- Email: [email protected]
Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing
Vaccine 34 (2016) 3935–3937
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Short communication
Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing Julia L. Livengood a, Saraswathi Lanka b, Carol Maddox c, Deepanker Tewari a,⇑ a
Pennsylvania Veterinary Laboratory, 2305 N Cameron Street, Harrisburg 17110, United States Veterinary Diagnostic Laboratory, University of Illinois College of Veterinary Medicine, 2001 S. Lincoln Avenue, Urbana, IL 61802, United States c Pathobiology Department, University of Illinois College of Veterinary Medicine, 2001 S. Lincoln Avenue, Urbana, IL 61802, United States b
a r t i c l e
i n f o
Article history: Received 16 January 2016 Received in revised form 21 April 2016 Accepted 7 June 2016 Available online 17 June 2016 Keywords: Streptococcus equi ssp. equi Strep. zooepidemicus-like protein (SzPSe) Pyrosequencing Strangles vaccine Modified live vaccine
a b s t r a c t Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is an important equine pathogen. Strangles is a highly contagious disease and a commercial modified live vaccine (MLV) is used for protection, which although effective, may also result in clinical signs of the disease. A rapid means to differentiate between the MLV and wild-type infection is crucial for quarantine release and limiting the disease spread. This study describes the use of a pyrosequencing assay targeting a single nucleotide deletion upstream of the SzPSe gene to distinguish between the wild-type and vaccine strains. A set of 96 characterized clinical specimens and isolates were tested using the assay. The assay was successful in differentiating between wild-type S. equi and the vaccine strains and in discriminating S. equi from other Streptococci. The vaccine strain was identified in 61.7% (29/47) of the strangles cases in horses with a history of MLV vaccination. Ó 2016 Elsevier Ltd. All rights reserved.
Infection with Streptococcus equi subspecies equi (S. equi) in equines is characterized by an initial onset of fever, followed by nasal discharge and lymph node enlargement [1]. These signs of infection, aptly named strangles, can become severe, leading to airway restriction and ultimately death [1]. S. equi is shed through nasal secretions after the onset of fever. S. equi may persist in the guttural pouches of horses, leading to a chronic carrier state where intermittent shedding of S. equi into the environment causes sporadic strangles outbreaks. In the United States, the most commonly used vaccine to prevent strangles is the PinnacleÒ I.N. vaccine (Zoetis, Florham park, NJ). It is an intranasally administered modified live vaccine (MLV) produced by N-methyl-N0 -nitro-N-nitrosoguanidine treatment of S. equi strain CF32, which was isolated in New York in 1981 [2]. The Pinnacle I.N. vaccine has been shown to cause fever, abscesses, and purpura hemorrhagica, especially in young equines [3,4]. In a recently vaccinated horse, or a horse of unknown vaccine status with abscesses, the ability to differentiate between vaccine and wild-type S. equi infection for disease control can be very useful. Culture, historically the gold standard of diagnosis, may initially appear to resolve between vaccine and wild-type strains, as vac-
⇑ Corresponding author. E-mail address: [email protected] (D. Tewari). http://dx.doi.org/10.1016/j.vaccine.2016.06.035 0264-410X/Ó 2016 Elsevier Ltd. All rights reserved.
