Immunosuppressive viral diseases pose a great threat to poultry industry because affected flocks respond poorly to administered vaccines and lead to heavy mortality and huge economic losses to poultry farmers. Some of the viral diseases affecting poultry lead to severe immunosuppression thus making the birds susceptible to secondary infections by multiple causative agents. Immunosuppression caused by viruses in poultry birds is directly related to the effect produced by viruses on lymphoid organs. Viruses follow various strategies to escape host immune surveillance and thus downregulate the immune response leading to immunosuppression¹. Some of the economically significant immunosuppressive viral diseases of poultry are Chicken Infectious Anemia (CIA), Infectious Bursal Disease (IBD), Marek‘s Disease (MD), Inclusion Body Hepatitis (IBH), Avian Reoviruses (ARV) and Retro virus infections²,³.

Chicken infectious anaemia is a re-emerging disease that leads to production losses due to mortality, morbidity, and condemnation at slaughter⁴. The immunosuppressive effects of CIA on broilers are economically significant as the affected birds show poor feed conversion and reduced weight gain resulting in increased condemnation rates at slaughter⁵. Infectious bursal disease (IBD) affects lymphoid tissues especially bursa of Fabricius and causes heavy economic losses in poultry industries due to immunosuppression in subclinical cases⁶, and heavy mortalities due to bursal damage in acute cases⁷. Avian Reoviruses (ARV) usually cause co-infection with other infectious pathogens like CIA and IBD under field conditions that result in increased pathological effects and economic losses⁸,⁹. Keeping in view, the importance of viral infections causing immunosuppression in the poultry flocks, the present study was undertaken to diagnose immunosuppressive viral diseases using immuno-histochemistry and to correlate the pathological changes with localization of viral antigens in lymphoid organs.

The present study was conducted on 100 cases presented for post mortem examination in the Poultry Disease Diagnostic Laboratory of the Department of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana and from three commercial poultry farms (Table 1).

Thorough postmortem of birds was conducted and gross lesions in affected organs were recorded. Tissue pieces of lymphoid organs including thymus, bursa, spleen and caecal tonsils were collected in 10% neutral buffered formalin for histopathology and immunohistochemistry. For histopathology, 5–6 μm thick paraffin-embedded tissue sections were cut and stained using routine hematoxylin and eosin (H&E) staining technique. The histopathological lesions in thymus, bursa and spleen were scored from 0–4, and in caecal tonsils from 0–3 as described previously¹⁰,¹¹. The average of five fields was taken per slide for scoring the lesions.

For immunohistochemical studies, tissue sections were mounted on Superfrost/Plus, positively charged microscopic slides (Fisher Scientific, USA) and stained using specific antisera against CAV, IBDV and ARV (Charles River Laboratories USA) at the dilution of 1:10000. The avidin biotin peroxidase complex (ABC) method for immunoperoxidase staining was carried out for localization of viral antigens using commercial Vectastatin ABC reagents (Vector Laboratories, Burlingame, USA). Briefly, heat induced epitope retrieval (HIER) was employed using EZ antigen retrieval solutions by EZ-Retriever TM System as per manufacturer‘s instructions (BioGenex Laboratories Inc., San Ramon, California, USA). The endogenous peroxidase was quenched in 3% H₂O₂ in methanol for 40 min at room temperature.The sections were incubated with 1:10 dilution of a normal goat serum (Vector Laboratories, Burlingame, USA) mixed with power block (1 drop in 1 ml) (Biogenex Laboratories Inc., San Ramon, California) to block non-specific protein binding for 60 min at room temperature. After incubation with primary antibody (1:10000) at 4°C for overnight, the sections were incubated with secondary biotinylated anti-chicken IgG (Vector Laboratories, Burlingame, USA) at a concentration of 1:400 for 30 min at room temperature. The sections were incubated in Vectastain ABC reagent (Vector laboratories, Burlingame, USA) for 30 min at room temperature. The antigen-antibody-peroxidase reaction was developed with a freshly prepared 3,3'-diaminobenzidine (DAB) solution by mixing 1 drop of DAB chromogen with 1 ml of DAB buffer supplied by the manufacturer (Vector Laboratories, Burlingame, USA) and counterstained with Gill‘s haematoxylin (Merck, Germany). ¹² For each antibody, a negative control was run by replacing primary antibody with PBS buffer.

