pcr

Photo courtesy Dr. Emma Adam.

As we discussed in a previous issue (PCR – what’s behind this commonly used acronym?) PCR, or the polymerase chain reaction, is an incredibly sensitive technique to detect DNA. The technique is used for parentage testing, in forensic science and is heavily relied upon in medical testing to diagnose diseases, such as those caused by bacteria and viruses.

Our desire for rapid and sensitive tests to diagnose infections has pushed us to expand the number of PCR tests available and validated for use. As we discussed before, PCR can be performed in a matter of hours, whereas other techniques can take days to weeks. So why do we even need to retain the more traditional techniques at all when we have this amazing modern age method in our toolkit?

Well, as anyone who has watched a crime show on TV can tell you, DNA is a very stable molecule and can be detected easily with modern PCR techniques. This can lead to minute traces of DNA being enough to find the culprit of the crime, but is it as accurate when we are looking for the culprit responsible for disease in an animal?

There are a few factors to consider here. The first factor is the incredible stability of the DNA molecule. DNA fragments can exist in the environment for some time and can still be detected via PCR testing if the fragment corresponds the right primer sequence added to the reagent mix as we discussed in the previous article. That brings us to our next considerations. Since we use short (~20 nucleotide) sequences unique enough to identify a specific bacteria or virus, how do we know if these sequences come from a fragment of the genome of interest or the presence of the whole genome? Additionally, do we know if that bacterium is still viable and capable of replicating and causing disease? The answers are not clear cut.

Bacteria and viruses can survive in the environment and remain viable for variable periods of time depending on conditions and the veracity of the agent itself. Viruses are obligate intracellular pathogens and, as a result of that, virus isolation techniques are quite involved but do remain the ‘gold standard.’ Techniques to isolate viruses require special lab equipment, skilled staff and can take days to weeks, even when there is considerable knowledge on how to culture that particular virus.

Bacteria have traditionally been considered easier to culture in the lab and we have a long history of research that has generated knowledge on how to grow certain types of bacteria, prevent overgrowth of fastidious bacteria by those that grow easily in vitro and so on. Bacteria can be difficult to grow if the animal has received antibiotics, present in small numbers and easily overgrown by other bacteria in the sample or prefers conditions that we don’t yet understand or replicate in vitro. As such, whilst culturing bacteria is a more accessible and quicker process than it might be with viruses, it’s not without issues. That said, the ability to demonstrate bacterial growth from a sample is the ultimate demonstration that the bacteria are present, viable and can replicate, making culture still the ‘gold standard.’

Let us consider two real world scenarios. Streptococcus equi subspecies equi, the causative agent of ‘strangles’ is not a commensal organism (a relationship in which one organism derives food or other benefits from another organism without hurting or helping it) and it is always considered a pathogen when identified by culture or PCR. A PCR test was performed on a mare entering a herd to make sure she was not a carrier of this dangerous organism. The test involved flushing sterile saline into her guttural pouches and catching it in a sterile cup as it ran out of her nostrils. This is a pretty standard technique that allows the saline to interact with the guttural pouch, pharynx and nasal passages. The PCR test came back positive, so the mare was examined and cultures were performed. These cultures turned out to be repeatedly negative, but the washes from her nose were positive on PCR. At a loss, finally the equipment was tested and it became apparent that the farm’s rope twitch was the source of the Streptococcus equi subspecies equi DNA. Biosecurity and disinfection measures were adjusted, and all was well. Not only does this demonstrate the sensitivity of PCR, but also the importance of using more than one testing modality (as well as a reminder about biosecurity being of paramount importance!)

A second scenario relates to the presentation of a placenta to the University of Kentucky Veterinary Diagnostic Laboratory that was thought to have an infection caused by a group of organisms collectively termed ‘nocardioform bacteria.’ The placenta had the characteristic physical changes seen in this disease process, the mare had had all the clinical signs consistent with the disease as diagnosed by her veterinarian and the microscopic exam of the tissues were also consistent with the diagnosis. However, nocardioform organisms could not be grown from the placental tissue. The placental sample was positive on PCR testing, however. So how did this occur when everything pointed to the bacterial infection? The deduction was that because the mare had received antibiotics, the bacteria were either dead or inhibited from growing in culture. We don’t know for sure, but this example is one seen in many situations where antibiotic therapy has been instituted and bacteria cannot be cultured.

These examples and considerations make it hugely important that we maintain and support labs capable of using advanced techniques to get to the bottom of such issues. Fortunately, we have the resources offered by UK’s Veterinary Diagnostic Laboratory and Gluck Equine Research Center, as well as the clinical expertise of our veterinary community, to ensure we get the best answers possible for our equine population. Each test and each examination are a piece of the jigsaw puzzle that we try to put together to understand disease. It is rare that one single test will deliver the answers 100% of the time.


| Emma Adam, DVM, PhD, DACVIM, DACVS, based at UK’s Gluck Center and Veterinary Diagnostic Lab, is responsible for research and serves as veterinary industry liaison. Jackie Smith, MSc, PhD, MACE, Dipl AVES, is an epidemiologist based at the UK Veterinary Diagnostic Lab.