To achieve a definitive diagnosis in equine respiratory infections, testing is essential. But have you ever had an in-depth look at each test characteristic ?

While timely answers are paramount in managing the biosecurity measures to be put in place, it may be legitimate to wonder if point-of-care testing can provide the same level of confidence as that obtained from an external laboratory.

This article presents the different diagnostic tools available in equine respiratory diseases, emphasizing their ability to deliver early and accurate diagnosis, and thereby ultimately improving the clinical outcome as well as mitigating the risk of a yard outbreak.

Modern molecular diagnostics detect the genetic material of pathogens, allowing a rapid and accurate identification of the infectious agent.

Before this can be done, the sample must be carefully prepared : the process of extraction consists of cell lysis to release DNA or RNA which is then purified to eliminate proteins, enzymes and other inhibitors. Extraction is a key step to the performance of the test. 

Before DNA amplification, it is essential to perform efficient isolation and purification steps to obtain high quality genetic material.

For this, cells must be first lysed to release their contents. This can be achieved through chemical lysis (using detergents and enzymes), thermal lysis (applying heat to disrupt cell structures), or mechanical lysis (physically breaking cells with tools like bead beaters or sonication).

Combining different lysis techniques ensures an effective elimination of unwanted cellular components while preserving as much DNA as possible.

Recent studies show that combining chemical and mechanical lysis can give very good results, allowing both a high DNA yield and a good preservation of its quality, as long as the mechanical action is carefully controlled. On the other hand, thermal lysis alone is often considered less efficient because it does not release as much usable DNA as chemical or mechanical methods.

Once the cells are lysed, DNA can be extracted and purified using methods such as silica column-based extraction or magnetic bead separation, both will help isolating and cleaning the DNA while keeping it intact and suitable for the amplification.

Polymerase chain reaction, also known as PCR, was invented in 1983 and is a sensitive and specific technique used to detect the presence of viral or bacterial DNA in a sample. It is particularly useful for detecting infectious agents that are difficult to culture, and can also identify antibiotic resistance genes.

PCR is the most effective and rapid method used to identify the pathogens responsible for respiratory diseases in horses.

The amplification process consists of multiple cycles of 3 steps controlled by temperature :

• Denaturation ~ 95°C : Separation of the two DNA strands.
• Annealing ~ 56°C : Binding of specific primers to the target strands.
• Extension ~ 72°C : Synthesis of new DNA by the polymerase enzyme.

The exact temperatures depend on the nucleotide composition of the targeted genetic material and are determined during the development process to guarantee high levels of specificity and sensitivity.

The final PCR reaction mix is prepared by combining the purified nucleic acid with DNA polymerase, specific primers, dNTPs, buffer, magnesium chloride and water.

The mix is then placed into a thermocycler which will raise and lower the temperature of the sample, thus performing the pre-programmed cycles needed for amplification.

Loop Mediated Isothermal Amplification, also known as LAMP, is an amplification technique for DNA which is carried out at a constant temperature (typically between 60°C and 65°C).

LAMP utilizes a set of four to six specially designed primers for one target, contrary to PCR which requires only two.

The mix contains the polymerase, the primers, dNTPs, buffer, and other necessary reagents.

LAMP amplification results in the generation of stem-loop structures. The loop regions contain repeated sequences derived from the target DNA or RNA sequence. These loop structures act as templates for the initiation of new DNA synthesis, leading to rapid and exponential amplification of the target sequence.

LAMP amplification can be visually detected with the addition of DNA-intercalating dyes that produce a color change or fluorescent signals when bound to the amplified DNA. Turbidity-based detection, where the accumulation of magnesium pyrophosphate during the reaction causes the solution to become turbid, can also be employed.

Bacterial culture is a traditional method where the pathogen is grown in the laboratory. It is particularly useful for confirming infections such as strangles, caused by Streptococcus equi subsp. equi, or bacterial pneumonia.

This technique not only identifies the exact bacterium involved but may also provide information about its sensitivity to antibiotics, helping the veterinarian choose the most effective treatment.

However, culture has important limitations. Due to the need for the bacteria to multiply, the results often take from two days up to a week, which can be too long in severe cases where rapid action is needed. Moreover, it only works for bacterial infections and cannot detect viruses, which are a major cause of respiratory diseases in horses. Even amongst bacteria, some species are difficult or impossible to grow in the lab, which can lead to inconclusive results.

For these reasons, bacterial culture is a valuable complementary tool in equine respiratory infections.

Serology consists of testing a horse’s blood to look for antibodies directed against specific infectious agents. The presence of these antibodies indicates that the horse has been in contact with the pathogen, either through infection or vaccination. This makes serology useful for detecting exposure to diseases such as equine influenza or equine herpesvirus.

However, serology alone cannot confirm an active infection. Because antibodies are produced during the days following the infection, and may persist long after exposure to the pathogen, or even appear after a vaccination, false positives are common. For this reason, serology should only be used as a complementary tool in very specific cases.