DIAGNOSTIC BIOMARKERS OF INFECTIOUS CANINE HEPATITIS IN EXPERIMENTALLY INFECTED PUPPIES

Infectious canine hepatitis (ICH) is a systemic disease of Canidae and Ursidae caused by canine adenovirus type 1 (CAV-1) produces acute necrohemorrhagic hepatitis. The aim of the present study was to throw the light on the humoral and molecular diagnosis by polymerase chain reaction (PCR) and biochemical changes that occur in puppies infected with infectious canine hepatitis virus. Liver functions. tests, hematological profile, in addition to histopathological changes were performed. The study was carried out on fourteen native breed puppies of about three to four months old, apparently healthy, free from internal and external parasites. They were fed balanced diet and adequate water and housed under hygienic conditions in special isolated kennels. The animals were not vaccinated, and found to be free from canine Diagnostic Biomarkers Of Infectious Canine Hepatitis In . Khaled Kahilo et. , al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 98 hepatitis antibodies as screened by serum neutralization test. Puppies were divided into two groups, group 1, composed of 11 puppies were experimentally infected group with ICHV, and group 2, composed of 3 puppies were kept as noninfected control group. The results showed that, CAV-1 was detected in eight infected puppies by PCR showing amplicon of 508 bp sizes at the 1 and 2 week post viral infection. The infected puppies showed an elevation of serum enzymes activities as ALT, AST, ALP and GGT throughout the experimental period, also serum total protein and serum albumin were decreased at the 3 and 4 week post infection. Concerning hematological profile the infected puppies showed a decrease of HB, RBCs, platelets and haematocrit throughout the experimental period leading to anemia. The infected puppies showed a decrease of the total leukocytic count throughout the experimental period whereas neutrophils count increased significantly at the 1 week post viral infection then decline to the normal level at the 2 week and finally decreased below the normal upper limits at the 3 and 4 weeks. The infected puppies showed a decrease of lymphocytes count at 1 week post viral infection and then rapidly increased at the 3 and 4 week post viral infection. Moreover, the mean values of monocytes and eosinophils count increased gradually throughout the experimental period. It was clearly observed from the present study that, PCR procedure is faster, sensitive and easier as diagnostic biomarkers for ICH especially at the acute stage. Moreover, liver functions tests as well as hematological indices were the best diagnostic biomarkers in the chronic stage of ICH. Diagnostic Biomarkers Of Infectious Canine Hepatitis In . Khaled Kahilo et. , al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 99 INTRODUCTION Infectious canine hepatitis is a systemic disease of Canidae and Ursidae caused by canine adenovirus type 1 (CAV-1), (Appel, 1987). It is classified as group I (dsDNA), family Adenoviridae, genus Mastadenovirus and species canine adenovirus-1 (carter and wise, 2006). Infectious canine hepatitis, involves the massive destruction of liver cells (hepatocytes), (Hoskins et al., 2000). It is one of the acute hepatitis which firstly described by Rubarth, (1947) as it is a viral disease of the liver which produces striking hepatic damage in dogs and which has the experimental advantage of being caused by a cultivatable adenovirus. Viral hepatitis is widespread in the canine population and is a common cause of death. It causes serious and sometimes fatal disease of dogs which severely harms infected animals. (Wang et al., 2007). Owing to the partial antigenic cross reactivity of CAV-1 with human adenoviruses subsequent investigation showed that, it is not known to produce disease in man. (Kapsenberg et al., 1959). In the same line, ICH has the potential to do a great damage to cells throughout the body, initial cellular injury of the liver, kidney and eye is associated with cytotoxic effect of virus which ends in respiratory and eye infections. The main route of infection is ingestion of urine, feces, or saliva of infected dogs, the primary mode of transmission is by direct contact with an infected dog. Contaminated runs, cages, boots can also serve as a source of transmission (Foster and Smith, 2007). Initial infection occurs in the tonsillar crypts and Peyer’s patches, followed by viremia which lasts for 4-8 days, and infection of endothelial cells in many tissues. (Green, 1998). It was shown that, clinical signs of ICH may range from anorexia and lethargy to ataxia, seizures, paralysis Diagnostic Biomarkers Of Infectious Canine Hepatitis In . Khaled Kahilo et. , al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 100 and death (Cabasso, 1981). All unvaccinated dogs of all ages are at risk and susceptible to ICH; however, the disease is most prevalent in dogs less than one year of age (Muller et al., 2010). The incubation period of the virus is from four to nine days (Buxton and Fraser, 1977). Higuchi, (1990) reported that, the spread of a disease organism through populations of domestic or wild animals can be monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be detected and monitored. The sub-types of an organism that were responsible for earlier epidemics can also be determined by PCR analysis. Hu et al., (2001) reported that, PCR procedure is faster and easier to perform than any of the other assays used for detecting canine adenovirus, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses. Pursuing this further, they make a study to develop a simple method of detecting CAV-1, after electrophoresis, under the amplification conditions, 508 bp PCR products were observed for CAV-1. Liver functions test are some of the most commonly performed blood tests. These tests can assess liver functions or liver injury. An initial step in detecting liver damage is a simple blood test to determine the presence of certain liver enzymes in the blood. Under normal circumstances, these enzymes rise within the cells of the liver. But when the liver is injured for any reason, these enzymes are spilled into the blood stream. (Sharp and Dohme, 2011). Through infectious canine hepatitis virus infection there is increased liver enzyme activities and abnormal liver function test results were the most consistent clinicopathologic changes (Crawford et al., 1985). Diagnostic Biomarkers Of Infectious Canine Hepatitis In . Khaled Kahilo et. , al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 101 ALT is an enzyme present in hepatocytes of liver cells, when a cell is damaged, it leaks this enzyme into the blood, where it is measured. ALT rises dramatically in acute liver damage, such as viral hepatitis. (Nyblom, 2004). AST is similar to ALT in that it is another enzyme associated with liver parenchymal cells. It is raised in acute liver damage, but is also present in red blood cells and cardiac and skeletal muscle and is therefore not specific to the liver. The ratio of AST to ALT is sometimes useful in differentiating between causes of liver damage. Elevated AST levels are not specific for liver damage, and AST has also been used as a cardiac marker (Nyblom, 2006). ALP is an enzyme in the cells lining the biliary ducts of the liver; its levels in plasma will rise with large bile duct obstruction, intrahepatic cholestasis or infiltrative disease of the liver (Mengel, 2005). Albumin is synthesized only in the liver; it is secreted across the sinusoidal surface of the hepatocyte into plasma from the Golgi apparatus. Hypoalbuminemia is noted in various liver disorders. Furthermore, albumin levels are decreased in acute and chronic liver disease, such as cirrhosis. The half-life of albumin is 18-20 days. Albumin alone is not considered to be an especial marker of liver synthetic function. (Martin, 1961). Dogs suffered from infectious canine hepatitis suffered from leukopenia, the hemostatic defect of ICH was characterized by thrombocytopenia, abnormal platelet function, and anemia. (Wigton et al., 1976). On histopathological examination, intranuclear inclusion bodies were visible in hepatocytes, in association with hepatocyte dissociation and necrosis. (Thompson et al., 2010). Diagnostic Biomarkers Of Infectious Canine Hepatitis In . Khaled Kahilo et. , al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 102 MATERIALS AND METHODS