Equine Health: Blood test can identify Strangles carriers

For the last four years, scientists at the Animal Health Trust, in Newmarket, have been analyzing the genetic structure of Streptococcus equi equi (S. equi), the bacterium which causes equine strangles. Their aim was to identify the antigens that are produced specifically by this bacterium. With funding from The Horse Trust, two antigens that are unique to S. equi, and which are targeted by the equine immune system following exposure to S. equi, have been identified. AHT bacteriologists have developed a new ELISA test which identifies antibodies to these S. equi specific antigens. Horses that have been exposed to S. equi will develop antibody titres to these antigens and have a positive result on the ELISA test.

As with any diagnostic test, proper use of the test and correct interpretation of the results are essential if the ELISA is to be used effectively. This hand-out is designed to help you interpret the results that you receive from us and to help you use the test in the best way possible.

Whenever the S. equi ELISA is used, it is important to remember the following points:

1) If the test result is positive for either or both antigens (antigens A and B), the horse is likely to have been exposed to S. equi in the recent past. This does not necessarily mean that it is currently infected with S. equi and could indicate any of the following:

* Exposure to S.equi and incubation of the disease
* Acute phase strangles in which case the horse may show clinical signs such as nasal discharge or abcessation of the lymph nodes of the head or neck
* Infection with S. equi in the previous 6 months, with or without clinical signs, followed by full recovery
* Infection with S.equi in the past, with or without clinical signs, resulting in immunity to the disease in the face of recent exposure
* Past infection with S.equi which has resulted in the horse becoming a carrier

2) It takes about 2 weeks for a horse to develop antibodies against each S. equi antigen. As a result, if a horse is tested in the acute stages of disease, a false negative result could be obtained.

3) Horses that recover from infection and have no residual carrier state can remain seropositive on the ELISA test for up to 6 months (due to the time it takes for antibody levels to naturally wane). It is important to remember that the ELISA test indicates exposure to S. equi only and does not determine whether the animal is actively shedding bacteria or not. To detect bacterial shedding further testing using culture or PCR is required. These techniques can be performed on nasopharyngeal swabs or guttural pouch lavage material.

4) The S. equi ELISA (as with most diagnostic tests) does not have a sensitivity and specificity of 100%. A false negative result can occur in the following circumstances:

* Late seroconversion
* Horse unable to sero-convert due to endogenous immuno-compromise
* True false negatives (of which we'd expect some 8%)

It is important to remember that the ELISA is an additional to test to use in the identification of S. equi carriers; it does not replace culture and PCR. If you are trying to determine if an animal is acutely infected with S. equi or if you are trying to verify that an ELISA positive animal is truly a S. equi carrier, we recommend using culture and PCR to identify organisms obtained via nasopharyngeal swabs or guttural pouch lavage. A series of 3 nasopharyngeal swabs, collected 1 week apart, will result in detection by a positive culture on at least one of the swabs in approximately 60% of carrier animals. Concurrent testing of these swabs by PCR increases the likelihood of detection to over 90% of carrier animals.

Below we have outlined two situations in which we envisage the S. equi ELISA test being used. We have outlined protocols for the use of the ELISA test in each situation. Both situations highlight the fact the S. equi ELISA should be used alongside PCR and culture to gain optimal results.

Situation 1 – Testing horses prior to their introduction into a new herd

In accordance with HBLB Codes of Practice, it is recommended that horses entering new premises are quarantined for 3-4 weeks in case they are incubating disease such as equine influenza virus or Strangles. Strangles obviously presents an additional problem due to the possibility of horses being sub-clinical carriers of S.equi i.e. they are shedding organism but have no outward signs of clinical disease. Because of the existence of a S. equi carrier state it is recommended that horses are tested while in quarantine to establish whether they are carriers or not. Until the introduction of the S. equi ELISA it was recommended that this was done by performing three nasopharyngeal swabs (at weekly intervals) and testing them via culture and PCR. A horse testing positive via culture or PCR was considered a S. equi carrier and thus required treatment to eliminate the carrier status before entering the new herd.

