Herd Health

Oral fluids show promise as an alternative to blood samples

Simple to do, they involve less labour, offer greater herd sampling opportunities, reduce overall cost and mean less chance of injury to workers and pigs.

by ERNEST SANFORD

About 12 years ago, Danish researchers developed a test to identify salmonella antibodies (Abs) in fluids from diaphragm muscle taken from slaughter hogs. They reasoned, correctly, that these fluids -- they called them “meat juice” -- would contain Abs to various organisms, including salmonella spp. (ED: OK?) to which the pigs had been previously exposed some time in the past.

The “meat juice” was obtained by harvesting portions of pigs’ diaphragm muscle at slaughter, freezing it, then allowing it to thaw at room temperature at a later time, just prior to running their tests. The test they developed is called a mix-Elisa test and it, or variations of it, are being used to test for salmonella in many countries around the world, including Canada.

Meat juice is currently undergoing validation for its suitability as a source to test for PRRS and porcine circovirus type 2 (PCV2), the cause of postweaning multisystemic wasting syndrome (PMWS), now called porcine circovirus disease (PCVD), a disease that has approached epidemic proportions in Ontario and Quebec in the last year and a half.

The above is one example of an alternative means of obtaining samples to conduct diagnostic tests, instead of bleeding pigs to get blood samples. Researchers are constantly looking for alternatives to our conventional method of bleeding pigs to attain samples for diagnostic testing for PRRS and many other diseases.

In a previous issue, I documented the innovative approach, developed by Dr. Darwin Reicks, of using the ear vein blood swab technique for testing boars for PRRS virus infection (Better Pork, February 2005). The same meat juice developed by the Danes for salmonella testing is currently being evaluated for its suitability for PRRS testing.

Yet another technique currently being worked on is the use of oral fluids (saliva) as an alternative to blood samples. These samples are being examined for their suitability for both PRRS virus (PRRSV) detection (e.g. PCR or virus isolation) and antibody titres (e.g. IDEXX Elisa).

Drs. (ED: first name?) Zhou and Jeff Zimmerman, researchers at Iowa State University, have been investigating the possibility of using oral fluids (saliva) as an alternative sample for diagnostic testing for PRRS. The results of their experiments have shown that PRRS virus can be found diagnostically in oral fluids.

The researchers demonstrated PRRSV antigen (Ag) using quantitative PCR in side-by-side comparison trials run on both oral fluid and serum (blood) samples. They went further and examined levels of antibodies (Abs) in oral fluid compared with levels in serum from pigs after they experimentally infected them with PRRSV. They sampled the experimental pigs at certain prearranged days post infection. They also maintained uninfected control pigs.

What they found was that, when they used the IDEXX PRRS Elisa test at the manufacturer’s recommended 1:40 dilution, all serum samples were positive for PRRS Abs, but all oral fluid samples were negative. However, when they diluted to a 1:2 dilution, the oral fluids were positive for PRRS Abs in the earlier days post-infection, but they declined to negative values over time.

They have since developed additional tests, which show PRRS Abs present in oral fluid samples at diagnostically significant levels for at least 60 days post-infection. More testing has to be done to confirm the repeatability of these results.

Dr. Zimmerman’s research team at Iowa State University is also experimenting with pen-based collection of oral fluid samples (saliva) to see if this method would provide samples acceptable for diagnostic testing for PRRSV and PRRSV Abs.

What the researchers do is attach a cotton rope in a pen of pigs for the pigs to chew on the rope. Cotton rope is not easy to find these days, but the researchers prefer cotton rather than nylon or any of the other synthetics because of its great absorbency.

The researchers report that the pigs are immediately attracted to the rope and usually start chewing on it pretty quickly. About 30 minutes of chewing, sufficient saliva has accumulated to obtain a decent sample for diagnostic testing. After the pigs have chewed on the rope for 30 minutes (or more), the rope is placed inside a plastic bag and the "juice" is squeezed into the plastic bag. Then a corner of the bag is cut and the sample poured into a blood sample tube.

The researchers report that the sample may need to be centrifuged if it contains lots of dirt and feed. PCR tests for PRRSV or tests for PRRS Abs can be run on the sample.

There are several advantages to having pen-based oral fluid samples for testing versus conventional blood sampling. They are simple to do, involve less labour, offer greater herd sampling opportunities, reduce overall cost (no needles, collection tubes, etc.) and the chance of injury to workers and pigs.

These techniques are not limited to PRRS diagnostics. Once refined, they can be used for many other current and future diseases. If they hold up we can certainly adapt saliva-based testing for PRRS in some way that is feasible.

It might not be applicable for identifying an acute PRRS outbreak, but might very well be suited for epidemiological work or population surveys for PRRS and other diseases. This would be a breakthrough not to have to repeatedly take blood samples to test for various diseases.

S. Ernest Sanford, DVM, Dip. Path., Diplomate ACVP, is a swine specialist with Boehringer Ingelheim Vetmedica (Canada) in Burlington BP