**4.1 Virus isolation**

Virus isolation is considered as a gold standard for any viral disease diagnosis. In case of CPV-2 different cell lines like CRFK (Crandell Rees feline kidney), MDCK (Madin-Darby canine kidney) and A-72 are used for the isolation and propagation of the virus. The adapted virus causes distinct cytopathic effect in infected cell lines as cell rounding, aggregation, and necrosis of the affected cells. This requires the presence of special laboratory and is laborious [54].

### **4.2 Electron microscopy**

It is an expensive technique for the detection of the virions by negative staining in the stool samples or culture isolated virus. Immunoelectron microscopy can also be done by using CPV-specific antibodies. The need of expensive electron microscope makes it out of reach for regular usage [55].

### **4.3 Haemagglutination test (HA)**

The property of the CPV to cause agglutination of the pig, cat or rhesus monkey red blood cells at 4°C is used for detection of the CPV. The reciprocal of the maximum dilution of virus exhibiting ample agglutination of erythrocytes (mat formation) is designated as HA titer. The HA titer of more than 1:32 is usually considered as specific for CPV-2 [56].

## **4.4 Counter-immunoelectrophoresis (CIEP)**

The use of electric current allows the rapid movement of antigen and antibody towards each other resulting into the formation of precipitation line quicker than

simple diffusion reaction. This technique is not commonly used but have been utilized for the prevalence of CPV infection in clinically suspected dogs [57].
