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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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Subject: Veterinary Epidemiology and Preventive Medicine × Type: Thesis ×
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    Molecular epidemiological studies of inclusion body hepatitis-hydropericardium syndrome in broiler chickens
    (LUVAS, 2007) Mittal, Dinesh; Khokhar, R.S.
    The present work was carried out to study the epidemiology of inclusion body hepatitis-hydropericardium syndrome (IBH/HPS) in broiler chicks in parts of Haryana as well as to characterize the fowl adenovirus (FAV) by polymerase chain reaction (PCR), restriction enzyme analysis (RE analysis) and sequencing. The data related to IBH/HPS outbreaks for the period from Jan, 1997 to Dec, 2006 was analyzed. During this period, 480 outbreaks of IBH/HPS were recorded in broiler chicks with overall morbidity, cumulative mortality and case fatality rate of 6.69%, 3.98% and 59.45%, respectively. The disease was observed through out the year and 74.58% outbreaks were in the age group of 21-40 days. A total of 40 tissue samples were collected for diagnosis of FAVs by histopathology, agar gel precipitation test (AGPT) and molecular techniques. Of the 40 samples, 22 showed basophilic intranuclear inclusion bodies on histopathological examination and 27 samples were positive by AGPT. Detection of FAVs by PCR was carried out in all the 40 field samples and two vaccines by amplifying the hexon gene. The FAVs could be detected in 33 field samples and both the vaccines as a single band of 900 bp was observed in the FAV positive samples. The seven field samples which were negative for PCR were also negative by histopathology and AGPT. The REA of 33 field isolates and both the vaccines was carried out by Sty 1, BsiWI, Mlu 1, Asp 1, Bgl 1 and Sca I enzymes after purification of the PCR products. The restriction fragment length polymorphism (RFLP) pattern generated by these enzymes indicated that 10 field samples belonged to serotype FAV-4, five to FAV-8, two each to FAV-2 and FAV-12 and, one each to FAV-5 and FAV-6. Twelve field samples were having more than two serotypes i.e. four had FAV-8 and FAV-5, two each had FAV-8 and FAV-7, FAV-8 and FAV-6, FAV-8 and FAV-12, one each had FAV-8 and FAV-4, FAV-4 and FAV-7. Both the vaccines had serotypes FAV-4 and FAV-8. The hexon A and B amplified and gel purified PCR products of four field isolates (HR 1, HR 2, HR 3 and HR 4) were cloned and sequenced. Nucleotide sequence analysis revealed that all the four field isolates had 67.6 to 95.2% similarity with in themselves. The field isolate HR 1 was having maximum similarity with FAV-2 (96.4%), HR 2 with FAV-4 (98.2%), HR 3 with FAV-8 (96.7%) and HR 4 with FAV-12 (97.7%) at nucleotide level. Amino acid sequence analysis revealed 64.6-95.3% sequence similarity between all the four field isolates. The isolate HR 1 had maximum amino acid similarity of 96.3% with FAV-2 reference strain. The similarity of HR 2 was highest with FAV-4 (97.3%), HR 3 with FAV-8 (94.3%) and HR 4 with FAV-12 (97.0%). Phylogenetic analysis based on nucleotide sequence revealed that all the four field isolates were placed in separate groups and were different serotypes. The field isolate HR 1 was grouped with FAV-2 reference strain, HR 2 with FAV-4, HR 3 with FAV-8 and HR 4 with FAV-12 reference strains. The RFLP pattern, nucleotide and deduced amino acid sequence analysis revealed the presence of FAV-2, FAV-4, FAV-5, FAV-6, FAV-7, FAV-8 and FAV-12 in the environment of Haryana.