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Survival of Streptococcus equi subsp. equi in Normal Saline Versus Phosphate-Buffered Saline and at Two Different Temperatures
Journal of Equine Veterinary Science 83 (2019) 102814
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Survival of Streptococcus equi subsp. equi in Normal Saline Versus Phosphate-Buffered Saline and at Two Different Temperatures Sara Frosth*, Susanna Sternberg Lewerin Faculty of Veterinary Medicine and Animal Science, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2019 Received in revised form 23 September 2019 Accepted 15 October 2019 Available online 23 October 2019
Streptococcus equi subsp. equi causes strangles in horses. Sampling to detect carriers is important for the control of the disease, and maximizing the sensitivity of this procedure is necessary. To provide a basis for the choice of sampling solution and transport temperature for samples, comparisons were made between the survival of Streptococcus equi subsp. equi in normal saline versus phosphate-buffered saline and at two different temperatures (cold and room temperature). At present, normal saline is used to sample the nasopharynx as well as the guttural pouches, and the sampling solution is transported without special cooling. The results revealed no significant difference in bacterial concentration levels between the two sampling solutions, but a significantly higher concentration of viable bacteria in the samples kept cold compared with room temperature. Hence, a change of sampling solution is not warranted, but maintaining the cold chain during storage and transport to the laboratory may be important for clinical samples. © 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Lavage Normal saline PBS Subclinical carriers Transport
1. Introduction Strangles is an upper respiratory disease in horses characterized by a sudden onset of fever followed by inflammation of the pharynx and later swelling and abscess formation of the lymph nodes [1]. Strangles is caused by the gram-positive bacterium Streptococcus equi subsp. equi [2]. After recovery from disease, some horses still carry the bacterium in their guttural pouches [3] or in their paranasal sinuses [4]. These are commonly referred to as long-term subclinical S. equi carriers [3]. Subclinical S. equi carriers can spread the disease to susceptible animals and serve as a reservoir of disease within the horse population [1]. A guttural pouch lavage, where the guttural pouches are washed with 50 mL normal saline (0.9% sodium chloride [NaCl]), followed by quantitative polymerase chain reaction (qPCR) is recommended to detect subclinical carriers [1]. Normal saline is also used in nasopharyngeal washes, which is
Animal welfare/ethical statement: This study did not require an ethical approval. Conflict of interest statement: The authors declare no conflicts of interest. * Corresponding author at: Sara Frosth, Faculty of Veterinary Medicine and Animal Science, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, P. O. Box 7036, SE-750 07 Uppsala, Sweden. E-mail address: [email protected] (S. Frosth).
an easier procedure than a guttural pouch lavage, but if it used alone, it can lead to false negative results because of intermittent bacterial shedding from the guttural pouch [1,5]. Hence, it is recommended to perform three consecutive nasopharyngeal washes to detect the presence or absence of S. equi carriers. In addition, a nasopharyngeal wash can be performed as a complement to a guttural pouch lavage not to miss potential S. equi residing in the paranasal sinuses. Identification of subclinical carriers is important for control of the disease, and it would be beneficial if the accurate detection of current infection could be further improved. Molecular approaches to detect and assess the viability of the pathogen have been explored [6]. Although qPCR can detect both viable and dead bacterial cells as well as free DNA, many sample preparation protocols rely on centrifugation to concentrate bacteria in liquid samples before qPCR analysis. Centrifugation is a quick and easy method, but free DNA and nonintact bacterial cells can be lost during the procedure; hence, viable bacterial cells are important also for qPCR. Optimal conditions for sampling and transport are important for bacterial viability and to prevent diagnostic sample degradation. For culturing purposes, viable bacteria are crucial, and although qPCR has proven more sensitive than culturing [6e8], further characterization of the bacterium, such as whole genome sequencing, requires previous isolation. Therefore, further
https://doi.org/10.1016/j.jevs.2019.102814 0737-0806/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2
S. Frosth, S.S. Lewerin / Journal of Equine Veterinary Science 83 (2019) 102814