cine strains typically exhibit a dry morphology, whereas wildtype strains are usually mucoid [5,1]. This is not, however, a reliable method of differentiation, as both colony types have been cultured from vaccine vials, and a vaccine strain may also revert from dry to mucoid [5]. Polymerase chain reaction assays are beneficial for identifying S. equi, but currently cannot differentiate between wild-type and vaccine strains [6]. Different methods have been described to differentiate wild-type and Pinnacle I.N. vaccine strains of S. equi including BiologTM MicroID System substrate utilization, pulsed-field gel electrophoresis, seM and SzPSe sequencing, and full genome sequencing [2,5]. These have proven to be reliable, but are labor intensive and time consuming for achieving strain differentiation [5]. The current study describes the application of a novel pyrosequencing assay targeting the described single base deletion 85 bp upstream of SzPSe in S. equi [5]. Pyrosequencing has been used in the past to accurately classify Streptococcus to the species level and more recently to identify nucleotide polymorphisms in viruses [7,8]. This study included 66 specimens (61 horses diagnosed with clinical strangles confirmed with isolation of S. equi and 5 healthy controls, that were either vaccinated or had no known history of vaccination) from the University of Illinois Veterinary Diagnostic Laboratory (VDL), and 30 nasal swabs from horses submitted to the Pennsylvania Veterinary Laboratory (PVL), where strangles was not on the differential list. Specimens positive for S. equi
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J.L. Livengood et al. / Vaccine 34 (2016) 3935–3937
included clinical nasal flushes, swabs, pus, and cultures of postvaccination abscesses. Control ATCC strains and Pinnacle I.N. (LOT#1391988) were also included for determining test specificity. DNA from the nasal flushes and swabs was extracted at the VDL using a Qiagen spin column extraction method according to manufacturer’s recommendations (QIAGEN, Inc., Valencia, CA). DNA was extracted from the bacterial isolates by a Chelex boiling method at the VDL (Bio-Rad, Hercules, CA). DNA from ATCC strains, Pinnacle I.N., and nasal swabs was extracted at the PVL using the Prepman Ultra Kit following manufacturer’s recommendations (Life Technologies, Carlsbad, CA). All specimens were screened for S. equi using both a real-time and a conventional PCR assay. The real-time assay targeting a portion of the seeI gene was carried out using the QuantiFast Pathogen + IC Kit (QIAGEN, Inc., Valencia, CA, USA), a final concentration of 0.4 lM each of SeeI-F and SeeI-R primers, 0.2 lM SeeI probe and 5 ll of DNA template in a final volume of 25 ll. The specimens were amplified and analyzed on an Applied Biosystems 7500 Fast Real Time PCR System (Life Technologies, Carlsbad, CA, USA) [6]. The conventional PCR assay targeting a 541 bp segment of the SeM protein gene was carried out in a 50 ll reaction with 10X PCR Buffer II, 0.1 unit of Taq Gold (Life Technologies, Carlsbad, CA, USA), a final concentration of 2 mM MgCl2, 0.2 mM dNTP’s, 0.4 lM each of ASW73 and ASW74 primers and 5 lL of DNA template. The specimens were amplified on a GeneAmp 9700 PCR System (Life Technologies, Carlsbad, CA, USA) as described previously [9]. Amplification of the full length SzPSe and SeM protein genes and sequencing of a 500 bp region that includes the targeted single nucleotide polymorphism (SNP) or deletion was carried out as previously described [5,10]. A SNP in the SeM protein gene and resulting change at the 63rd codon from Arginine to Glycine has also been useful in differentiating certain wild-type strains of S. equi from the Pinnacle I.N. strain [10]. For pyrosequencing, PCR targeting a 360 bp region surrounding the single guanine deletion upstream of SzPSe was carried out with Taqman Fast Universal PCR Master mix (Life Technologies, Carlsbad, CA, USA), FS (50 -CCGCAATGACTCAAAACTAATCGA-30 ) and RSbio (biotinylated-50 -TGCCATTTTATTCCCCTTTTGTTT-30 ) primers at a final concentration of 0.2 lM, and 1 lL of DNA template (including 1:10 dilution) in a final volume of 25 lL. Samples were amplified on a Veriti Fast Thermocycler with conditions: 95 °C for 1 min, 45 cycles of 95 °C for 0 s, 62 °C for 15 s (Life Technologies, Carlsbad, CA, USA). Subsequently, the amplified DNA templates were immobilized on streptavidin-coated Sepharose beads using the Vacuum workstation of the PyroMark Q24 according to manufacturer’s recommendations (QIAGEN, Inc., Valencia, CA, USA). DNA templates were released into an annealing mixture containing 0.3 lM of S1 sequencing primer (50 -CGGTTTAATATTGACCT-30 ), which was designed to be complementary to a short region directly upstream of the targeted guanine deletion. The mixture was incubated at 80 °C for 2 min, followed by a 5 min cooling period at room temperature. Specimens were pyrosequenced using Pyromark Gold reagents with an AQ assay and a dispensation order of: GACTGCAT (QIAGEN, Inc., Valencia, CA, USA). Specimens were classified as wild-type or vaccine based upon the presence of the double guanine or single guanine at the targeted location, respectively [5]. The ATCC control S. equi (#33398), Pinnacle I.N., Streptococcus agalactiae (#13813), Streptococcus pneumoniae (#49619), Streptococcus equisimilis (#35666) and Streptococcus zooepidemicus (#43079) were used for pyrosequencing assay verification. Sixty-one specimens that were positive for S. equi by isolation and SeM PCR (including 29 clinical specimens and 32 S. equi isolates) were also positive by the pyrosequencing PCR (Table 1).
