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Outbreak of upper respiratory disease in horses caused by Streptococcus equi subsp. zooepidemicus ST-24
Veterinary Microbiology 166 (2013) 281–285
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Outbreak of upper respiratory disease in horses caused by Streptococcus equi subsp. zooepidemicus ST-24 Susanne B. Lindahl a,b,*, Anna Aspa´n a, Viveca Ba˚verud a, Romain Paillot c, John Pringle b, Nicola L. Rash c, Robert So¨derlund a, Andrew S. Waller c a
Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, PO Box 7054, SE-750 07 Uppsala, Sweden c Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, United Kingdom b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 March 2013 Received in revised form 17 May 2013 Accepted 18 May 2013
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is generally considered a commensal and an opportunistic pathogen of the upper airways in horses. Establishing whether certain strains of S. zooepidemicus can cause upper respiratory disease as a hostspecific pathogen of horses, and if there are certain genogroups of S. zooepidemicus that are more virulent than others is of major clinical importance. In this study, we describe an outbreak of upper respiratory disease in horses that was associated with S. zooepidemicus. Upper respiratory samples were cultured, analyzed by real-time PCR for S. zooepidemicus and S. equi, and genetically differentiated by sequencing of the SzP protein gene and multilocus sequence typing (MLST). Serum samples were analyzed for antibodies against S. equi and common viral respiratory pathogens. The ST-24 strain of S. zooepidemicus was isolated from all horses with clinical signs of disease, while the healthy horses carried other strains of S. zooepidemicus. Bacteriological, molecular and serological analyses strongly suggest that a single strain (ST-24) was responsible for the disease outbreak, and that certain strains of this presumed commensal may be more virulent than others. ß 2013 Elsevier B.V. All rights reserved.
Keywords: S. equi subsp. zooepidemicus MLST SzP Respiratory disease Equine
1. Introduction Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is considered an opportunistic pathogen of the upper airways in horses (Anzai et al., 2000; Timoney, 2004; Webb et al., 2008), unlike the closely related biovar S. equi subsp. equi (S. equi), an obligate pathogen that causes the important equine specific respiratory disease strangles (Timoney, 2004). Since S. zooepidemicus is generally regarded an opportunistic pathogen, the importance of this bacterium in disease outbreaks may be neglected. S. zooepidemicus is genetically diverse (Barquero et al., 2010;
* Corresponding author at: Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden. Tel.: +46 18 67 40 00; fax: +46 18 67 40 93. E-mail address: [email protected] (S.B. Lindahl). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.05.006
Chanter et al., 1997; Holden et al., 2009; Webb et al., 2008), and the importance of particular evolutionary events to the pathogenicity of different strains is currently being investigated (Newton et al., 2008; Paillot et al., 2010a; Webb et al., 2008). 2. Materials and methods 2.1. Study population An outbreak of upper respiratory disease occurred in a herd of 17 Icelandic horses in a loose housing system outdoors in Sweden in late September 2009 and lasted approximately six weeks. Clinical signs were: normal to slightly elevated temperature (37.5–38.5 8C), serous to purulent nasal discharge, lymphadenopathy in the head and neck region, cough and depression. The initial clinical assessment was that the signs were consistent with an
S.B. Lindahl et al. / Veterinary Microbiology 166 (2013) 281–285
282
Fig. 1. Timeline for collection of bacteriological samples and blood samples during an outbreak of respiratory disease (Samplings 0 and I), and after the horses had recovered (Samplings II and III). NP = nasopharyngeal. *sampling performed by the practicing clinician. **one horse, not included in the bacteriological sampling group of 12 horses, was not available for blood collection at the second blood sampling occasion.