The S. equi ELISA can be used as an adjunctive test in this situation. As explained above, an animal who is a S. equi carrier would be expected to have a positive ELISA result. We would recommend performing the ELISA test on all animals when they enter quarantine and repeating it 3 weeks later (while the horses are still in quarantine). If all horses in quarantine are negative on both tests then there is no indication that any of the horses are S. equi carriers and they should be able to safely enter the new herd. If any of the horses are positive, on the first or second test, then further testing, using culture and PCR, is needed in order to determine whether the positive animals are true carriers. We recommend the following steps be taken:

1 – All horses in the group should remain in isolation while further testing is occurring (additional animals could have been exposed to S. equi and may yet seroconvert).

2 – Perform three nasopharyngeal swabs (at weekly intervals) or guttural pouch endoscopy on all ELISA positive horses and test samples via culture and PCR. If the affected animals have no organisms identified (i.e. culture and PCR negative on all samples) then the results indicate that they are not shedding S.equi and are unlikely to be carriers. If culture or PCR are positive, the horse is a S.equi carrier and all animals in the group are considered ‘in contacts’. Testing of all animals in the group should then be carried out using culture and PCR to identify animals which have been affected. Positive animals should be treated to eliminate carrier state and retested to ensure treatment was successful.

3 – Horses should only leave quarantine once it has been established that none of the animals in the group are S. equi carriers.

Situation 2 - At the end of a Strangles outbreak

During an outbreak of Strangles, culture and PCR are the most useful tests to use to determine if a horse is acutely affected with disease; ELISA testing is less useful as horses can be negative in the acute stages of the disease due to the time needed for seroconversion. The ELISA can, however, play a role after the outbreak is over. When a Strangles outbreak occurs, most affected animals will clear the infection within 30 days. However some horses will develop a carrier state and remain a source of infection to other animals although they outwardly appear healthy. The S. equi ELISA can be useful in identifying these animals.

Following an outbreak, the best time to detect a carrier animal is a minimum of 30 days after the last clinical signs are seen. Shedding usually ends rapidly after recovery although it may be intermittent. Prior to and during the testing period all affected horses and in contacts should be kept in strict isolation with the highest possible standards of hygiene. After 30 days, testing can occur to determine which horses, if any, have developed carrier status. No convalescent horse or in-contact can be considered free from infection until further testing has occurred. The protocol for further testing varies depending if horses are known to have been affected by Strangles or not. Prior to and during the testing period all affected horses and their in-contacts should be kept in strict isolation with the highest possible standards of hygiene.

* If a horse had clinical signs of Strangles during the outbreak, 30 days after the outbreak is over the horse would still be expected to have antibodies to S. equi, even if the horse has not become a S. equi carrier. In these cases, to demonstrate that the horse is not a S. equi carrier, culture and PCR testing should be performed on nasopharyngeal swabs (or guttural pouch lavage fluid). As previously mentioned, three nasopharyngeal samples should be obtained over a period of 2 weeks.

* During an outbreak there are likely to be several horses who have had no clinical signs of Strangles but who have been in contact with affected animals. These horses could be sub-clinical S. equi carriers despite showing no signs of disease. These animals can be tested using the S. equi ELISA to determine if they have evidence of an antibody response to S.equi. If any of these animals are positive via ELISA, they should go through the same culture/PCR testing process that is outlined above to determine if they are truly carriers. If a S. equi carrier is identified, this animal should be isolated and all in-contact horses retested 2-3 weeks after their last possible exposure to the carrier animal.

Any animal identified as a carrier should undergo treatment to eliminate carrier status and further testing to demonstrate clearance of infection. Diagnosis of S. equi carrier status requires that horse owners are committed to follow-up with appropriate veterinary care and management to either isolate the carrier(s) permanently or, preferably, isolate the carrier(s) and treat to eliminate the infection. Failure to test for carriers after an outbreak, or failure to treat or isolate known carriers, implies acceptance of the possibility that some of the convalescent cases and their in-contacts continue to present a danger of transmitting S. equi to other horses.