Gene-based strangles testing assessed in American study

Colonies of Streptococcus equi on a blood agar plate. Photo: Stefan Walkowski CC BY-SA 4.0 via Wikimedia Commons
Colonies of Streptococcus equi on a blood agar plate. Photo: Stefan Walkowski CC BY-SA 4.0 via Wikimedia Commons

Up to 20% of horses who outwardly recover from a strangles outbreak are persistent carriers of the organism in their guttural pouches, posing a challenge for veterinarians to identify these animals.

Guttural pouches are sacs of air that expand from the eustachian tube, with one on each side of the horse’s head. They lie beneath the ear and each pouch can hold as much as a coffee mug in a fully grown horse.

Streptococcus equi subsp equi (S. equi) is the pathogen behind strangles, which is a highly infectious upper respiratory disease in horses. It is sometimes called equine distemper.

Horses are generally treated with antibiotics to tackle the infection and prevent a carrier state as quickly as possible to allow the release of the animal from quarantine. But how can veterinarians reliably test to establish that animals are clear of the infection?

University of Pennsylvania researchers Ashley Boyle, Darko Stefanovski and Shelley Rankin examined this question in a study, the findings of which have been published in BMC Veterinary Research.

The trio said detection of S. equi was influenced by the site of specimen collection, the method of sampling, and type of diagnostic test performed.

They tested two gene-seeking tests. The first was a loop-mediated isothermal amplification (LAMP) test that targeted the eqbE gene, which is specific to S. equi. The second was a realtime polymerase chain reaction (PCR) test for the seeI gene, also specific to S. equi.

The study team hypothesized that the LAMP test would be more sensitive than the PCR test, and that the LAMP testing of specimens obtained by lavage (washing out) of the guttural pouch would be more sensitive in identifying carriers than the same test applied to samples from the nasopharyngeal region.

They described the taking of samples from 44 convalescent horses, through swabbing of the nasopharyngeal region, a nasopharyngeal wash, and collection through a lavage of the guttural pouch. A handful of samples were not tested, for various reasons, including the loss of three during transport.

The LAMP test was performed on all available specimens, while the PCR test and aerobic cultures were performed only on the guttural pouch specimens.

Specimens were positive using the LAMP test  in 1 of 41 nasopharyngeal swabs, 6 of the 38 nasopharyngeal washes, and 24 of the 44 guttural samples. By comparison, 18 of the 44 guttural pouch specimens were positive by PCR test and S. equi was isolated from 4 out of 44 of these specimens.

Detection of S. equi DNA was 51 times more likely from the guttural pouch samples than the nasopharyngeal samples, they reported.

When the guttural pouch samples were positive through the LAMP test, it was eight times more likely that the guttural pouch had an abnormality visible through an endoscopic examination, and almost 20 times more likely that mild pus accumulation was found.

A positive LAMP test also made it eight times more likely that the PCR test would be positive for S. equi DNA.

The study, they said, provided strong evidence that the LAMP test performed with guttural pouch specimens was more sensitive for the detection of S. equi than using a nasopharyngeal flocked swab or a nasopharyngeal wash taken from the same horse at the same time.

The LAMP test was comparable to the PCR test, they concluded.

“The 2005 American College of Veterinary Internal Medicine Consensus Statement recommendation for the detection of S. equi carriers is to obtain 3 negative nasopharyngeal washes or swab PCR samples over a 3-week period in order for an animal to be considered free of the organism.

“If the animal is found to be positive, endoscopic examination of the guttural pouch is strongly recommended.

“The data presented here show that a single guttural pouch lavage nucleic acid amplification test in conjunction with visual examination of the guttural pouches in the convalescent period provides a cost and time efficiency to determine S. equi status of an animal.”

The guttural pouch was the preferred anatomic site to sample to detect S. equi DNA in outwardly healthy convalescent horses using the LAMP test, they concluded.

“When used in conjunction with visual examination of the guttural pouch it provides evidence to eliminate the need for repeat testing with nasopharyngeal washes or swabs, thus saving time and money.

Since the LAMP test had acceptable agreement with the PCR test used, field verification was warranted of the LAMP test in a point-of-care device. This, they said, would enable stall-side testing performed by the field veterinarian.

The study, published under a Creative Commons License, can be read here

Comparison of nasopharyngeal and guttural pouch specimens to determine the optimal sampling site to detect Streptococcus equi subsp equi carriers by DNA amplification
Ashley G. Boyle, Darko Stefanovski and Shelley C. Rankin
BMC Veterinary Research 2017 13:75 DOI: 10.1186/s12917-017-0989-4

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