Molecular and pathological investigations of hemorrhagic syndrome associated viral infection in broilers and sasso breeds in Dakahlia Governorate

Molecular and pathological investigations of hemorrhagic syndrome associated viral infection in broilers and sasso breeds in Dakahlia Governorate

^*Rania, I. Mohamed and ^**Enas, M.A. Hammad

^*Department of Pathology, Animal Health Research Institute, Agriculture Research Center, EGYPT, Mansoura Provincial, Lab.

^**Department of Poultry Diseases, Animal Health Research Institute, Agriculture Research Center, EGYPT, Mansoura Provincial, Lab.

ABSTRACT

Chicken infectious anemia (CIA) is a worldwide, highly contagious immunosuppressive viral disease of young chickens 4-6 week of age caused by chicken anemia virus (CAV). It is implicated in several field problems of economic significance after maternal antibodies have waned. CAV threaten poultry industry alone or either coinfection with infectious bursal disease virus (IBDV) or inclusion body hepatitis (IBH). This paper aimed to understand the pathogenesis of this existing condition. Three hundred chickens from fifteen selected commercial broiler flocks (7 Cubb and 8 Native breeds) were collected from different areas of Dakahlia Governorate Egypt during the period from October 2019 to September 2020. Necropsy was carried out on freshly dead or diseased birds beside blood samples for hematological changes. Tissue samples were subjected to PCR assay for detection of CAV DNA, IBDV RNA and IBH DNA. Conventional PCR test indicated that all tissue samples collected form Cubb and Native breed farms were either PCR positive for CAV and IBDV coinfected with CAV while all samples were negative for IBH virus. Hematological findings showed a significant decrease (p<0.05) in RBCs count, Hb concentration and PCV in CIA and coinfected chickens. Grossly, emaciation was characteristic in carcasses moreover, lymphoid organs were congested and atrophied beside several multifocal hemorrhagic areas in skeletal muscles accompanied with pale bone marrow. Microscopically, lesions noticed were more intense and more severe in CIA coinfection with IBDV. Generalized lymphoid cell depletion in lymphoid organs and clusters of bacterial aggregations were common. Hemopoietic elements of bone marrow showed necrosis beside extensive depletion. It could be concluded that disseminated hemorrhages mainly in skeletal muscles and viscera beside lymphoid depletion of immune organs were responsible for high mortalities in CIA infected flocks which became higher in intensity in coinfection with IBDV. Presence of bacterial colonies in various examined organs following immunosuppression. Great economic losses from concomitant and subclinical infection by CIA beside carcass condemnation so, CIA vaccination of broiler chickens is recommended.

INTRODUCTION

High production pressure in poultry industry directly affected chickens which became more susceptible for many diseases, secondary infections and immunosuppression. This recent huge industry may be resulted in improper vaccination programs. Immunosuppressive viruses cause many clinical or subclinical conditions in poultry (Lütticken, 1997) The mechanism of this state of immunosuppression, is due to immune failure that recognize numerous signals (Babiuk et al. 2003).

From these viruses Infectious Bursal Disease (IBDV), Chicken Infectious Anemia (CIAV) and Inclusion Body Hepatitis (IBHV) viruses cause severe economic losses (Islam et al. 2002). These three viruses cause destruction of lymphoid tissues, poor response and depletion of immune cells which responsible for this state of immunosuppression beside mortalities and areas of congestion and hemorrhage in muscles and visceral organs (Dhama et al. 2002).

Chicken Anemia virus (CAV) is considered one of the smallest, non- enveloped, single stranded, circular negative sense DNA virus (Li et al., 2017a; Rosario et al. 2017). The virus DNA encodes viral proteins of VP1, VP2 and VP3 (Ducatez et al. 2008) belonging to the genus Gyrovirus of the family Cicroviridae, and has 23-25 nm in size. It causes Chicken Infectious Anemia (CIA) which is an acute highly contagious disease of young chickens, after an incubation period of 1 to 14 days with clinical symptoms in young chickens while it is a sub-clinical disease in older ones, which is the only natural host of the virus (Cardona et al., 2000b). CIA characterized by severe anemia, weakness, anorexia, ruffled feathers, stunted growth, generalized lymphoid atrophy and increased mortalities (5%-10%) up to 60% (Todd 2004 Dhama et al. 2008 McNeilly et al. 1991).

CIA is mainly noticed in broiler chicks of up to 3–4 weeks of age, which usually acquire the infection vertically (Pope 1991 Todd 2000 Dhama et al. 2008) but after 3 weeks of age the susceptibility to clinical disease decline. CIA infected birds developing an immunosuppression in the presence of other viruses such as Fowl adenovirus (FAV) (Toro et al. 2001) and NDV (De Boer et al. 1994). Beside attenuated immune response against several viral vaccines, resulting in vaccination failures (Todd 2000 Schat 2003 Toro et al. 2006 Dhama et al. 2008). Maternal antibodies prevent the clinical signs of disease but do not prevent infection and transmission of the virus or its immunosuppression effect (Sommer and Cardona, 2003). Among the histopathological changes, severe depletion of cortical thymocytes and erythroblastoid cells in the bone marrow which leads to immunodeficiency and anemia (Noteborn and Koch 1995). Diagnosis of CAV infection depends on history, clinical signs, hematological, pathological findings: gross and histopathological lesions which were confirmed by PCR as a developed tool of diagnosis (Gowthaman et al. 2014 Al-Ajeeli et al. 2020).

