Elsevier

Veterinary Microbiology

Volume 159, Issues 3–4, 12 October 2012, Pages 406-410
Veterinary Microbiology

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi

https://doi.org/10.1016/j.vetmic.2012.04.014Get rights and content

Abstract

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1 ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1 ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1 ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1 cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100 cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.

Introduction

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles (Sweeney et al., 2005). Asymptomatic carriers in the population may result in the spread of disease via introduction of S. equi to naïve populations. Bacterial culture and polymerase chain reaction (PCR) of nasopharyngeal (NP) washes and guttural pouch (GP) lavages have been used for both diagnostic testing and for the detection of S. equi in carrier animals but no definitive or gold standard test method has been shown to be optimal (Holland et al., 2006, Timoney and Artiushin, 1997, Sweeney et al., 2005, Verheyen et al., 2000). The sensitivity and specificity of S. equi PCR from NP swabs have been documented to range from 45% to 50% and 71%, respectively and the sensitivity and specificity of culture from NP swabs range from 18% to 45% and 94%, respectively (Gronbaek et al., 2006, Newton et al., 1997, Newton et al., 2000).

Recently, flocked swabs have been introduced in human diagnostics and have been shown to increase the sensitivity of bacterial culture and PCR via improved specimen collection and efficient release of the specimen material (Chernesky et al., 2006, Goldfarb et al., 2009, Van Horn et al., 2008). We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate (Method 2 > Method 1) (Table 1), (ii) centrifugation of a 1 ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples respectively (Methods 3 and 4 > Method 1; Methods 5 and 7 > Method 6), (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone. (Methods 5 and 7 > Methods 1 and 2), and finally (iv) PCR of a 1 ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1 ml saline (Methods 5 and 7 > Method 8). In this in vitro study, our goal was to optimize and determine the limit of detection of S. equi by aerobic culture and PCR in 0.9% NaCl using 4 bacterial culture techniques and 3 DNA amplification techniques.

Section snippets

Materials and methods

All testing was performed at the University of Pennsylvania New Bolton Center Microbiology Laboratory. This lab is fully accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD).

Results

The ability of each method to detect S. equi of known cfu/ml in 0.9% saline by eight (seven different) laboratory methods was examined (Table 1). Each method was replicated five times for each ten-fold serial dilution tested. The LOD for direct PCR (Methods 5 and 7) and flocked swab culture (Method 2) was determined at 1 cfu/ml. The LOD for assays, except for rayon culture swab (Method 1) and flocked swab PCR (Method 8), were reproducible at 30 cfu/ml or higher. LOD for Methods 1 and 8 were

Discussion

This preliminary in vitro study was performed to determine the optimal method for culture and/or PCR for NP and GP lavage washes from horses. The sensitivity and specificity of flocked swab culture and direct PCR are currently being compared in an funded field trial to assess how each of these assays perform with NP and GP wash samples from horses.

In the human and veterinary medical literature there are many examples that demonstrate that PCR is more sensitive than traditional culture methods.

Conclusion

To the author's knowledge, the pre-processing techniques for bacterial culture and PCR of S. equi in 0.9% saline had not been previously examined. A funded field trial will compare Method 2 (flocked swab culture), Method 8 (flocked swab PCR) with the current “gold standard” used in the lab (as represented by Methods 5 and 7) 1 ml aliquot PCR in the presence of background “noise” (commensal bacteria, epithelial cells, and white blood cells). This field trial will test NP wash and GP lavage

Conflict of interest statement

None. There is no relationship between the authors and Copan Diagnostics, Incorporated, manufacturers of Copan™ flocked swabs.

Acknowledgments

This work was supported by the Raymond Firestone Trust Research Grant, Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania. Study sponsor approved study design proposal and had no role in the collection, analysis and interpretation of data, writing the manuscript or the decision to submit the manuscript for publication.

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