Table 1 Comparison of PCR, sequencing, and pyrosequencing for the detection and typing of Streptococcus equi ssp. equi in specimens collected from horses. Specimen (s)
Vaccinationa
SeM & seeI PCR
SeM/SzPSe Seq.b,c
SzPSe Pyrosequencingc
# of clinical specimens (nasal flushes, swabs and abscess) tested 6 Vaccinated + wt wt 10 Vaccinated + vx vx 11 Unknown + wt wt 2 Unknown + vx vx 35 Unknown d ND – # of culture isolates (from abscess and nasal swabs) tested 12 Vaccinated + wt 19 Vaccinated + vx 1 Unknown + vx
wt vx vx
wt = Wild-type strain; vx = vaccine strain; ND = not determined. a Horses received vaccination with PinnacleÒ I.N or had unknown vaccination status. b SeM gene sequencing indicated a substitution of glycine for arginine at the 63rd amino acid residue of the M-like protein for the vaccine strain [9]. c Sequencing upstream of SzPSe indicated the presence of a single guanine at the targeted single guanine deletion location for the vaccine strain [5]. d 1 specimen seeI PCR positive but SeM, SzPSe and pyrosequencing negative.
These results were confirmed by seeI PCR. One specimen reacted with seeI primers, but not with SeM or the pyrosequencing primers. The fact that the seeI gene resides on a mobile genetic element and can be exchanged with other bacteria may explain the discrepancy, but this could not be confirmed further as bacterial isolation was not attempted for this specimen [6,11]. Pyrosequencing of the 29 S. equi positive clinical specimens, which consisted of nasal and abscess swabs collected from both vaccinated and unvaccinated horses, showed 58.6% to contain the GG genotype, leading to a classification as wild-type. The remaining 12 clinical specimens (41.4%) were identified as vaccine strains with a genotype of –G (Table 1). The second subset of specimens was comprised of S. equi isolates cultured from post-vaccination abscesses or nasal swabs from horses vaccinated with Pinnacle I. N. Of the 32 S. equi isolates, 12 isolates (37.5%) had a genotype that correlated with wild-type S. equi, while 20 isolates (62.5%) were identified as the vaccine strain by pyrosequencing. The pyrosequencing results were confirmed with gene sequencing (Table 1). The sensitivity and specificity of the pyrosequencing assay were determined to be 100%. Overall, the vaccine strain was identified in 61.7% (29/47) of the specimens from horses with clinical strangles that had a history of Pinnacle I.N. vaccination (Table 1). The time elapsed since vaccination was not always known, but the vaccine strain was detected up to 5 weeks post-vaccination. A recent vaccination history could explain the higher detection rate for the vaccine strain, although the vaccine itself has also been reported to cause the disease, which would contribute to vaccine strain detection [2]. The predominance of wild-type strains (78.5%) in horses with strangles that had no vaccine history (11/14) was as expected, but in some such cases the vaccine strain was also recovered (Table 1). To better understand shedding of the vaccine strain, vaccination efficacy with the MLV and the disease epidemiology, adding S. equi strain typing for routine diagnosis would be very useful. The pyrosequencing assay described in this study rapidly and accurately distinguishes between wild-type and vaccine types of S. equi. Wild-type S. equi strains consistently displayed the GG genotype in the pyrograms. A sequence of ATTGCAAT was consistently found in vaccine strains, with pyrograms obtained within an hour of the start of the pyrosequencing assay (Fig. 1). Sequencing upstream of SzPSe has previously shown the vaccine progenitor to have a GG genotype at the study location [3]. Thus, the pyrosequencing assay will also identify the progenitor strain as a wildtype strain with a GG genotype, not as a vaccine strain.