Immunohistochemical scoring was done by counting the number of cells showing positive reactivity usinga 40X objective lens of the light microscope¹³. Score 0 depicted <5 stained cells for no infection, 1 for mild infection (5–50 stained cells), 2 for medium infection (50150 stained cells), 3 for heavy infection (over 150 stained cells). The average of five fields was taken per slide.

The data was analysed using SPSS software and the correlation of histopathological changes and localization of viral antigens in lymphoid organs was determined using Pearson‘s technique. Chi-square test was applied to find the farm wise and disease wise incidence of immunosuppressive viral diseases under field conditions.

Postmortem findings revealed general paleness of carcass in affected birds and noticeable lesions in thymus, bursa, spleen, caecal tonsils, bone marrow and other visceral organs. In most of the cases of IBD, bursa was oedematous, enlarged with congestion, hemorrhages on serosal as well as mucosal surfaces (Fig. 1A), and atrophy of bursa (Fig. 1B) was also evident in advanced stage of the disease. Thymus in cases of CIA showed remarkable changes that included hemorrhages, congestion and atrophic thymic lobes (Fig. 1D) distributed on both sides along the neck. Most of the affected birds showed splenic enlargement and hemorrhages (Fig. 1E). The pale and fatty bone marrow (Fig. 1F), atrophied lymphoid organs and anemic visceral organs were suggestive of CIA (28/100) and changes in bursa along with presence of haemorrhages on breast muscles (Fig. 1C) indicated infection with IBDV (12/100).

Histopathological lesions in thymus of CIA affected birds constituted focal or diffuse, mild to moderate lymphoid depletion and presence of eosinophilic intranuclear inclusions in the lymphoblasts mainly inthe cortex. In some cases indistinct boundary between cortex and medulla of thymus was observed (Fig. 2A, B). The bursal lesions in cases of IBD exhibited mild to moderate depletion of lymphoid cells and atrophy of the bursal follicles with infiltration of mixed inflammatory cells (Fig. 2C). In advanced cases of IBD, marked atrophy of bursal follicles along with inter follicular proliferation of fibrous tissue and cystic degeneration of bursal follicles was observed (Fig. 2D). Lymphoid depletion was also prominent in spleen and caecal tonsils along with hyperplasia of reticulo-endothelial cellsin the splenic parenchyma (Fig. 2E, F). Bone marrow of CIA affected birds showed marked depletion of hematopoietic cells with noticeable replacement by adipocytes.

Immnunohistochemical localization of viral antigens in the lymphoid tissues revealed moderate to strong reactivity for CAV, IBDV and ARV antigens in the cytoplasm, as well as in the nucleus of lymphoid cells in thymus, bursa, spleenand caecal tonsils (Figs. 3, 4, 5). On the basis of immunoreactivity of various viral antigens, a total of eight cases were found to be positive for CAV alone and two cases for IBDV alone. Mixed infection of both CAV and ARV was observed in six cases, CAV and IBDV in seven cases, CAV, IBDV and ARV in nine cases, and only one case was found positive for IBDV and ARV (Table 2).

The disease wise, histo-pathological and immunohistochemical score of lymphoid organs and their correlation is given in the Table 3. Overall, a significant positive correlation was obtained between histopathological score and IHC score of thymus (0.60; P<0.05), spleen (0.63; P<0.05), bursa (0.51; P<0.05) and caecal tonsils (0.80; P<0.05) as depicted in Table 4. In thymus, asignificant positive correlation was obtained in CAV+IBDV (0.98; P<0.05) and CAV+ARV (0.80; P<0.05) mixed infections. However, no significant correlation in thymus was found in individual cases of CAV (0.37) and co-infection of CAV with IBDV+ARV (0.55). Significant positive correlation was obtained between histopathological score and IHC score of spleen (0.75; P<0.05) and bursa (0.66; P<0.05) in mixed infections of CAV+IBDV+ARV. Where as in birds infected with individual CAV, mixed infection of CAV and IBD, and mixed infection of CAV and ARV non-significant correlation was obtained for spleen and bursa. In caecal tonsils, a significant positive correlation was observed in individual CAV infection (0.94; P<0.05), mixed CAV+ARV (0.92; P<0.05) and co-infection of CAV with IBDV+ARV (0.62; P<0.05). However, for birds infected with CAV+IBDV (−0.67) negative, non-significant correlation was obtained in the caecal tonsils (Table 4).