assessment of the standard sampling solution and the need for additional measures to maintain a cold chain are warranted. At present, normal saline is used to sample the nasopharynx as well as the guttural pouches, and the sampling solution is transported without special cooling. The ideal sampling solution must be easy to obtain and promote bacterial survival but not be harmful to the mucous membranes of the nares and guttural pouch. Phosphatebuffered saline (PBS) was suggested as a potentially superior sampling solution to the commonly used normal saline because of its buffering ability. PBS has been used as the sampling solution in strangles cases [8], and we have found no data to support the current recommendation to use normal saline [1]. The aim of this study was to compare the survival of S. equi in PBS and in normal saline and at cold (5 C) compared with room temperature (approximately 20 C). We hypothesized that 1-week survival of S. equi would be better (1) in PBS than in normal saline and (2) at 5 C than 20 C. An increased survival of S. equi in guttural pouch lavages and nasopharyngeal washes during transport to the laboratory and up until analysis could improve diagnosis. 2. Material and Methods Three different S. equi isolates were used in this study: two clinical isolates, S1 and S2, and one reference isolate, CCUG 23255T (Culture Collection, University of Gothenburg, Sweden). The clinical isolate S1 was received from the National Veterinary Institute, Uppsala, Sweden. The second clinical isolate S2 was isolated from the environment of a Swedish horse stable in 2017, which 1 month earlier had had a diagnosed case of strangles. All isolates had been stored at 70 C in Brain Heart Infusion (BHI) broth (CM1135; Oxoid, Basingstoke, UK) with 15% glycerol and were subcultured twice on 5% bovine blood agar (BA) plates (B341960; National Veterinary Institute) at 37 C with 5% CO2 for 24 hours before use. One colony from each of the three subcultured isolates was isolated with a 1 mL loop and inoculated into 25 mL BHI (Oxoid) in a 50 mL centrifuge tube, and subsequently incubated at 37 C for 20 hours. Twenty milliliters of each cultured broth was divided into two 15 mL centrifuge tubes (10 mL in each), which were then
centrifuged at 1680 g for 10 minutes. The pellets were washed three times by the addition of 10 mL peptone saline (0.1% peptone in 0.85% NaCl) followed by centrifugation at 1680 g for 10 minutes. After the final wash, the duplicate pellets of each isolate were resuspended in 10 mL of normal saline (0.9% NaCl) pH 5.8 and PBS pH 7.4 (P5368; Sigma Aldrich, St. Louis, MO), respectively. The normal saline and PBS resuspensions for each isolate were diluted 1000 times (high spike) followed by a subsequent 100 times dilution (low spike), respectively. Four replicates were created by aliquoting each dilution into eight 50 mL centrifuge tubes and placing half of the tubes in a cooling incubator (5 C), whereas half of the tubes were kept on a laboratory bench at room temperature (approximately 20 C). Viable counts were performed on the BHI cultures and on the dilutions made after washing and resuspension of the bacterial cells in normal saline and PBS at the start of the experiment (Day 0). All samples (four replicates and 96 samples in total) were analyzed by viable count every 24 hours for 7 subsequent days. This was done by plating 100 mL of each culture or sample directly or in 10-fold serial dilutions of each sample on BA plates with the aim of receiving a countable number of colonies. All plates were incubated at 37 C with 5% CO2 for 24 hours. The initial bacterial concentrations of the 20 hours BHI cultures were 1.3 108, 4.4 108, and 2.3 108 CFU/mL for S1, S2, and CCUG 23255T, respectively, according to the viable count. Bacterial concentrations after washing, resuspension, and dilution in normal saline and PBS, respectively, for each S. equi isolate at the start of the experiment (Day 0) can be seen in Fig. 1. The results were analyzed by plotting the means of the four replicates and visual comparison of the resulting curves. In addition, the paired t-test was used for statistical comparison of the bacterial concentrations in the two sampling solutions and the two temperatures, respectively. 3. Results and Discussion The results from the viable counts for all three isolates in normal saline and PBS and at cold and room temperature, respectively, at Days 0e7 are shown in Fig. 1. The statistical analysis compared the
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Fig. 1. Mean bacterial concentrations (CFU/mL) of three S. equi strains during 7 days (0e7) in two different sampling solutions and at two different temperatures. The S. equi isolates are color coded as follows: S1 ¼ red, S2 ¼ green, and CCUG23255 ¼ blue. The left y-axis shows the samples with the initially high concentrations (solid line), and the right y-axis shows the samples with the initially low concentrations (dotted line). The unit of the x-axis is days. Different symbols (# or @) indicate significant differences (P < .05, paired t-test).