Infectious bursal disease (IBD) or Gumboro disease is an acute highly contagious disease of young chickens, characterized by severe immunosuppression in young chicks of 3-6 weeks of age (Sachan et al. 2019). Gumboro disease is caused by IBD virus (IBDV), is a nonenveloped double stranded RNA virus transmitted by oral fecal route (Smith et al. 2015). IBDV is belonging to family Birnaviridae, genus Avibirnavirus, which is responsible for major economic losses in poultry industry worldwide.

The virus has two serotypes: serotype1(pathogenic strain) which is classified into classical (intermediate and very virulent strains) (Jayasundara et al. 2017) and serotype 2(non- pathogenic one). IBDV causes cytolysis of dividing cells in the bursa of Fabricius in the infected chicks resulting in severe immunosuppression (Ciccone et al. 2017). This is severe state of immunosuppression due to apoptosis of B cell of the bursa and cytokine storm which considered the main factors of the severity of Gumboro that, may responsible for secondary infections beside failure of vaccination (Cubas-Gaona, et al. 2018). Gumboro disease is controlled either by live attenuated or inactivated (killed) IBDV vaccines which is preferred than live attenuated one. High levels of humeral immunity occurred by using killed vaccine for chickens at pre laying stage for inducing antibodies for a period of two weeks. Infectious Bursal Disease (IBD) causes a state of immunosuppression for infected chickens (Hon et al. 2008). Outbreaks occurred among the vaccinated flocks resulted in a chick that more susceptible to secondary infections by either viral or bacterial agents (Sachan et al. 2019). Hemorrhages in skeletal muscles, high mortalities and poor weight gain that considered as fatal complications of the disease (Zachar et al. 2016). Clinical diagnosis of Gumboro (acute form) is based on disease evolution in the form of mortality peak followed by recovery within 5-7 days (Van den Berg et al. 2000). Also, noticed symptoms, characteristic pathognomonic lesions in bursa of Fabricius beside histopathological investigations together with demonstration of viral antigen by immunohistochemistry (Balamurugan and Kataria, 2006). RT-PCR allows rapid identification of IBDV virus (Toroghi et al. 2003).

Inclusion Body Hepatitis (IBH) is one of immunosuppressive disease of poultry, caused by Fowl adeno virus (FAV). IBHV is a nonenveloped virus belonging to the adenoviridae family (Balamurugan et al. 2001) which mainly affects hepatic, endothelial and lymphoid cells. This disease affects poultry of 3-6 weeks old chicks, characterized by sudden onset, enlarged and mottled friable liver, that showing intranuclear inclusion bodies (IBHs) in the hepatocytes (Gowda and Satyanarayana, 1994) with high mortalities. FAV and CAV are transmitted vertically (Toro et al. 2001). There is a relationship between occurrence of IBH and presence of IBD and CIA viruses (Shane and Jaffery, 1997). FAV needs impairment of immune system of the bird to produce its pathogenic effect. So, IBD and CIA viruses predispose to FAV by their immunosuppression effect on chickens. Recently, IBH virus has reported as a virulent virus that is able to cause IBH as a primary disease without previous predisposing infection by IBDV and CIAV. (Susantha et al. 2006). Microscopically, it causes severe depletion of lymphocytes in medullae of bursal follicles. Molecular technique as PCR is a specific diagnostic method for demonstration of adenovirus (Balamurugan and Kataria, 2006).

The aim  of the present study to focus light on etiology and pathologic findings of widespread hemorrhagic syndrome of broiler chickens among some flocks (foreign and native) breeds reared in Dakahlia, Egypt in the period between October 2019 to September 2020.

MATERIAL AND METHODS

Chickens

Three hundred chickens were examined from fifteen commercial broiler flocks; (7 Cubb and 8 Native sasso) breeds were used for detection of CAV and coinfection with IBDV and IBHV during October 2019 to September 2020 in this study. Broiler Cubb farms aged 3-6 and 4-8 weeks old for sasso farms in different localities of Dakahlia Governorate, Egypt. All chicken flocks had been vaccinated against IBD, Newcastle, Avian Influenza and Infectious Bronchitis diseases. All birds with a history of mild respiratory manifestations, off food, depression, diarrhea with morbidity rate (20-70) % and low mortalities were subjected to study. The selected flocks were reared under cage system with standard managemental conditions.

Hematological changes

Blood samples were collected from wing vein of diseased and control healthy birds in tubes containing anticoagulant (dipotassium salt of EDTA 1mg/ 1ml blood) for RBCs count and determination of hemoglobin contents and Packed cell volume (PCV) using the automatic cell counter system XT (2000 IX) according to (Feldman et al. 2000).

Detection of CAV DNA, IBDV RNA, and IBHV DNA by Conventional PCR

Twenty pooled tissue specimens represented five selected examined flocks (4 birds selected from each flock), including bursa of Fabricious (for detection of IBDV RNA), liver, thymus and spleen (for detection of CAV DNA and IBHV DNA). Specimens were homogenized and subjected for conventional PCR. Tissue samples transported on ice from field to Animal Health Research (Provinicial-Lab Mansoura) and were labelled and stored at -20 C until used for PCR testing for detection of presence of CAV, IBDV and IBHV genome in the Biotechnology unit Reference lab for veterinary quality control on poultry production (Animal health research institute, Dokki, Giza, Egypt).

Nucleic acid extraction. Whole nucleic acid extraction from samples was performed using the QIAamp minielute virus spin kit (Qiagen, Germany, GmbH). Briefly, 200 µl of the sample suspension was incubated with 25 µl of Qiagen protease and 200 µl of AL lysis buffer at 56^OC for 15 min. After incubation, 250 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer.

Oligonucleotide Primers.

Supplied from (Metabion Germany) are listed in table (1).