In the present study significant difference in incidence of immuno suppressive viral diseases was found between four different farms with respect to CIA (X2=34.38; P<0.001), IBD (X2=25.11; P<0.001) and ARV (X2=29.95; P<0.001). The incidence of CIA was significantly higher in farm 1 (80%) than farm 2 (30.2%) and farm 3 (11.7%). Incidence of IBD (55.0%) and ARV (50.0%) was significantly higher in farm 1. Significant difference (X2=9.23; P<0.05) was found between immunosuppressive viral diseases with respect to the incidence. CIA was found with highest incidence (31%). The difference between incidence of IBD (18%) and ARV (15%) was insignificant (Table 5).

The present study was conducted to diagnose immunosuppressive viral diseases using immunohisto- chemistry and to correlate the pathological changes with localization of viral antigens in lymphoid organs. The viral diseases were tentatively diagnosed on the basis of gross and histopathological lesions as reported earlier¹⁴,¹⁵-¹⁶ and the immunosuppressive effect of viruses might be attributed to the destructive effect of viruses on lymphoid organs thus leading to impaired immune response. However, on the basis of gross and histopathological lesions exact etiology of immunosuppression could not be ascertained and differential diagnosis of immunosuppressive viral diseases could not be achieved because of similarities in clinical presentation of these diseases, as reported earlier¹⁷. In the present study confirmatory diagnosis of immunosuppressive viral diseases was doneby immunohistochemical localization of CAV, IBDV and ARV antigens in the lymphoid organs, and immunohistochemistry was found to be an effective technique in diagnosing the viral infections causing immunosuppression either due to individual infection or mixed viral infections under field conditions. We were able to diagnose individual infection of CAV and its co-infection with IBD and ARV by localizing the viral antigens in formalin-fixed, paraffin-embedded lymphoid tissue sections. Earlier researchers also diagnosed immunosuppressive viral diseases by immunolocalizing CAV18, IBDV7 and ARV19 antigens in lymphoid and visceral organs of poultry.

In the present study, overall significant positive correlation was observed between histopathological changes and localization of viral antigens in the lymphoid organs indicating that these viral antigens causes immunosuppression by targeting the lymphoid organs¹⁷. Furthermore, in the present study the incidence of mixed immunosuppressive viral infections (CAV, IBDV, ARV) was found to be more than that of individual viral infections as reported in the previous studies¹⁷,¹⁹. Moreover, combined infections of CAV, IBDV and ARV resulted in increased incidence and severity of lesions in lymphoid organs when compared with birds infected with individual virus infection, lending support to the similar earlier obsrvations²⁰.

CAV infection and co-infection of CAV with other viruses under field conditions was found in birds between 6–12 weeks age which might be due to decrease in maternal antibodies around 3 weeks of age as also reported in earlier studies²¹.

Farm wise (34.38%) as well as disease wise (31%) incidence of CIA was found highest in the present study, and all the cases which were positive for IBDV or ARV infections were also positive for CAV infections suggesting CAV as a potent immunosuppressive viral agent causing co-infections with other viral agents under field conditions thus increasing the economic lossesto the poultry farmers²².

The present study suggested as documented earlier that the mixed infections of immunosuppressive viral diseases are common under field conditions, and also the synergic pathological effects, produced by co-infection of CAV with IBDV and ARV, are more as compared to individual virus infection, reinforcing the concept of immunosuppressive potential of CAV.

The authors are thankful to Dr. Geeta D Leishangthem and Dr. Neeraj Kashyap for helping in statistical analysis of data in the present study. The financial assistance received from the university to undertake this research work is highly acknowledged.

*level of significance P<0.05, HP - Histopathological score, IHC-immunohistochemistry score, PCC-Pearson Correlation coefficient, CAV-chicken anaemia virus, IBDV-infectious bursal disease virus, ARV-avian reovirus

^a,b,c,abThe values designated with different letters are statistically significant (P<0.05) ^aThe values designated with same letters are statistically insignificant.(P<0.05)

^a,bThe values designated with different letters are statistically significant (P<0.05) ^aThe values designated with same letters are statistically insignificant.(P<0.05)

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