S. Frosth, S.S. Lewerin / Journal of Equine Veterinary Science 83 (2019) 102814
two variables of interest (sampling solution and temperature, respectively) for all strains and all time points studied. There was a significant difference between the bacterial concentration levels at cold and room temperature (P ¼ .04), but the difference between the bacterial concentration levels in normal saline and PBS were not statistically significant (P ¼ .68). The bacterial concentration at Day 0 was lower for the S1 isolate than the S2 and CCUG 23255T isolates. This could possibly be explained by the loss of bacterial cells during the washing procedure because the initial BHI culture concentrations were similar for all three isolates. The survival of S. equi was not better in PBS than in normal saline; therefore, a change of sampling solution is not warranted. However, cold storage (5 C) had a positive effect on the survival regardless of the sampling solution used (PBS or normal saline), particularly for the latter half of the study period (Fig. 1). To keep clinical samples cooled during transport can be expensive and not always convenient, but based on the results of this study, it may be important to prevent sample degradation and ensure optimal bacterial survival during transport and before analysis. If a cold chain during transport cannot be ascertained, samples should at least be kept cold before transport and up until analysis. In addition, as for all culture samples and supported by the temporal decline observed in this study, the time between sampling and analysis should be kept at a minimum. The period examined in this study reflects the span that can be expected for postal delivery in Sweden, unless special courier is used, the transport time will exceed 48 hours. As many viable bacteria as possible in the sample is important for diagnostic sensitivity, particularly when looking for subclinical S. equi carriers, where bacterial numbers in the sample are expected to be lower, and when culture of the bacterial isolate is needed for further characterization, such as whole genome sequencing. 4. Conclusions The results revealed no significant difference in bacterial concentration levels between the two sampling solutions, but a
3
significantly higher concentration of viable bacteria in the samples kept at cold compared with room temperature. Hence, the results do not support a change of sampling solution, but maintaining the cold chain during storage and transport to the laboratory may be important for clinical samples.
Acknowledgments €m, Department of The authors thank Lise-Lotte Fernstro Biomedical Sciences and Veterinary Public Health, for valuable support in the laboratory. Financial Disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1] Boyle AG, Timoney JF, Newton JR, Hines MT, Waller AS, Buchanan BR. Streptococcus equi infections in horses: guidelines for treatment, control, and prevention of strangles-revised consensus statement. J Vet Intern Med 2018;32: 633e47. [2] Timoney JF. The pathogenic equine streptococci. Vet Res 2004;35:397e409. [3] Newton JR, Wood JL, Dunn KA, DeBrauwere MN, Chanter N. Naturally occurring persistent and asymptomatic infection of the guttural pouches of horses with Streptococcus equi. Vet Rec 1997;140:84e90. [4] Tremaine WH, Dixon PM. Diseases of the nasal cavities and paranasal sinuses. In: Lekeux P, editor. Equine Respiratory Diseases. Ithaca, NY: International Veterinary Information Service; 2002. [5] Boyle AG, Stefanovski D, Rankin SC. Comparison of nasopharyngeal and guttural pouch specimens to determine the optimal sampling site to detect Streptococcus equi subsp equi carriers by DNA amplification. BMC Vet Res 2017;13:75. [6] Pusterla N, Leutenegger CM, Barnum SM, Byrne BA. Use of quantitative realtime PCR to determine viability of Streptococcus equi subspecies equi in respiratory secretions from horses with strangles. Equine Vet J 2018;50: 697e700. [7] Newton JR, Verheyen K, Talbot NC, Timoney JF, Wood JL, Lakhani KH, et al. Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi. Equine Vet J 2000;32:515e26. [8] Timoney JF, Artiushin SC. Detection of Streptococcus equi in equine nasal swabs and washes by DNA amplification. Vet Rec 1997;141:446e7.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Survival of Streptococcus equi subsp. equi in Normal Saline Versus Phosphate-Buffered Saline and at Two Different Temperatures Sara Frosth*, Susanna Sternberg Lewerin Faculty of Veterinary Medicine and Animal Science, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2019 Received in revised form 23 September 2019 Accepted 15 October 2019 Available online 23 October 2019