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J.L. Livengood et al. / Vaccine 34 (2016) 3935–3937
Fig. 1. (A) Pyrogram showing GG genotype (double peak) highlighted in grey for S. equi wild-type strain. (B) Pyrogram showing G genotype (single peak) highlighted in grey for S. equi vaccine strain.
--
l
- - - - l - - -- - l GGTTTAATA
----l----l
- ---l----l
S1 Sequencing Primer
AAC
TTGACCTA
S. equi Wild Strain
AAC
GGTTTAATA
TTGACCTAAT
TGGCAATAAC
S. equi Vaccine Strain
AAC
GGTTTAATA
TTGACCTAAT
T--GCAATAAC
U04620.1 S. zooepidemicus W60
AAC
CGGTTTAATA
TTGATTTAAT
TGGCAATAAC
CP001129 S. zooepidemicus MGCS10565
AAC
CGGTTTAATA
TTGATTTAAT
TGGCAATAAC
CP002904.1 S. zooepidemicus ATCC 35246
AAC
AGGTTTAATA
TTGATTTAAT
TGGCAATAAC
FM204884.1 S.zooepidemicus H70
C
CCCTTTTGTT
TTTATCTA
GACATGATG
CP006770.1 S. zooepidemicus CY
C
CCCTTTTGTT
TTTATCTA
GACATGATG
Fig. 2. Pyrosequencing primer alignment with S. equi (wild-type and vaccine strain) and select isolates of S. zooepidemicus.
It was noteworthy that although S. zooepidemicus showed amplification in the PCR for pyrosequencing due to sequence identity with the FS and RS-bio primers, the sequencing primer chosen in our assay binds to S. equi DNA due to sequence identity (Fig. 2). Generally, a two base mismatch towards the 30 end of a primer can easily prevent non-specific binding and any additional amplification, thus making the pyrosequencing assay more specific [12]. In conclusion, these findings show that pyrosequencing targeting the described single base deletion can be used to classify S. equi strains from nasal flushes, swabs, and bacterial isolates as either vaccine type or wild-type. Pyrosequencing is particularly valuable in this differentiation, as a more rapid diagnosis will allow for the most appropriate management of strangles cases. References [1] Sweeney CR, Timoney JF, Newton JR, Hines MT. Streptococcus equi infections in horses: guidelines for treatment, control, and prevention of strangles. J Vet Intern Med 2005;19:123–34. [2] Cursons R, Patty O, Steward KF, Waller AS. Strangles in horses can be caused by vaccination with Pinnacle IN. Vaccine 2015;33(30):3440–3. [3] Al-Ghamdi GM. Characterization of strangles-episodes in horses experiencing post-vaccinal reaction. J Anim Vet Adv 2012;11:3600–3.