outbreak of strangles (S. equi) and the farm was quarantined. Two horses, of which one also had a ruptured submandibular abscess, were sampled for bacteriological analysis by a nasal swab and an abscess sample respectively (sampling point 0, Fig. 1). Twelve horses (including the two index cases), of which ten horses displayed clinical signs of respiratory disease and two horses were clinically healthy, were included in the study (Table 1). 2.2. Clinical samples The horses were sampled for bacteriological analysis on three occasions: sampling I during the outbreak was performed according to Lindahl et al. (2013) by using two types of nasal swabs, a nasopharyngeal swab and a nasopharyngeal wash. Samplings II and III (four and eight months after the outbreak) were performed using two nasal ESwabs (Copan, Brescia, Italy) and a nasopharyngeal
wash. At sampling points II and III, none of the horses showed any clinical signs of respiratory disease. Blood samples were collected from all 17 horses in the herd three and seven weeks after the onset of the outbreak (Fig. 1). 2.3. Culture, biochemical fermentation and real-time PCR Swabs and wash samples were cultured on Colistin Oxalinic Acid Blood Agar and b-hemolytic colonies were subcultured on 5% horse blood agar (Lindahl et al., 2013). Isolates from the index cases were recovered from frozen stocks (70 8C) and cultured on 5% horse blood agar. DNA was extracted from cultures and directly from swabs/wash samples as previously described (Lindahl et al., 2013). Real-time PCR was performed, using sodA and seeI as target genes (Baverud et al., 2007), on all samples from sampling I, and on culture of the abscess strain from the index case. For samples collected at samplings II and III, b-hemolytic
Table 1 Twelve horses (#1–12) sampled in an outbreak of upper respiratory disease. The results for bacteriologic analysis (Sez = S. zooepidemicus), sequencing of the szP gene and multi-locus sequence typing (MLST) are given for sampling points I-III. szP sequences are labeled according to their GenBank accession numbers. MLST sequence types (ST) were assigned using http://pubmlst.org/szooepidemicus/. Sampling I was performed during the outbreak. Two horses (#11 and 12) were clinically normal. Samplings II and III were performed after the outbreak had resolved; none of the horses showed any clinical signs of upper airway disease at these sampling points. Horse ID
Age (year)
Sampling I October 16th, 2009
Sampling II February 24th, 2010
Culture
szP
MLST
Culture
Diseased horses 1 1 2 2
Sez Sez
AF519488 (n = 5) AF519488 (n = 3)
ST-24 ST-24
NS NG
3 4 5 6 7 8 9 10
1 1 15 8 9 3 2 3
Sez Sez Sez Sez Sez Sez Sez Sez
AF519488 AF519488 AF519488 AF519488 AF519488 AF519488 AF519488 AF519488
(n = 4) (n = 5) n = 2) (n = 6) n = 1) (n = 5) (n = 4) (n = 4)
ST-24 ST-24 ST-24 ST-24 ST-24 ST-24 ST-24 ST-24
Sez NG NG NG NG NG NG NG
Healthy horses 11 2 12 13
Sez Sez
AF519478 (n = 4) AF519475 (n = 4)
ST-70 ST-39
NG Sez
szP
Sampling III June 16th, 2010 MLST
Culture NG Sez
AF519475 (n = 4)
AF519475 (n = 5)
ST-39
ST-39
NG NG Sez NS NS Sez Sez NS
szP
MLST
AF519488 (n = 1) AF519474 (n = 1)
ST-24 ST-238
AF519478 (n = 2)
ST-43
AF519478 (n = 2) AF519474 (n = 2)
ST-70 ST-238
NS NS
n = number of strains investigated. NS = not sampled, the horse had moved to another stable or was unavailable for sampling due to other reasons. NG = no growth of S. zooepidemicus on bacteriological culture.
S.B. Lindahl et al. / Veterinary Microbiology 166 (2013) 281–285
streptococci were subtyped by biochemical fermentation (Quinn et al., 1994).
283
analyzed using Quansoft software (Techne). Mitogenic activities of S. zooepidemicus culture supernatants were measured as previously described (Paillot et al., 2010a,b).