Streptococcus equi subsp. equi causes strangles in horses. Sampling to detect carriers is important for the control of the disease, and maximizing the sensitivity of this procedure is necessary. To provide a basis for the choice of sampling solution and transport temperature for samples, comparisons were made between the survival of Streptococcus equi subsp. equi in normal saline versus phosphate-buffered saline and at two different temperatures (cold and room temperature). At present, normal saline is used to sample the nasopharynx as well as the guttural pouches, and the sampling solution is transported without special cooling. The results revealed no significant difference in bacterial concentration levels between the two sampling solutions, but a significantly higher concentration of viable bacteria in the samples kept cold compared with room temperature. Hence, a change of sampling solution is not warranted, but maintaining the cold chain during storage and transport to the laboratory may be important for clinical samples. © 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Lavage Normal saline PBS Subclinical carriers Transport
1. Introduction Strangles is an upper respiratory disease in horses characterized by a sudden onset of fever followed by inflammation of the pharynx and later swelling and abscess formation of the lymph nodes [1]. Strangles is caused by the gram-positive bacterium Streptococcus equi subsp. equi [2]. After recovery from disease, some horses still carry the bacterium in their guttural pouches [3] or in their paranasal sinuses [4]. These are commonly referred to as long-term subclinical S. equi carriers [3]. Subclinical S. equi carriers can spread the disease to susceptible animals and serve as a reservoir of disease within the horse population [1]. A guttural pouch lavage, where the guttural pouches are washed with 50 mL normal saline (0.9% sodium chloride [NaCl]), followed by quantitative polymerase chain reaction (qPCR) is recommended to detect subclinical carriers [1]. Normal saline is also used in nasopharyngeal washes, which is
Animal welfare/ethical statement: This study did not require an ethical approval. Conflict of interest statement: The authors declare no conflicts of interest. * Corresponding author at: Sara Frosth, Faculty of Veterinary Medicine and Animal Science, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, P. O. Box 7036, SE-750 07 Uppsala, Sweden. E-mail address: [email protected] (S. Frosth).
an easier procedure than a guttural pouch lavage, but if it used alone, it can lead to false negative results because of intermittent bacterial shedding from the guttural pouch [1,5]. Hence, it is recommended to perform three consecutive nasopharyngeal washes to detect the presence or absence of S. equi carriers. In addition, a nasopharyngeal wash can be performed as a complement to a guttural pouch lavage not to miss potential S. equi residing in the paranasal sinuses. Identification of subclinical carriers is important for control of the disease, and it would be beneficial if the accurate detection of current infection could be further improved. Molecular approaches to detect and assess the viability of the pathogen have been explored [6]. Although qPCR can detect both viable and dead bacterial cells as well as free DNA, many sample preparation protocols rely on centrifugation to concentrate bacteria in liquid samples before qPCR analysis. Centrifugation is a quick and easy method, but free DNA and nonintact bacterial cells can be lost during the procedure; hence, viable bacterial cells are important also for qPCR. Optimal conditions for sampling and transport are important for bacterial viability and to prevent diagnostic sample degradation. For culturing purposes, viable bacteria are crucial, and although qPCR has proven more sensitive than culturing [6e8], further characterization of the bacterium, such as whole genome sequencing, requires previous isolation. Therefore, further
https://doi.org/10.1016/j.jevs.2019.102814 0737-0806/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2
S. Frosth, S.S. Lewerin / Journal of Equine Veterinary Science 83 (2019) 102814