[4] Borst LB, Patterson SK, Lanka S, Barger AM, Fredrickson RL, Maddox CW. Evaluation of a commercially available modified-live Streptococcus equi subsp equi vaccine in ponies. Am J Vet Res 2011;72:1130–8. [5] Lanka S, Borst L, Patterson S, Maddox C. A multiphasic typing approach to subtype Streptococcus equi subspecies equi. J Vet Diagn Invest 2010;22:928–36. [6] Baverud V, Johansson SK, Aspan A. Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi supsp. zooepidemicus. Vet Microbiol 2007;124:219–29. [7] Innings A, Krabbe M, Ullberg M, Herrmann B. Identification of 43 Streptococcus species by pyrosequencing analysis of the rnpB gene. J Clin Microbiol 2005;43:5983–91. [8] Tewari D, Del Piero F, Cieply S, Feria W, Acland H. Equine herpesvirus 1 (EHV1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing. J Virol Methods 2013;193:371–3. [9] Kelly C, Bugg M, Robinson C, Mitchell Z, Davis-Poynter N, Newton JR, et al. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. J Clin Microbiol 2006;44:480–6. [10] Borst LB, Patterson SK, Lanka S, Suemoto MM, Maddox CW. Zebrafish (Danio rerio) as screen for attenuation of lancefield group C streptococci and a model for streptococcal pathogenesis. Vet Pathol 2013;50:457–67. [11] Holden MTG, Heather Z, Paillot R, Steward KF, Webb K, Ainslie F, et al. Genomic evidence for the evolution of Streptoccocus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathog 2009;5 (3):e1000346. http://dx.doi.org/10.1371/journal.ppat.1000346. [12] Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. PrimerBLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 2012;13:134.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Short communication
Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing Julia L. Livengood a, Saraswathi Lanka b, Carol Maddox c, Deepanker Tewari a,⇑ a
Pennsylvania Veterinary Laboratory, 2305 N Cameron Street, Harrisburg 17110, United States Veterinary Diagnostic Laboratory, University of Illinois College of Veterinary Medicine, 2001 S. Lincoln Avenue, Urbana, IL 61802, United States c Pathobiology Department, University of Illinois College of Veterinary Medicine, 2001 S. Lincoln Avenue, Urbana, IL 61802, United States b
a r t i c l e
i n f o
Article history: Received 16 January 2016 Received in revised form 21 April 2016 Accepted 7 June 2016 Available online 17 June 2016 Keywords: Streptococcus equi ssp. equi Strep. zooepidemicus-like protein (SzPSe) Pyrosequencing Strangles vaccine Modified live vaccine
a b s t r a c t Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is an important equine pathogen. Strangles is a highly contagious disease and a commercial modified live vaccine (MLV) is used for protection, which although effective, may also result in clinical signs of the disease. A rapid means to differentiate between the MLV and wild-type infection is crucial for quarantine release and limiting the disease spread. This study describes the use of a pyrosequencing assay targeting a single nucleotide deletion upstream of the SzPSe gene to distinguish between the wild-type and vaccine strains. A set of 96 characterized clinical specimens and isolates were tested using the assay. The assay was successful in differentiating between wild-type S. equi and the vaccine strains and in discriminating S. equi from other Streptococci. The vaccine strain was identified in 61.7% (29/47) of the strangles cases in horses with a history of MLV vaccination. Ó 2016 Elsevier Ltd. All rights reserved.
Infection with Streptococcus equi subspecies equi (S. equi) in equines is characterized by an initial onset of fever, followed by nasal discharge and lymph node enlargement [1]. These signs of infection, aptly named strangles, can become severe, leading to airway restriction and ultimately death [1]. S. equi is shed through nasal secretions after the onset of fever. S. equi may persist in the guttural pouches of horses, leading to a chronic carrier state where intermittent shedding of S. equi into the environment causes sporadic strangles outbreaks. In the United States, the most commonly used vaccine to prevent strangles is the PinnacleÒ I.N. vaccine (Zoetis, Florham park, NJ). It is an intranasally administered modified live vaccine (MLV) produced by N-methyl-N0 -nitro-N-nitrosoguanidine treatment of S. equi strain CF32, which was isolated in New York in 1981 [2]. The Pinnacle I.N. vaccine has been shown to cause fever, abscesses, and purpura hemorrhagica, especially in young equines [3,4]. In a recently vaccinated horse, or a horse of unknown vaccine status with abscesses, the ability to differentiate between vaccine and wild-type S. equi infection for disease control can be very useful. Culture, historically the gold standard of diagnosis, may initially appear to resolve between vaccine and wild-type strains, as vac-
⇑ Corresponding author. E-mail address: [email protected] (D. Tewari). http://dx.doi.org/10.1016/j.vaccine.2016.06.035 0264-410X/Ó 2016 Elsevier Ltd. All rights reserved.