2.4. Serological analyses Serum was separated from the blood samples and stored at 70 8C until further analysis by iELISA (Animal Health Trust, Newmarket, UK) for the presence of antibodies against S. equi: Ninety-six well Immulon 2HB Flat bottom Microtitre plates were coated overnight at 4 8C with 200 ng of antigen A (Robinson et al., 2013) or antigen B per well. Plates were washed with phosphate buffered saline containing 0.05% Tween 20 (PBST) and blocked with PBST containing 1% non-fat milk (PBSTN) for 1 h at 37 8C. Plates were washed with PBST and 100 mL of test or control serum diluted 1/800 in PBSTN was added to the appropriate well for 1 h at 37 8C. Assays were run in duplicate. Plates were washed with PBST and 100 mL of peroxidaseconjugated goat anti-horse IgG (Kirkegaard and Perry Laboratories) diluted 1/600 in PBSTN was added to each well and the plates incubated for 1 h at 37 8C. Plates were washed with PBST, 100 mL of SureBlue reserve peroxidase substrate 3,30 ,5,50 -tetramethylbenzidine (Kirkegaard and Perry Laboratories) was added and the plate incubated for 10 min at 21 8C. 100 mL of 0.18 M sulphuric acid was added and the optical density at 450 nm (OD450 nm) measured. An OD450 nm of 0.5 was considered to be a positive result for antigen A and an OD450 nm of 1.0 was considered to be a positive result for antigen B iELISAs and for the combined assay a sample was considered to be positive if either assay generated a positive result. Serum samples from the 12 horses included in the bacteriological sampling were also analyzed for the presence of antibodies against equine herpes viruses type 1 and 4 (EHV-1/-4) by complement fixation test, equine arteritis virus (EAV) by serum neutralization test, and equine influenza virus A (equine influenza) by hemagglutination inhibition assay (National Veterinary Institute, Uppsala, Sweden). 2.5. Molecular characterization of S. zooepidemicus isolates Molecular characterization of the isolates was performed by sequencing part of the szP gene (Anzai et al., 2002; Baverud et al., 2007) and by multi-locus sequence typing (MLST) (Webb et al., 2008). Between one and six colonies per horse and sampling occasion were selected for sequencing of the szP gene. MLST was performed on one isolate from each horse, except for horse #2 where two isolates were analyzed (Table 1). MLST and szP sequences were analyzed using BioNumerics 6.5 (Applied Maths, Sint-Martens-Latem, Belgium). Sequence types (STs) were determined using the PubMLST database http://pubmlst.org/szooepidemicus/ (Jolley et al., 2004). Superantigens (sAgs) are believed to be of importance in streptococcal disease (Cole et al., 2011; Commons et al., 2008). Therefore, 20 isolated strains from samplings I-III, including the abscess sample from the index case, were analyzed for presence of S. zooepidemicus superantigens szeF, szeN and szeP, and S. equi superantigens seeH, seeI, seeL and seeM according to Paillot et al. (2010a,b). Data were
3. Results 3.1. Bacteriology All 12 horses sampled during the outbreak (samplings 0 and I) were positive for S. zooepidemicus by real-time PCR and biochemical fermentation. The colonies were mucoid and had a wide zone of b-hemolysis. All isolates fermented sorbitol and lactose, but not trehalose, except for the abscess isolate that only fermented lactose. At sampling point II, two of 11 sampled horses were positive for S. zooepidemicus (sorbitol ve, lactose +ve, trehalose ve), while at sampling point III four of seven sampled horses were positive for S. zooepidemicus (sorbitol +ve, lactose +ve, trehalose ve) (Table 1). 3.2. Multi-locus sequence typing (MLST) and szP sequencing The szP gene was amplified from all isolates of S. zooepidemicus. Isolates from sampling I (outbreak) showed identical szP sequences (GenBank acc. no. AF519488) in all b-hemolytic colonies (n = 39) recovered from the ten horses with signs of clinical disease. The S. zooepidemicus isolates from healthy horses displayed sequences of szP (AF519475 and AF519478) that differed from the sequence of the isolates from diseased horses. The abscess strain (sampling 0) also displayed an AF519475 sequence; however, the isolates from the nasopharyngeal sampling of this horse (#2) at sampling point I displayed an AF519488 sequence (Table 1). MLST analysis identified a single sequence type (ST-24) in all isolates from diseased horses. The isolates recovered from the two healthy horses were identified as ST-70 and ST-39 (Table 1). The abscess isolate was also identified as ST-39. At sampling points II and III (recovered/healthy horses), only two and four horses respectively were positive for S. zooepidemicus. All, except horse #2, displayed STs and szP types not found in isolates from diseased horses during the outbreak. Horse # 2 was the sole animal in this study to have two different strains of S. zooepidemicus (sampling III) (Table 1). MLST STs and szP-types generally corresponded, except one in horse, #5, at sampling III (Table 1). 