ANNEX U.K. MANUAL OF DIAGNOSTIC PROCEDURES, SAMPLING METHODS AND CRITERIA FOR THE EVALUATION OF THE RESULTS OF LABORATORY TESTS FOR THE CONFIRMATION AND DIFFERENTIAL DIAGNOSIS OF SWINE VESICULAR DISEASE

CHAPTER IV U.K. Sampling procedures in a holding with clinical suspect pigs

1. When the presence of swine vesicular disease virus in a holding is suspected as clinical signs of disease have been observed, appropriate samples from representative groups of pigs showing these signs must be collected for disease confirmation and differential diagnosis with foot-and-mouth disease. U.K.

2. In these holdings the preferred samples for diagnosis are epithelium and vesicular fluid from unruptured or freshly ruptured vesicles collected from pigs showing the typical signs of disease, in which swine vesicular disease virus, its antigens or genome can be detected. It is recommended that about five or six of these pigs are sampled. U.K.

3. Even if fresh epithelial tissue and vesicular fluid in sufficient quantity (1g or more) are available, the following samples must also be collected: U.K.

(a)

blood samples from the suspected pigs and in-contact pigs for serological testing; and

(b)

faecal samples from suspected pigs and from the floor of their pen and of adjacent pens for virological testing.

4. The samples must be collected and transported in accordance with the following procedures: U.K.

(a)

for epithelium samples and vesicular fluid:

• if possible, at least 1g of epithelium tissue from an unruptured or recently ruptured vesicle must be collected. It is recommended that pigs are sedated before samples are collected both to avoid injury to personnel as well as for pig welfare,

• if transport to the national laboratory is carried out immediately (less than three hours), epithelial samples can be transported dry and kept refrigerated. However, if the time taken is likely to exceed three hours, the samples must be placed in a small volume of transport medium consisting of equal amounts of glycerol and 0,04 M phosphate buffer or other equivalent buffer (hepes), so that the pH is maintained in the optimal range for foot and mouth disease virus survival (pH from 7,2 to 7,6). The transport medium must contain antibiotics for additional anti-microbial activity. Suitable antibiotics and their concentration per ml final are:

  (i)

  penicillin 1000 IU,

  (ii)

  neomycin sulphate 100 IU,

  (iii)

  polymyxin B sulphate 50 IU,

  (iv)

  mycostatin 100 IU,

• if vesicular fluid can be collected from an unruptured vesicle, this must be kept undiluted in a separate container;

(b)

for blood samples:

• blood samples can be collected for serological or virological tests. However, generally they are collected only from pigs suspected to have recovered from clinical or subclinical infection for antibody detection, as epithelium, vesicular fluid and faecal samples from pigs showing clinical signs of disease are more suitable for virus detection than blood samples. It is recommended that whole blood samples are taken using vacutainers with no anticoagulant and that the vacutainers are transported unopened;

(c)

for faecal samples:

• faecal samples from the floor of premises suspected to contain, or to have contained, pigs infected with swine vesicular disease or faecal swabs and faecal samples from suspected live pigs must be placed in strong, leak-proof containers.

Containers of suspected samples must be disinfected on the outside before being transported to the laboratory. Suitable disinfectants are:

• sodium hydroxide (1:100 dilution),

• formalin (1:9 dilution of a solution of formalin containing a minimum of 34 % formaldehyde), and

• sodium hypochlorite (2 % available chlorine).

These disinfectants must be handled with care.