cine strains typically exhibit a dry morphology, whereas wildtype strains are usually mucoid [5,1]. This is not, however, a reliable method of differentiation, as both colony types have been cultured from vaccine vials, and a vaccine strain may also revert from dry to mucoid [5]. Polymerase chain reaction assays are beneficial for identifying S. equi, but currently cannot differentiate between wild-type and vaccine strains [6]. Different methods have been described to differentiate wild-type and Pinnacle I.N. vaccine strains of S. equi including BiologTM MicroID System substrate utilization, pulsed-field gel electrophoresis, seM and SzPSe sequencing, and full genome sequencing [2,5]. These have proven to be reliable, but are labor intensive and time consuming for achieving strain differentiation [5]. The current study describes the application of a novel pyrosequencing assay targeting the described single base deletion 85 bp upstream of SzPSe in S. equi [5]. Pyrosequencing has been used in the past to accurately classify Streptococcus to the species level and more recently to identify nucleotide polymorphisms in viruses [7,8]. This study included 66 specimens (61 horses diagnosed with clinical strangles confirmed with isolation of S. equi and 5 healthy controls, that were either vaccinated or had no known history of vaccination) from the University of Illinois Veterinary Diagnostic Laboratory (VDL), and 30 nasal swabs from horses submitted to the Pennsylvania Veterinary Laboratory (PVL), where strangles was not on the differential list. Specimens positive for S. equi
3936
J.L. Livengood et al. / Vaccine 34 (2016) 3935–3937
included clinical nasal flushes, swabs, pus, and cultures of postvaccination abscesses. Control ATCC strains and Pinnacle I.N. (LOT#1391988) were also included for determining test specificity. DNA from the nasal flushes and swabs was extracted at the VDL using a Qiagen spin column extraction method according to manufacturer’s recommendations (QIAGEN, Inc., Valencia, CA). DNA was extracted from the bacterial isolates by a Chelex boiling method at the VDL (Bio-Rad, Hercules, CA). DNA from ATCC strains, Pinnacle I.N., and nasal swabs was extracted at the PVL using the Prepman Ultra Kit following manufacturer’s recommendations (Life Technologies, Carlsbad, CA). All specimens were screened for S. equi using both a real-time and a conventional PCR assay. The real-time assay targeting a portion of the seeI gene was carried out using the QuantiFast Pathogen + IC Kit (QIAGEN, Inc., Valencia, CA, USA), a final concentration of 0.4 lM each of SeeI-F and SeeI-R primers, 0.2 lM SeeI probe and 5 ll of DNA template in a final volume of 25 ll. The specimens were amplified and analyzed on an Applied Biosystems 7500 Fast Real Time PCR System (Life Technologies, Carlsbad, CA, USA) [6]. The conventional PCR assay targeting a 541 bp segment of the SeM protein gene was carried out in a 50 ll reaction with 10X PCR Buffer II, 0.1 unit of Taq Gold (Life Technologies, Carlsbad, CA, USA), a final concentration of 2 mM MgCl2, 0.2 mM dNTP’s, 0.4 lM each of ASW73 and ASW74 primers and 5 lL of DNA template. The specimens were amplified on a GeneAmp 9700 PCR System (Life Technologies, Carlsbad, CA, USA) as described previously [9]. Amplification of the full length SzPSe and SeM