assessment of the standard sampling solution and the need for additional measures to maintain a cold chain are warranted. At present, normal saline is used to sample the nasopharynx as well as the guttural pouches, and the sampling solution is transported without special cooling. The ideal sampling solution must be easy to obtain and promote bacterial survival but not be harmful to the mucous membranes of the nares and guttural pouch. Phosphatebuffered saline (PBS) was suggested as a potentially superior sampling solution to the commonly used normal saline because of its buffering ability. PBS has been used as the sampling solution in strangles cases [8], and we have found no data to support the current recommendation to use normal saline [1]. The aim of this study was to compare the survival of S. equi in PBS and in normal saline and at cold (5 C) compared with room temperature (approximately 20 C). We hypothesized that 1-week survival of S. equi would be better (1) in PBS than in normal saline and (2) at 5 C than 20 C. An increased survival of S. equi in guttural pouch lavages and nasopharyngeal washes during transport to the laboratory and up until analysis could improve diagnosis. 2. Material and Methods Three different S. equi isolates were used in this study: two clinical isolates, S1 and S2, and one reference isolate, CCUG 23255T (Culture Collection, University of Gothenburg, Sweden). The clinical isolate S1 was received from the National Veterinary Institute, Uppsala, Sweden. The second clinical isolate S2 was isolated from the environment of a Swedish horse stable in 2017, which 1 month earlier had had a diagnosed case of strangles. All isolates had been stored at 70 C in Brain Heart Infusion (BHI) broth (CM1135; Oxoid, Basingstoke, UK) with 15% glycerol and were subcultured twice on 5% bovine blood agar (BA) plates (B341960; National Veterinary Institute) at 37 C with 5% CO2 for 24 hours before use. One colony from each of the three subcultured isolates was isolated with a 1 mL loop and inoculated into 25 mL BHI (Oxoid) in a 50 mL centrifuge tube, and subsequently incubated at 37 C for 20 hours. Twenty milliliters of each cultured broth was divided into two 15 mL centrifuge tubes (10 mL in each), which were then
centrifuged at 1680 g for 10 minutes. The pellets were washed three times by the addition of 10 mL peptone saline (0.1% peptone in 0.85% NaCl) followed by centrifugation at 1680 g for 10 minutes. After the final wash, the duplicate pellets of each isolate were resuspended in 10 mL of normal saline (0.9% NaCl) pH 5.8 and PBS pH 7.4 (P5368; Sigma Aldrich, St. Louis, MO), respectively. The normal saline and PBS resuspensions for each isolate were diluted 1000 times (high spike) followed by a subsequent 100 times dilution (low spike), respectively. Four replicates were created by aliquoting each dilution into eight 50 mL centrifuge tubes and placing half of the tubes in a cooling incubator (5 C), whereas half of the tubes were kept on a laboratory bench at room temperature (approximately 20 C). Viable counts were performed on the BHI cultures and on the dilutions made after washing and resuspension of the bacterial cells in normal saline and PBS at the start of the experiment (Day 0). All samples (four replicates and 96 samples in total) were analyzed by viable count every 24 hours for 7 subsequent days. This was done by plating 100 mL of each culture or sample directly or in 10-fold serial dilutions of each sample on BA plates with the aim of receiving a countable number of colonies. All plates were incubated at 37 C with 5% CO2 for 24 hours. The initial bacterial concentrations of the 20 hours BHI cultures were 1.3 108, 4.4 108, and 2.3 108 CFU/mL for S1, S2, and CCUG 23255T, respectively, according to the viable count. Bacterial concentrations after washing, resuspension, and dilution in normal saline and PBS, respectively, for each S. equi isolate at the start of the experiment (Day 0) can be seen in Fig. 1. The results were analyzed by plotting the means of the four replicates and visual comparison of the resulting curves. In addition, the paired t-test was used for statistical comparison of the bacterial concentrations in the two sampling solutions and the two temperatures, respectively. 3. Results and Discussion The results from the viable counts for all three isolates in normal saline and PBS and at cold and room temperature, respectively, at Days 0e7 are shown in Fig. 1. The statistical analysis compared the
PBS 20°C #
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Fig. 1. Mean bacterial concentrations (CFU/mL) of three S. equi strains during 7 days (0e7) in two different sampling solutions and at two different temperatures. The S. equi isolates are color coded as follows: S1 ¼ red, S2 ¼ green, and CCUG23255 ¼ blue. The left y-axis shows the samples with the initially high concentrations (solid line), and the right y-axis shows the samples with the initially low concentrations (dotted line). The unit of the x-axis is days. Different symbols (# or @) indicate significant differences (P < .05, paired t-test).
