Changes

===Laboratory Tests===

===Laboratory Tests===

 

In many areas of the world, bluetongue in sheep, and especially in other ruminants, is subclinical and, therefore, laboratory confirmation based on virus isolation in embryonated chicken eggs, susceptible sheep, or cell cultures, or the identification of viral RNA by PCR is necessary. The identity of isolates may be confirmed by the group-specific antigen-capture ELISA, immunofluorescence, immunoperoxidase, serotype-specific virus neutralization tests, or hybridization with complementary gene sequences of group- or serotype-specific genes. For virus isolation, blood (10-20 mL) is collected as early as possible from febrile animals into an anticoagulant such as heparin, sodium citrate, or EDTA and transported at 4°C to the laboratory. For longterm storage where refrigeration is not possible, blood is collected in oxalate-phenol-glycerin (OPG). Blood to be frozen should be collected in buffered lactose peptone and stored at or below -70°C. Blood collected at later times during the viremic period should not be frozen, as lysing of the RBC or thawing releases the cell-associated virus, which may then be neutralized by early humoral antibody. The virus does not remain stable for long at -20°C. In fatal cases, specimens of spleen, lymph nodes, or red bone marrow are collected and transported to the laboratory at 4°C as soon as possible after death. A serologic response in ruminants can be detected 7-14 days after infection and is generally lifelong. Current recommended serologic techniques for the detection of bluetongue virus antibody include agar gel immunodiffusion and competitive ELISA. The latter is the test of choice and does not detect cross-reacting antibody to other orbiviruses, especially anti-EHDV (epizootic hemorrhagic disease virus) antibody. Various forms of virus neutralization test, including plaque reduction, plaque inhibition, and microtiter neutralization can be used to detect type-specific antibody.

6.12 It is essential for the diagnosis of BT that the most appropriate specimens are

carefully collected and properly transported. The following specimens are required for

the diagnosis of BT.

· Two 10 mL samples of blood from the jugular vein of each of up to six sheep with

high (in excess of 40.5°C) temperatures.

- One 10 mL of blood is run into a sterile bottle and allowed to clot to provide a

Technical Review - Bluetongue: The Virus, Hosts and Vectors

16.

- A second 10 mL is added to an anticoagulant, in vacutainers or commercially

prepared disposable tubes. EDTA is the anticoagulant of choice if blood is to be

tested by PCR, but heparin is suitable for most purposes.

present, sera should be collected from in-contact sheep.

· Sera from in-contact cattle, ideally yearlings, and from other ruminants.

· Cardiac and skeletal muscle (especially if abnormal) in formol saline.

6.13 Sera may be transported frozen at –20oC. Other specimens should be submitted

on wet ice and MUST NOT BE FROZEN. If ice blocks are used, extreme care should

be taken to ensure specimens do not contact the blocks. Direct contact with ice causes

freezing which lyses the blood cells thereby releasing, the predominantly cellassociated

virus that will be inactivated if antibody is present. Whole blood should be

held at 4°C.

6.14 A full history and identification of samples is necessary.

6.15 Diagnostic tests currently available at Pirbright, what each detects and the time

required to obtain the results are shown in Table 1, below:

===Pathology===

===Pathology===