protein genes and sequencing of a 500 bp region that includes the targeted single nucleotide polymorphism (SNP) or deletion was carried out as previously described [5,10]. A SNP in the SeM protein gene and resulting change at the 63rd codon from Arginine to Glycine has also been useful in differentiating certain wild-type strains of S. equi from the Pinnacle I.N. strain [10]. For pyrosequencing, PCR targeting a 360 bp region surrounding the single guanine deletion upstream of SzPSe was carried out with Taqman Fast Universal PCR Master mix (Life Technologies, Carlsbad, CA, USA), FS (50 -CCGCAATGACTCAAAACTAATCGA-30 ) and RSbio (biotinylated-50 -TGCCATTTTATTCCCCTTTTGTTT-30 ) primers at a final concentration of 0.2 lM, and 1 lL of DNA template (including 1:10 dilution) in a final volume of 25 lL. Samples were amplified on a Veriti Fast Thermocycler with conditions: 95 °C for 1 min, 45 cycles of 95 °C for 0 s, 62 °C for 15 s (Life Technologies, Carlsbad, CA, USA). Subsequently, the amplified DNA templates were immobilized on streptavidin-coated Sepharose beads using the Vacuum workstation of the PyroMark Q24 according to manufacturer’s recommendations (QIAGEN, Inc., Valencia, CA, USA). DNA templates were released into an annealing mixture containing 0.3 lM of S1 sequencing primer (50 -CGGTTTAATATTGACCT-30 ), which was designed to be complementary to a short region directly upstream of the targeted guanine deletion. The mixture was incubated at 80 °C for 2 min, followed by a 5 min cooling period at room temperature. Specimens were pyrosequenced using Pyromark Gold reagents with an AQ assay and a dispensation order of: GACTGCAT (QIAGEN, Inc., Valencia, CA, USA). Specimens were classified as wild-type or vaccine based upon the presence of the double guanine or single guanine at the targeted location, respectively [5]. The ATCC control S. equi (#33398), Pinnacle I.N., Streptococcus agalactiae (#13813), Streptococcus pneumoniae (#49619), Streptococcus equisimilis (#35666) and Streptococcus zooepidemicus (#43079) were used for pyrosequencing assay verification. Sixty-one specimens that were positive for S. equi by isolation and SeM PCR (including 29 clinical specimens and 32 S. equi isolates) were also positive by the pyrosequencing PCR (Table 1).
Table 1 Comparison of PCR, sequencing, and pyrosequencing for the detection and typing of Streptococcus equi ssp. equi in specimens collected from horses. Specimen (s)
Vaccinationa
SeM & seeI PCR
SeM/SzPSe Seq.b,c
SzPSe Pyrosequencingc
# of clinical specimens (nasal flushes, swabs and abscess) tested 6 Vaccinated + wt wt 10 Vaccinated + vx vx 11 Unknown + wt wt 2 Unknown + vx vx 35 Unknown d ND – # of culture isolates (from abscess and nasal swabs) tested 12 Vaccinated + wt 19 Vaccinated + vx 1 Unknown + vx
wt vx vx
wt = Wild-type strain; vx = vaccine strain; ND = not determined. a Horses received vaccination with PinnacleÒ I.N or had unknown vaccination status. b SeM gene sequencing indicated a substitution of glycine for arginine at the 63rd amino acid residue of the M-like protein for the vaccine strain [9]. c Sequencing upstream of SzPSe indicated the presence of a single guanine at the targeted single guanine deletion location for the vaccine strain [5]. d 1 specimen seeI PCR positive but SeM, SzPSe and pyrosequencing negative.
