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Oral fluids sampling kit

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In these last years, new, quicker, easier and cheaper pig sampling, monitoring and diagnosis tools have been developed and validated.

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One of these techniques is the sampling and use of oral fluids (saliva) as a sample for diagnosis in pigs. This technique has already been experimentally validated for PRRSV, PCV2 and influenza, and it is being used routinely in USA farms for the detection of PRRSV and influenza at a population level. This technique is based on hanging a cotton rope in a pen, so the pigs bite it whilst they leave their saliva on it (Prickett et al., 2008).

The kit contains the following items:

100%-cotton rope (not treated with chemical products), a plastic bag (Zipplog ® style), a sterile plastic tube with a conical or rounded base and with enough capacity for the sample volume, and a pair of latex gloves.

1. Key points:

  • The number of ropes needed so the sampling is representative will depend on the population size, the arrangement of the buildings, and the size of the pens (it is deemed that in single-floor buildings with 1,000 animals, 6 ropes are enough to detect a 10% prevalence with a 95% confidence level (Detmer et al., 2010).
  • The diameter of the rope and the height at which it must be placed will depend on the age of the animals, but it must be easy to reach for all the group.
  • A 30-minute period is enough so 75% of the animals come into contact with the rope in a pen with 25-30 pigs, this figure increasing to 90% if we hang two ropes for the same length of time.
  • Before recovering the rope, check that it is saturated with saliva (this will help to decide if the period has been enough).

2. Sample collection:

The wet area of the rope is cut off and introduced in a clean plastic bag. The saliva is obtained as shown in the pictures, and the sample is decanted in the sterile tubes.

Warnings: The saliva extraction must be carried out as soon as possible after collecting the ropes, and care must be taken to handle the rope as little as possible directly with the hands to avoid cross contaminations. Refrigerate the samples quickly.

Oral fluids samples

3. Preservation:

- The dirt in the samples (artifacts) can interfere with the diagnosis. Due to this, the saliva must be centrifuged (1,000-2,000 rpm/30 min) or allow, at least, that for 60 min the dirt particles present in the sample settle in the bottom of the tube. Test or keep the supernatant.

- The samples must kept in a fridge (4-10°C). At 4°C, they may be kept up to 12 days, without this entailing a variation in the detection of PRRSV by PCR. To preserve the samples, they must be frozen (-20°C or -80°C).

4. Testing:

The detection of viral and bacterial pathogens is carried out through the polymerase chain reaction (PCR). The viral RNA of PRRS and influenza can be detected in saliva at 24 h post-infection. The detection of antibodies against PRRSV and PCV2 can be carried out through an ELISA test, and it is an alternative versus PCR to reduce the diagnosis costs. Nevertheless, the viral isolation of PRRSV and PCV2 is not performed yet, and in the case of influenza, the percentage of isolation from PCR-positive oral fluids is 51%.

5. Advantages and limitations of the technique:

The monitoring of a population through oral fluids can result in a lower diagnostic cost, whilst the number of sampled pigs increases, and in the future the number of pathogens that can be detected will increase too.

The collection of samples does not need a specialised staff. The pigs’ curiosity facilitates the sampling, because they interact and play with the rope whilst they place their saliva on it.

With the oral fluids samples, we obtain a higher population representation, and the sensitivity to detect low-prevalence infections (≤ 10%) is equal or lower than the traditional 30 individual samples.

The oral fluids samples are representative, at a population level, but they can never replace the individual samples for the isolation of a microorganism.

It is very important, in order to avoid false negatives, to avoid pooling samples and to verify that the samples are processed in a laboratory that is familiar with the techniques for the diagnosis in saliva samples.

To obtain more information, check:

http://vetmed.iastate.edu/vdpam/disease-topics/oral-fluids
http://www.vdl.umn.edu/submission-guidelines/oral-fluids/index.htm

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