Herd Health: Oral fluids for diagnostic tests - a progress report

Tuesday, June 2, 2009

Research suggests that, while they may not be suitable for an accurate, immediate diagnosis of an acute or new infection, they seem well suited for disease surveillance and field studies

by S. ERNEST SANFORD

Since I made my initial report on using oral fluids (saliva) to replace or reduce the dependence on blood samples for diagnostic tests (Better Pork, October 2006), this technique has progressed. It is now at the point where it is being tested and tried in the field by veterinarians, who hope eventually to use it as a routine tool.

Oral fluids are a mixture of saliva and oral mucosal transudate. Saliva (spit) is produced by the salivary glands. Oral mucosal transudate is fluid squeezed out of blood vessels in the lips, tongue, cheeks, floor and roof of the mouth into the oral cavity.

Oral fluids contain, among other things, both pathogens (bacteria and viruses) that may be circulating in the animal's blood and antibodies (Abs) to pathogens to which the animal has been exposed. In humans, oral fluids have long been used to test for many infections, such as HIV (AIDS), the hepatitis viruses (A, B, C), SARS virus and measles.
So far, in swine populations oral fluids have been used to detect PRRS virus (PRRSV), PCV2, Swine influenza virus (SIV), Mycoplasma hyopneumoniae (Mhyo) and Actinobacillus pleuropneumoniae (APP).

Over the last two years, researchers have been testing the sensitivity, specificity and overall reliability of oral fluid samples when compared with the routine blood sample tests that we currently use. They have been testing them both for their reliability in identifying different infectious agents like PRRSV, PCV2 and Mhyo as well as for detecting the presence and level of Abs.

Their findings so far indicate that these fluids can be used on a herd or population basis, but not for an accurate, immediate diagnosis of an acute or new infection. Routine diagnostics still have to be done in the case of a brand new disease outbreak.

The researchers used ELISA and PCR tests under experimental conditions to compare oral fluids against our conventional serum (blood) tests for PRRSV and PRRSV Ab levels in order to determine if oral fluids could be used reliably as routine diagnostic tests.  PRRSV was identified in serum for five weeks after experimental infection and for four weeks in oral fluid samples. Pig age at the time of experimental inoculation of pigs had no effect on the quantity or duration of virus in the oral fluid samples. Only low levels of PRRSV Abs were detected in the oral fluid samples.

From these results, they concluded that oral fluids would not be the best for routine diagnostic testing, but would hold up well for population surveys and retrospective work, or for seroprofiling herds over time.

After conducting further studies, the researchers determined that testing oral fluids for Abs might need some adjustments to the currently available commercial test kits if they were to give reliable results. They found that the commercial ELISAs could be for use with oral fluid samples by modification of the test protocol. Specific assay modifications may include incubation times, level of sample dilution, detection threshold (cut-off) and other alterations.

Testing field samples. Applying these tests to field samples has given somewhat equivocal results to date. At the Centralia Swine Research Update (CSRU) in January this year, Dr. Greg Wideman of Maitland Veterinary Services presented the early results of field trials he and his Listowel colleagues have been doing to validate the usefulness of the oral fluid samples for diagnostic work.

The results so far are running similar to other field applications of the technique. That is, for quick identification of early infection in a population of pigs, it is proving to be less sensitive than the conventional blood samples. They have not yet tried to use the technique for serological sampling with ELISA Ab tests.

I have been getting fairly similar feedback from other veterinarians testing oral samples in the field. However, at the annual meeting of the American Association of Swine Veterinarians in Dallas, Tex., in March 2009, Dr. John Prickett of Iowa State University reported on an extensive field study comparing results between oral fluid samples from a large commercial swine system and blood samples from pigs in the same system.

Their work indicates that when the PRRS ELISA test is modified to be compatible with running oral fluid samples, the diagnostic sensitivity is 80 per cent and diagnostic specificity is 100 per cent. These are very good results and workable for field samples if they continue to hold up at these levels.

Collecting oral fluids. Here's a quick rundown of how oral fluid samples are collected. The good news is that, with very little training, any producer can collect these fluids from their pigs in very short order.

A single length of rope is hung at the pig's shoulder height in a pen of pigs. Installing a bracket with a hole in the horizontal part of the plate works well as a support for the rope.
Place the bracket away from feed, water and feces so that the sample will be less contaminated

A 1.3-centimetre diameter rope is used for nursery pigs and a 1.6-centimetre rope for grow-finishing pigs. The rope is knotted at one end through the hole in the horizontal plate of the bracket and left to hang in the pen. 

The pigs are immediately attracted to the rope and start chewing on it. In about half an hour, enough fluid has accumulated to harvest a suitable sample. 

The rope is placed in a plastic bag or plastic booty. Fluid is manually squeezed out of the rope into the bag, which is tilted so that the fluid accumulates in one corner. 

Snip the corner of the plastic and drain the fluid into a sample tube. A four-millilitre sample is sufficient for most tests. If the sample is contaminated, centrifuge for 10 minutes and then pour into another clean tube.

Rope alternatives. Nearly all ropes today are made of nylon or some other synthetic material. Unlike synthetics, however, cotton ropes are highly absorbent and are thus well suited for collection of the oral fluids. Cotton ropes can be sourced via the internet. A simple Google search brings up several sources of cotton rope.

One alternative to rope is to plait strands of a floor mop head together. This should be equally absorptive as cotton rope, maybe even more so. Another possibility is to use towels or some naturally absorbent cloth material. One question that arises, of course, is the possibility that the pigs would rip and shred towels to bits and negate the purpose of attaining fluid samples. However, I believe that the short time needed to gather the appropriate volume of oral fluid samples (30 minutes or less) would allow the samples to be collected before the material is shredded by the pesky pigs.

So what does the future hold for oral fluid sampling technology?

When this new tool of oral fluids is refined and becomes usable as replacement for tests currently done with blood samples, here are a few things that we can expect to see unfold:

• already available in humans for HIV, HBV, H. pylori, malaria, mononucleosis, and the like.)
• Real-time disease monitoring in your herd.
• Immediate analysis of data in the context of the herd's historical productivity data.
• Epidemiological studies on dynamics of various diseases.
• Monitoring the infection and shedding status of diseases like PRRS.
• Informed decision making.

In short, oral fluids are starting to become a viable alternative to conventional blood sampling for diagnostic testing. They can provide a very cost-effective tool with which to make management and health decisions and conduct field studies. Oral fluids appear to be well suited for disease surveillance and for investigating the disease dynamics for within-herd and between-herd populations. BP 

S. Ernest Sanford, DVM, Dip. Path., Diplomate ACVP, is a swine specialist with Boehringer Ingelheim Vetmedica (Canada) in Burlington. Email: ernest.sanford@boehringer-ingelheim.com