3.3. Superantigens and mitogenic activity S. zooepidemicus sAgs szeF, szeN and szeP were not present in the ST-24 isolates, nor were the S. equi sAgs seeH, seeI, seeL and seeM. The ST-39 isolate from the lymph node abscess did not possess any sAgs or mitogenic activity in vitro. Mitogenic activity was detected in ST-70 and ST-238 strains, which agreed with the presence of sAgs szeN and szeP in these isolates. 3.4. Serology One horse (#4) showed serologic evidence of previous exposure to S. equi; this horse was re-sampled after two
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months and tested negative on the iELISA. The remaining horses tested negative for antibodies against S. equi on paired samples. None of the 12 horses in the sampling group were positive for antibodies against EHV-1/-4 or EAV. Ten of the 12 horses were positive for antibodies against equine influenza. However, there was no evidence of seroconversion and nine of the ten positive horses had been vaccinated against equine influenza the previous year. The remaining seropositive horse (#1) was unvaccinated, but had a very low antibody titer (1:8 on both samples). 4. Discussion In this outbreak a single clone (ST-24) of S. zooepidemicus, as determined by sequencing of the szP gene and MLST, appeared to have selective pathogenic potential. There are 13 isolates with ST-24 previously described in the PubMLST database. Eleven of these isolates, as well as all four isolates in the database that are single locus variants of ST-24 (ST-79, ST-84, ST-161) were recovered from the respiratory tract of horses, suggesting that the ST24 group of S. zooepidemicus may be more adapted to infect this niche. An ST-24 strain was also isolated from horse #2 eight months after the outbreak. This suggests that either the ST-24 strain may persist in the respiratory tract of convalescent horses facilitating transmission to naı¨ve animals, or a separate incursion of an ST-24 strain had occurred. No superantigen genes or mitogenic activity were detected in any of the ST-24 isolates. This result is supported by a recent prevalence study that highlighted the absence of association between superantigen genes and upper-respiratory infection (N. Rash, personal communication). Although our evidence points to the pathogenic potential of this strain of S. zooepidemicus, more studies are needed to determine what mechanisms influence the pathogenicity of this strain. The discrepancy between the lymph node abscess isolate and the ST of the nasal samples from cases of respiratory disease highlights that these two conditions were not linked and were due to different strains of S. zooepidemicus. Usually isolates of S. zooepidemicus recovered from non-strangles lymph node abscesses contain coding sequences for superantigens (p = 0.0006) or have mitogenic activity in vitro (p = 0.003) (Paillot et al., 2010a) and so the abscess isolate (ST-39) is unusual, but not unique, in that it did not. As the majority of the ill horses displayed moderate clinical signs similar to strangles and one horse had a ruptured abscess, the clinical diagnosis of strangles could not be ruled out at the time of the outbreak. Significantly, involvement of S. equi in the clinical course of these horses can be fully ruled out on the basis of the uniformly negative bacteriological results from multiple samplings of each horse, and all but one horse (which had a declining titer) showed seronegativity to S. equi. Furthermore, the seronegativity to EAV, EHV-1 and EHV-4, along with the vaccinal associated seropositivity to influenza virus, strongly suggests that the S. zooepidemicus strain (ST-24/ szP type AF519488), identified solely in horses with clinical signs, was the primary pathogen.
