Streptococcus equi / Strangles Culture and PCR Testing

General Information

Diagnostic testing to detect shedding of the bacteria which causes Strangles in horses, Streptococcus equi, currently includes bacterial isolation by aerobic culture and subsequent biochemical identification, and bacterial DNA detection by the polymerase chain reaction (PCR) test.

See references at the end of this fact sheet for additional information.

Bacterial culture: Live Strep equi organisms must be recovered by a swab or wash of the nasopharynx, nasal passages, or draining abscesses in order to get a positive culture result. Samples with large amounts of mucopurulent discharge have been found to be negative on culture even if positive for Strep equi. If clinical signs suggest strangles, a repeat test should be done. Our normal turnaround time for culture results is 2-3 days. Additionally, the modified live vaccine strain of Strep equi (currently used in the only licensed intranasal strangles vaccine) will grow in culture, and cannot be differentiated from the field/wild pathogenic strains by routinely employed microbiological techniques. Sequencing techniques at the University of Kentucky Gluck Equine Research Center will allow for differentiating the wild strain from the vaccine strain.

A bacterial culture will be reported positive if a beta-hemolytic Streptococcus species is grown and identified specifically as the Strep equi (formal name is Streptococcus equi subspecies equi). A culture which does not grow a beta-hemolytic Streptococcus sp. within 48 hours on appropriate media is reported as negative for Strep equi. A culture which grows a beta-hemolytic Streptococcus sp, will be subjected to appropriate procedures to further identify the isolate, requiring at least an additional 24 hours of time to grow the organism, and sometimes longer, depending on the growth characteristics of the isolate

PCR (Polymerase Chain Reaction) has been extensively used for detection of Strep equi since the species specific M protein (SeM) sequence in this organism was reported by Timoney et al (1997). Strep equi PCR targeting this specific M protein has been proven to be quite specific.

The Animal Health Diagnostic Center (AHDC) at Cornell University employs Strep equi PCR with primers targeting this M protein sequence. Strep equi PCR is also sensitive and rapid. Strep equi PCR is more sensitive than culture, and due to its fast processing, results could be available on the same day that samples are submitted if STAT is requested

Our normal PCR turn-around time is 2-3 working days. STAT testing (same day results) for strangles PCR is available at the AHDC, but prior arrangements must be made with the laboratory. A STAT fee will be charged and a special weekend fee applies if results are needed on the weekend.

Sample Collection Techniques for PCR or Bacterial Culture

Nasopharyngeal wash: see link to AAEP guidelines. This procedure covers a large area of the nasal passages thus increasing the chance of recovery of the organism if present.

Nasal swabs or draining lymph nodes: swabs or material obtained from draining abscesses can be cultured for bacterial growth and/ or for PCR testing. When obtaining nasal swabs, it is acceptable to use routine length culture swabs. Two swabs can be introduced into the nares at the same time and kept in contact with the mucosa for a minimum of 20 seconds to insure good contact. One swab can be used for bacterial culture and the other for PCR.

Guttural pouch washes: Endoscopic examination and washes of both guttural pouches should be performed on any horse that has had positive PCR or culture. Three negative consecutive guttural pouch washes should be done 7 days apart for a period of 21 days to confirm negative status. Some veterinarians can submit samples from blind guttural pouch washes for testing, but to rule out carrier status visualization of the pouches should be done along with PCR as per the recommendations of the ACVIM. See link in reference section.

PCR Samples - Handling and Shipping

Guttural pouch washes and/nasopharyngeal wash samples should be submitted in a sterile plain red top tube or in a leak proof container. These samples must be shipped chilled and for overnight delivery.

PCR samples on swab: swabs should be placed in a sterile red top blood tube or a container that contains 1-2 drops of sterile saline to keep swab slightly moist. Bacterial transport media is not acceptable under any circumstance. These samples should be shipped chilled and for overnight delivery.

Bacterial Culture - Handling and Shipping

The same sample types obtained for PCR testing can be used for culturing, but swabs should be put in bacterial transport media such as Amies Media. Swabs or liquids shipped without a bacterial transport media may be cultured, but must be shipped chilled for overnight delivery. If more than 24 hours will lapse before arrival to laboratory, washes or swabs should be put into a transport media to assist in recovery of bacteria. A sterile swab should be saturated with the wash and placed into transport media. The bacteria may die without transport media after 24 hours; thus, causing a false negative.

Endoscopic/guttural pouch wash Bacterial Culture Sample Handling: If bacterial culture is requested from the guttural pouch wash, the liquid may be sent for direct culture if received chilled in a leak proof container within 24 hours post collection. If sample will not arrive within 24 hours, a sterile swab should be saturated with the wash material and placed in a suitable bacterial transport media such as Amies transport media or Stuart’s media

Interpretation of Results

Positive results by Strep equi PCR means that Strep equi DNA was detected in the sample. DNA can be detected from live, dead or modified live vaccine origin bacteria. Therefore, scheduling Strep equi PCR testing after administration of antibiotic treatments or intranasal vaccination should be carefully planned. The Zoetis (formerly Pfizer) drug company, manufacturers of Pinnacle IN (the intranasal strangles vaccine), is currently telling equine veterinarians that the Strep equi culture may be positive on routine culture up to 36 hours post administration of intranasal vaccine.

PCR tests after vaccination with the intranasal vaccine, Pinnacle, may be positive for up to 30 days. If a determination of whether the Strep equi isolated by culture is the wild or vaccine strain is required, sequencing can be done. If this is needed, the laboratory needs to be informed so as to keep the isolated Strep for further testing.

PCR testing may result in a SUSPECT result. This indicates that a positive signal was detected from the sample in a range near the limit of detection for the assay. While the signal detected in this range is most often specific, it can sometimes be the result of non-specific amplification. Any animal with exposure risk that has a SUSPECT result on PCR should be considered positive until proven otherwise.

References

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