These results were confirmed by seeI PCR. One specimen reacted with seeI primers, but not with SeM or the pyrosequencing primers. The fact that the seeI gene resides on a mobile genetic element and can be exchanged with other bacteria may explain the discrepancy, but this could not be confirmed further as bacterial isolation was not attempted for this specimen [6,11]. Pyrosequencing of the 29 S. equi positive clinical specimens, which consisted of nasal and abscess swabs collected from both vaccinated and unvaccinated horses, showed 58.6% to contain the GG genotype, leading to a classification as wild-type. The remaining 12 clinical specimens (41.4%) were identified as vaccine strains with a genotype of –G (Table 1). The second subset of specimens was comprised of S. equi isolates cultured from post-vaccination abscesses or nasal swabs from horses vaccinated with Pinnacle I. N. Of the 32 S. equi isolates, 12 isolates (37.5%) had a genotype that correlated with wild-type S. equi, while 20 isolates (62.5%) were identified as the vaccine strain by pyrosequencing. The pyrosequencing results were confirmed with gene sequencing (Table 1). The sensitivity and specificity of the pyrosequencing assay were determined to be 100%. Overall, the vaccine strain was identified in 61.7% (29/47) of the specimens from horses with clinical strangles that had a history of Pinnacle I.N. vaccination (Table 1). The time elapsed since vaccination was not always known, but the vaccine strain was detected up to 5 weeks post-vaccination. A recent vaccination history could explain the higher detection rate for the vaccine strain, although the vaccine itself has also been reported to cause the disease, which would contribute to vaccine strain detection [2]. The predominance of wild-type strains (78.5%) in horses with strangles that had no vaccine history (11/14) was as expected, but in some such cases the vaccine strain was also recovered (Table 1). To better understand shedding of the vaccine strain, vaccination efficacy with the MLV and the disease epidemiology, adding S. equi strain typing for routine diagnosis would be very useful. The pyrosequencing assay described in this study rapidly and accurately distinguishes between wild-type and vaccine types of S. equi. Wild-type S. equi strains consistently displayed the GG genotype in the pyrograms. A sequence of ATTGCAAT was consistently found in vaccine strains, with pyrograms obtained within an hour of the start of the pyrosequencing assay (Fig. 1). Sequencing upstream of SzPSe has previously shown the vaccine progenitor to have a GG genotype at the study location [3]. Thus, the pyrosequencing assay will also identify the progenitor strain as a wildtype strain with a GG genotype, not as a vaccine strain.
3937
J.L. Livengood et al. / Vaccine 34 (2016) 3935–3937
Fig. 1. (A) Pyrogram showing GG genotype (double peak) highlighted in grey for S. equi wild-type strain. (B) Pyrogram showing G genotype (single peak) highlighted in grey for S. equi vaccine strain.
--
l
- - - - l - - -- - l GGTTTAATA
----l----l
- ---l----l
S1 Sequencing Primer
AAC
TTGACCTA
S. equi Wild Strain
AAC
GGTTTAATA
TTGACCTAAT
TGGCAATAAC
S. equi Vaccine Strain
AAC
GGTTTAATA
TTGACCTAAT
T--GCAATAAC
U04620.1 S. zooepidemicus W60
AAC
CGGTTTAATA
TTGATTTAAT
TGGCAATAAC
CP001129 S. zooepidemicus MGCS10565
AAC
CGGTTTAATA
TTGATTTAAT
TGGCAATAAC
CP002904.1 S. zooepidemicus ATCC 35246
AAC
AGGTTTAATA
TTGATTTAAT
TGGCAATAAC
FM204884.1 S.zooepidemicus H70
C
CCCTTTTGTT
TTTATCTA
GACATGATG
CP006770.1 S. zooepidemicus CY
C
CCCTTTTGTT
TTTATCTA
GACATGATG
Fig. 2. Pyrosequencing primer alignment with S. equi (wild-type and vaccine strain) and select isolates of S. zooepidemicus.
It was noteworthy that although S. zooepidemicus showed amplification in the PCR for pyrosequencing due to sequence identity with the FS and RS-bio primers, the sequencing primer chosen in our assay binds to S. equi DNA due to sequence identity (Fig. 2). Generally, a two base mismatch towards the 30 end of a primer can easily prevent non-specific binding and any additional amplification, thus making the pyrosequencing assay more specific [12]. In conclusion, these findings show that pyrosequencing targeting the described single base deletion can be used to classify S. equi strains from nasal flushes, swabs, and bacterial isolates as either vaccine type or wild-type. Pyrosequencing is particularly valuable in this differentiation, as a more rapid diagnosis will allow for the most appropriate management of strangles cases. References [1] Sweeney CR, Timoney JF, Newton JR, Hines MT. Streptococcus equi infections in horses: guidelines for treatment, control, and prevention of strangles. J Vet Intern Med 2005;19:123–34. [2] Cursons R, Patty O, Steward KF, Waller AS. Strangles in horses can be caused by vaccination with Pinnacle IN. Vaccine 2015;33(30):3440–3. [3] Al-Ghamdi GM. Characterization of strangles-episodes in horses experiencing post-vaccinal reaction. J Anim Vet Adv 2012;11:3600–3.
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