Clinico-Pathological and Hemato-Biochemical Assessment of Field Originated Chicken Anemia Virus in Experimentally Challenged Broiler Chicken

Chicken infectious anemia (CIA) is an important devastating viral disease of commercial poultry industry caused by chicken infectious anemia virus (CIAV), which is placed in Circoviridae family and belongs to genus Gyrovirus. Since its first isolation in 1979 in Japan, this virus has been isolated in many countries (Oluwayelu, 2010). CIAV can be diagnosed by detecting virus antigens and specific antibodies. Although the chickens of all ages are prone to CIAV, young birds (10-14 days) are mostly infected. The infected birds showed poor feed conversion ratio, decreased hematocrit values, anemia, increased mortality rate and lymphoid atrophy with aplasia of bone marrow (Schat et al., 2011; Vaziry et al., 2011). Atrophied lymphoid organs particularly thymus may result in immunosuppression of chickens leading to high risk of diseases with reduced weight gain. With the passage of time, birds may develop resistance against the virus and become carrier and shed the virus throughout the life. Among the distinguished features of CIAV, the most prominent is its both horizontal and vertical transmission which is mostly observed in birds between 3 to 4 weeks of age (Li et al., 2017).
        
Previously, antibodies in serum samples of poultry flocks produced either by infection or vaccination were detected by virus neutralization tests and indirect immunofluorescent tests which have been outdated by ELISA. For diagnosing CIAV on commercial level, ELISA is being used by most diagnostic laboratories in unvaccinated flocks. Very less published data is available about CIAV infection and its effect on different organs and hematology from commercial chicken industry of Pakistan. This study was planned to evaluate the tissue tropism, histopathological changes and haemato-biochemical parameters in broilers experimentally infected with local field isolated CIAV.

Experimental scheme
 
One hundred (n=100) 1-day-old chicks were procured and kept under standard housing conditions in experimental shed of university of veterinary and animal sciences, Lahore, Pakistan.  The blood samples were collected to assess the status of anti-CIAV-antibodies through ELISA on 7^th day of  age. After ensuring the seronegative status of birds, the birds were categorized into two groups (A and B), with 50 birds in each group. Group A served as negative control while birds of group B were exposed to CIAV (100 CID₅₀) on 7^th day of age through intramuscular route. The endpoint estimation for 100 CID50 was clinical signs and hemoglobin estimation. At 7^th, 14^th, 21^st, 28^th and 35^th day post infection (dpi), 5 chicks from each group were slaughtered for collection of blood and tissues (heart, liver, spleen, thymus and bursa of Fabricius). Tissue samples were preserved in 10% neutral buffered formalin for histopathological (Karimi et al., 2010) and immunohistochemistry (Haridy et al., 2009) studies by commercially available IHC kit according to manufacturer instructions (HRP/DAB Detection IHC kit, Abcam, Japan). Scoring of pathological changes in dead birds were made according to criteria as described by Najm-ul-Islam et al. (2013). To validate the IHC results, tissue samples were tested for the presence of CIAV through PCR using primers targeting the specific regions overlapping VP1-VP2 and VP2-VP3, CAV-1 (5-CTAAGATCTGCAACTGCGGA-3) and CAV-2 (5-CCTTGGAAGCGGATAGTCAT-3) to amplify a 419 bp DNA fragment (Hermann et al., 2012) as shown in Fig 1.
 

 
Haemato-biochemical profile
 
Blood samples were divided in EDTA and gel containing vacutainers for hematological and serum biochemistry analysis respectively. The vacutainers were centrifuged at 3,000 rpm for ten minutes to collect the serum, labeled properly and stored at -20°C for further processing. Hematology of the samples were performed as descried by Al-Hassani (2011). Liver function tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, albumin, total protein and renal function test including creatinine and urea level were measured according to protocol as described by Khanam et al. (2016).
 
Statistical analysis
 
Data were presented in mean ± standard deviation form and analyzed by applying t-test on statistical package for the social sciences (SPSS) version 23.0 software.

