Lateral flow molecular assay for horse strangles

Recipient Organization
BIOHELIX CORPORATION
32 TOZER ROAD
BEVERLY,MA 01915

Performing Department
(N/A)

Non Technical Summary
This proposal seeks to develop a simple, and sensitive molecular diagnostic assay for the detection of Streptococcus equi; the causative agent of strangles. Outbreaks of this disease at racetracks and training establishments caused major disruptions, and economic losses to owners, and trainers in the United States in recent years. The control of the spread of strangles relies on a combination of diagnostics, quarantine, vaccination, and in some cases on antibiotic therapy. A diagnostic test is needed to help veterinarians identify convalescent animals that are still infectious after clinical recovery because up to 10% of affected animals continue to shed Streptococcus equi sporadically for prolonged periods, after clinical signs have resolved. The ``gold standard'' for the detection of S. equi is culture of nasal swabs and washes. However, as nasal wash specimens often contain other beta hemolytic streptococci, like Streptococcus zooepidemicus, a diagnostic test must be highly specific. Molecular testing is highly specific. In addition, the assay proposed is easier to implement than other molecular testing technologies because it does not require costly instrumentation.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3810	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1100 - Bacteriology;

Keywords

streptococcus equi,

upper respiratory disease

infectious disease,

isothermal dna amplification,

lateral flow test,

strangles.

Goals / Objectives
We propose to develop, and evaluate an inexpensive test for S. equi that can be performed by the veterinarian, in a low budget diagnostic laboratory, or even on farm to yield results in less than 2 hours. In Phase I, we will develop kits, and protocols for the detection of S. equi in specimens from horses. In Phase II, we will perform a prospective, multi-site clinical study with specimens collected from sick, convalescent, and healthy horses from farms where strangles outbreaks are occurring or have occurred in the recent past. We expect the assay will be simple to use, and affordable enough for clinical veterinary use.

Project Methods
Nasal washes will be used to innoculate culture media, briefly cultured, and nucelic acids will be isolated from the culture media by boiling a diluted suspension of media. The diluted media will be used to seed an isothermal, helicase dependent amplification (HDA) of an S. equi specific gene (se18.9). Reaction products will be detected using a hybridization probe specific to S. equi, followed by a colorimetric visualization of the result using a disposable device. The disposable device is a container that encases the amplification reaction vessel such as to protect the laboratory from contamination by amplicons. It also contains a sharp knife such that the reaction vessel can be punctured to release amplicons as the closed container is closed. The amplification reaction products are then separated on a lateral flow strip encased in the container, and detected by colorimetric visualization (like a pregnancy test). The sensitivity and specificity of the HDA assay will be compared to that of the polymerase chain reaction assay developed by Dr. Timoney's laboratory at the Gluck Equine Research Center at the University of Kentucky.

Progress 06/01/08 to 01/31/09

Outputs
OUTPUTS: BioHelix has entered into a subcontract agreement with the University of Kentucky (UK). We supplied them with kits for the detection of Streptococcus equi manufactured at Biohelix, as well as with BESt detection cassettes. UK used these to test randomly selected S. equi and S. zooepidemicus isolates were grown from single colonies in THB liquid media. Following treatment of culture pellets with mutanolysin, DNA's were isolated using ZR Genomic DNA II KitTM (Zymo Research). Portions of samples equivalent to 10,000 cfu were analyzed by HDA, nested PCR specific to seM gene and 16S RNA specific PCR (control for the presence of S. equi/zoo DNA). Culture and SeM specific nested PCR negative nasal washes, which contained typical quantity of Gram+ bacteria (1000-100000 cfu/ml) were spiked with culture of S. equi CF32. Following treatment of nasal washes with mutanolysin, DNA's were isolated using ZR Genomic DNA II KitTM (Zymo Research). The sensitivity of the HDA assay was comparable with nested PCR and apparently was not depend on the contamination of specimens by other bacteria. Fifteen culture positive and 6 culture negative nasal washes from two outbreaks of strangle were used to evaluate sensitivity and specificity of the HDA assay. S. zooepidemicus MB13 and two other HDA positive S. zooepidemicus isolates revealed identical sequence for HDA generated amplicon. Positive reaction was occurred only when high excess of S. soo DNA were used. 13 of 15 (87%) culture positive specimens were also positive in HDA and PCR assays, whereas 1 of 21 (5%) specimens showed positive response in HDA assays only. Thus modification of primer/s may be considered to increase specificity of the assay. Concentration of specimen by centrifugation or filtration may also increase sensitivity of the assay. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The S equi assay is not being manufactured by BioHelix. We approached IDEXX to see if they might be interested in distributing the test. We have not received a positive reply to this offer.

Publications

A manuscript is in preparation (2009)