S. Frosth, S.S. Lewerin / Journal of Equine Veterinary Science 83 (2019) 102814
two variables of interest (sampling solution and temperature, respectively) for all strains and all time points studied. There was a significant difference between the bacterial concentration levels at cold and room temperature (P ¼ .04), but the difference between the bacterial concentration levels in normal saline and PBS were not statistically significant (P ¼ .68). The bacterial concentration at Day 0 was lower for the S1 isolate than the S2 and CCUG 23255T isolates. This could possibly be explained by the loss of bacterial cells during the washing procedure because the initial BHI culture concentrations were similar for all three isolates. The survival of S. equi was not better in PBS than in normal saline; therefore, a change of sampling solution is not warranted. However, cold storage (5 C) had a positive effect on the survival regardless of the sampling solution used (PBS or normal saline), particularly for the latter half of the study period (Fig. 1). To keep clinical samples cooled during transport can be expensive and not always convenient, but based on the results of this study, it may be important to prevent sample degradation and ensure optimal bacterial survival during transport and before analysis. If a cold chain during transport cannot be ascertained, samples should at least be kept cold before transport and up until analysis. In addition, as for all culture samples and supported by the temporal decline observed in this study, the time between sampling and analysis should be kept at a minimum. The period examined in this study reflects the span that can be expected for postal delivery in Sweden, unless special courier is used, the transport time will exceed 48 hours. As many viable bacteria as possible in the sample is important for diagnostic sensitivity, particularly when looking for subclinical S. equi carriers, where bacterial numbers in the sample are expected to be lower, and when culture of the bacterial isolate is needed for further characterization, such as whole genome sequencing. 4. Conclusions The results revealed no significant difference in bacterial concentration levels between the two sampling solutions, but a
3
significantly higher concentration of viable bacteria in the samples kept at cold compared with room temperature. Hence, the results do not support a change of sampling solution, but maintaining the cold chain during storage and transport to the laboratory may be important for clinical samples.
Acknowledgments €m, Department of The authors thank Lise-Lotte Fernstro Biomedical Sciences and Veterinary Public Health, for valuable support in the laboratory. Financial Disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1] Boyle AG, Timoney JF, Newton JR, Hines MT, Waller AS, Buchanan BR. Streptococcus equi infections in horses: guidelines for treatment, control, and prevention of strangles-revised consensus statement. J Vet Intern Med 2018;32: 633e47. [2] Timoney JF. The pathogenic equine streptococci. Vet Res 2004;35:397e409. [3] Newton JR, Wood JL, Dunn KA, DeBrauwere MN, Chanter N. Naturally occurring persistent and asymptomatic infection of the guttural pouches of horses with Streptococcus equi. Vet Rec 1997;140:84e90. [4] Tremaine WH, Dixon PM. Diseases of the nasal cavities and paranasal sinuses. In: Lekeux P, editor. Equine Respiratory Diseases. Ithaca, NY: International Veterinary Information Service; 2002. [5] Boyle AG, Stefanovski D, Rankin SC. Comparison of nasopharyngeal and guttural pouch specimens to determine the optimal sampling site to detect Streptococcus equi subsp equi carriers by DNA amplification. BMC Vet Res 2017;13:75. [6] Pusterla N, Leutenegger CM, Barnum SM, Byrne BA. Use of quantitative realtime PCR to determine viability of Streptococcus equi subspecies equi in respiratory secretions from horses with strangles. Equine Vet J 2018;50: 697e700. [7] Newton JR, Verheyen K, Talbot NC, Timoney JF, Wood JL, Lakhani KH, et al. Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi. Equine Vet J 2000;32:515e26. [8] Timoney JF, Artiushin SC. Detection of Streptococcus equi in equine nasal swabs and washes by DNA amplification. Vet Rec 1997;141:446e7.