On day 7 PI, clinical signs developed in the infected birds were lethargic appearance, depressed, stunting growth, drooping of both wings and pale comb and wattle due to anemia. Only 06/50 birds (12.0%) were died throughout the whole time period of the study. On necropsy, hemorrhages on thigh and breast muscles, yellow fatty bone marrow, enlarged liver and atrophied lymphoid organs were observed. On 7^th DPI, mild (+) subcutaneous and cutaneous hemorrhages were found in the pectoral and breast muscles, heart, liver and skin underlying feathers which become further worse on day 14^th PI as moderate (++) to severe (+++) on day 28^th PI (Table 1). However, the severity in cutaneous and subcutaneous hemorrhages got reduced by 35^th DPI. The finding observed in this study are in concordance with the results reported earlier (Spackman et al., 2002; Najm-ul-Islam et al., 2013). Similar to those reported previously (Kuscu and Gürel, 2008), the histopathological changes in both lymphoid and non-lymphoid organs were observed on 7^th DPI in experimental birds. Histopathological changes in non-lymphoid organs such as in liver were mild hepatic necrosis and vascular degenerations (Fig 2a). In case of heart, multifocal myocarditis and a few mononuclear cell infiltrates were seen (Fig 2b). Lymphocyte depletions were seen in lymphoid follicles in bursa of Fabricius (Fig 2c). Necrotic areas were seen in spleen with lymphopenia in white pulp (Fig 2d). Necrosis was observed in the thymus in the cortical and medullary regions along with lymphocytic depletion especially in cortico-medullary regions (Fig 2e). Although histopathological changes in non-lymphoid organs were mild by day 14^th DPI, these changes were moderate in lymphoid organ. Karyomegaly in infected cells was obvious along with necrosis and lymphocytic depletions were mostly detectable in the white pulp area. On day 21 PI, histopathological changes both in lymphoid and non-lymphoid organ were similar to that of observed at day 14^th PI. Few bloody spots were also apparent in some regions of thymic lobules. Some detectable inclusion bodies were present in spleen. On day 28 PI, microscopic changes were absent in non-lymphoid organs (liver, heart), while in lymphoid organs such as bursa, thymus, spleen, mild microscopic lesions were observed with mild necrosis with lymphocytes depletions. Karyomegaly and inclusion bodies in infected cells were obvious. At day 35^th post-infection, microscopic changes were absent both in lymphoid organs and non-lymphoid organs. Our findings are supported by  similar studies by McNulty (1991) and Haridy et al., (2009). There were no microscopic lesions observed in organs tested in the non-infected birds.


 

        
At day 7^th, 14^th and 21^st PI, CIA antigen was present mostly in portal zone of liver, while at 28^th and 35^th DPI, no evidence of CIA antigen either in hepatocytes or in sinusoids was seen. On day 7^th, 14^th and 21^st post-infection, presence of CIA antigens was detectable by tan colored immune complex in the epicardium and myocardium of the heart. In bursa of Fabricius, CIA antigens were seen by day 7^th, 14^th, 21^st and 28th post-infection. Chicken infectious anemia antigen was more obvious in the cortical region of lymphoid follicles as compared to medullary area. In spleen, at day 7^th, 14^th, 21^st and 28^th post-infection, CIA antigen was detectable in white pulp as compared to red pulp, while no CIA antigen was seen in any region of spleen at day 35 post-infection. In thymus, at day 7^th, 14^th, 21^st and 28^th post-infection, CIA antigens were prominently present in cortical regions of thymic lobules as compared to medullary areas. Similar immunohistochemical findings have been reported earlier (Haridy et al., 2009). It was further analyzed and confirmed that all IHC positive were found positive through PCR.
        
The significant hematologic changes (Table 2) observed were more prominent especially on day 7^th to 21^st PI in the experimental group. The PCV in the challenged group was found in the range from 20.86±2.39-27.33±0.84 indicating anemia in a pattern similar to reported previously by Goodwin et al., (1992a). The hemoglobin concentration showed  decreased pattern starting from 7^th DPI (7.42±0.34) that continued until 14^th DPI (7.06±0.59). However, it was interesting to note that increasing pattern in the concentration of Hb on 21^st DPI although the concentration remained in the anemic range (7.18±0.96-.9.58±0.43) in the virus exposed group (7.42 to 9.58 g/dL). This might be due to development of immunity against the virus that could have decreased the virus load in infected group. All the data obtained from hematological findings showed that the pathological changes might be different, even between the birds infected with the same dose of CAV. Therefore, it is difficult to identify the bird having the subclinical form of the infection. Previous studies suggested that anemia and atrophy of lymphoid organs are not enough criteria to diagnose CIAV infections. Leukocytes (67.5%) and platelets percentage (74.3%) were found significantly (P< 0.05) lower than the control group (Goodwin et al., 1992b). The differences between the values of heterophils, lymphocytes, monocytes and eosinophils were statistically non-significant (P>0.05) throughout the study period.
 

 
The values of biochemical parameters have been given in Table 2. For ALT, there was non-significant difference (P>0.05) between the groups at day 7^th, while at day 14^th, 21^st, 28^th and 35^th, the difference regarding the ALT values between the groups was found significant (P<0.05). In the challenged group significantly, elevated values of AST (P<0.05) were noted than control group on 07^th, 14^th, 28^th and 35^th DPI, however; on 21^st DPI, no significant difference (P>0.05) was observed. A non-significant difference among the infected and control groups was observed on day 07, 14, 21, 28 and 35 PI with respect to alkaline phosphatase values. These findings agree to what has been reported previously by Krishan et al. (2015). For bilirubin, the difference between infected and control group was significant (P<0.05) at day 14^th and 28^th post-infection. Difference between infected and control group with respect to total protein was significant (P<0.05) at day 07^th, 14^th, 21^st, 28^th and 35^th post-infection. No significant difference was observed at day 07^th, 14^th, 21^st and 35^th post-infection between the infected and control groups with respect to albumin level. However, difference between infected and control groups regarding albumin level was significant (P<0.05) at day 28^th post-infection. No significant difference was observed between the infected and control groups with respect to creatinine level.