dpi as determined by quantitative real time PCR.

Figure 2 .
Figure 2. Blastogenic responses of peripheral blood mononuclear cells (PBMCs) of the chicks (Groups A to E) at different intervals of post infection to the non specific antigen ConA (A) and specific antigen (B) stimulation as determined by LTT using MTT dye.Values are represented as mean ± 1SD; * p ≤ 0.05.

Figure 3 .
Figure 3. Amplified PCR products of DNA extracted from pooled tissues (liver, thymus, spleen and bone marrow) of CIAV infected (Groups A to D) and control (Group E) chicks on 12 th dpi using VP2 gene specific primers.Lane M: 1 kb DNA ladder; Lanes 1 to 4: VP2 (651 bp) amplified product from Groups A to D chicks, respectively; Lane 5: uninfected control (group E); Lane 6: positive control (known CIAV DNA).
Figure 4. DNA laddering/fragmentation assay for the DNA extracted from thymus of CIAV infected and control chicks at 7 dpi.Lane M: 100 bp DNA ladder; Lanes 1 to 4: DNA extracted from chicks of Groups A to D, respectively; Lane 5: uninfected control (Group E).

Figure 5 .
Figure 5.DNA laddering/fragmentation assay for the DNA extracted from thymus of CIAV infected and control chicks.Lane M 1 : 100 bp DNA ladder; Lane M 2 : 1 kb ladder; Lanes 1 to 4: DNA extracted from chicks of Groups A to D at 10 dpi; Lanes 6 to 9: DNA extracted from chicks of Group A to D at 14 dpi; Lanes 5 and 10: uninfected control (Group E).

Figure 6 .
Figure 6.Histopathological changes in various tissues from the CIAV infected chicks at 12 dpi.Mild degeneration and focal lymphoid aggregation in liver (A and B), mild depletion of lymphocytes in the thymic cortex and medulla (C and D), mild depletion of lymphocytes in the bursal cortex and medulla (E and F); mild to moderate depletion of lymphocytes in the spleen (G and H).

Figure S2 .
Figure S2.Gross lesions in thymus, spleen, liver and bone marrow in CIAV infected chicks on 12 dpi indicating atrophied thymus and spleen, pale liver and whitish pale bone marrow.

Table 2 .
List of primers used for amplification of different genes by real time PCR.