Systematic Veterinary Virology

LABORATORY DIAGNOSIS

• Isolation and identification: The following are preferred clinical materials.
• Vesicular fluid
• Epithelium covering a vesicle
• Flaps of epithelial tissue still attached
• 5 ml of blood with anticoagulant during viraemic phase
• Esophageal—pharyngeal (OP) fluid from convalescent cattle, sheep, or goats.
• Serum
• From dead animals - samples of epithelial lesions, lymph nodes, thyroid, adrenal gland, kidney, and heart
• Specimens placed in ice should be delivered to a laboratory within 24 hours.
• When delivery may take longer, samples may be quick-frozen and should not thaw during transit.
• Isolation should be attempted in primary bovine thyroid cells, primary pig, calf or lamb kidney cells and cell lines, such as BHK-21 (baby hamster kidney) and IB-RS-2 cells. The CPE appears after 24-48 hours and the presence of virus should be confirmed by ELISA or PCR.
• Immunological methods of identification of virus
• ELISA: The preferred procedure for the detection of FMD viral antigen and identification of viral serotype is the ELISA as per the World Referral Laboratory for FMD (Refer General Characters Point 6). It is an indirect sandwich test in which different rows in multiwell plates are coated with rabbit antisera to each of the seven serotypes of FMD virus. These are the ‘capture’ sera. Test sample suspensions are added to each of the rows, and appropriate controls are also included. Guinea-pig antisera to each of the serotypes of FMD virus are added next, followed by rabbit anti-guinea-pig serum conjugated to an enzyme. Extensive washing is carried out between each stage to remove unbound reagents. A colour reaction on the addition of enzyme substrate, indicates a positive reaction. The ELISA is preferable to the complement fixation (CF) test because it is more sensitive and it is not affected by pro- or anti-complementary factors.
• Complement fixation test (CFT): Antisera to each of the seven types of FMD virus are diluted in veronal buffer diluent (VBD) in 1.5-fold dilution steps from an initial 1/16 dilution to leave 25 µl of successive antiserum dilutions in U-shaped wells across a microtitre plate. To these are added 50 µl of 3 units of complement, followed by 25 µl of test sample suspension(s). The test system is incubated at 37°C for 1 hour prior to the addition of 25 µl of 1.4% standardised sheep red blood cells (SRBC) in VBD sensitised with 5 units of rabbit anti-SRBC. The reagents are incubated at 37°C for a further 30 minutes and the plates are subsequently centrifuged and read. Appropriate controls for the test suspension(s), antisera, cells and complement are included. CF titres are expressed as the reciprocal of the serum dilution producing 50% haemolysis. A CF titre greater than or equal to 36 is considered to be a positive reaction. Titre values of 24 should be confirmed by retesting an antigen that has been amplified through tissue culture passage.
• Nucleic acid recognition methods
• PCR: The polymerase chain reaction is commonly performed to identify virus in diagnostic materials. Specific primers have been designed to distinguish between each of the seven serotypes. 
• In situ hybridisation: This technique is used to identify viral RNA in tissue samples.