The study was approved by the Swedish Ethical Committee on Animal Experiments (C199/8). Acknowledgements This work was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS). The authors thank Helena Ljung (National Veterinary Institute, Uppsala, Sweden) for excellent support during sampling of the horses and in the laboratory. Olga Stephansson (National Veterinary Institute) is acknowledged for performing sequencing of the szP gene. Sofia Lindstro¨m (National Veterinary Institute) is acknowledged for performing MLST. Thomas Manske, DVM, PhD, is acknowledged for collecting the blood samples. Dr. Keith Jolley (Department of Zoology, University of Oxford) is acknowledged for maintaining the MLST database. The authors also express their gratitude to the horse owner for cooperation in examination and sampling. References Anzai, T., Timoney, J.E., Kuwamoto, Y., Wada, R., Oikawa, M., Higuchi, T., 2002. Polymerase chain reaction-restriction fragment length polymorphism analysis of the SzP gene of Streptococcus zooepidemicus isolated from the respiratory tract of horses. Am. J. Vet. Res. 63, 1298– 1301. Anzai, T., Walker, J.A., Blair, M.B., Chambers, T.M., Timoney, J.F., 2000. Comparison of the phenotypes of Streptococcus zooepidemicus isolated from tonsils of healthy horses and specimens obtained from foals and donkeys with pneumonia. Am. J. Vet. Res. 61, 162–166. Barquero, N., Chanter, N., Laxton, R., Wood, J.L., Newton, J.R., 2010. Molecular epidemiology of Streptococcus zooepidemicus isolated from the respiratory tracts of Thoroughbred racehorses in training. Vet. J. 183, 348–351. Baverud, V., Johansson, S.K., Aspan, A., 2007. Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Vet. Microbiol. 124, 219–229. Chanter, N., Collin, N., Holmes, N., Binns, M., Mumford, J., 1997. Characterization of the Lancefield group C streptococcus 16S-23S RNA gene intergenic spacer and its potential for identification and subspecific typing. Epidemiol. Infect. 118, 125–135. Cole, J.N., Barnett, T.C., Nizet, V., Walker, M.J., 2011. Molecular insight into invasive group A streptococcal disease. Nat. Rev. Microbiol. 9, 724–736. Commons, R., Rogers, S., Gooding, T., Danchin, M., Carapetis, J., RobinsBrowne, R., Curtis, N., 2008. Superantigen genes in group A streptococcal isolates and their relationship with emm types. J. Med. Microbiol. 57, 1238–1246. Holden, M.T., Heather, Z., Paillot, R., Steward, K.F., Webb, K., Ainslie, F., Jourdan, T., Bason, N.C., Holroyd, N.E., Mungall, K., Quail, M.A., Sanders, M., Simmonds, M., Willey, D., Brooks, K., Aanensen, D.M., Spratt, B.G., Jolley, K.A., Maiden, M.C., Kehoe, M., Chanter, N., Bentley, S.D., Robinson, C., Maskell, D.J., Parkhill, J., Waller, A.S., 2009. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathog. 5, e1000346. Jolley, K.A., Chan, M.S., Maiden, M.C., 2004. mlstdbNet - distributed multilocus sequence typing (MLST) databases. BMC Bioinformatics 5, 86. Lindahl, S., Baverud, V., Egenvall, A., Aspan, A., Pringle, J., 2013. Comparison of sampling sites and laboratory diagnostic tests for S. equi subsp. equi in horses from confirmed strangles outbreaks. J. Vet. Intern. Med. 27, 542–547. Newton, J.R., Laxton, R., Wood, J.L., Chanter, N., 2008. Molecular epidemiology of Streptococcus zooepidemicus infection in naturally occurring equine respiratory disease. Vet. J. 175, 338–345. Paillot, R., Darby, A.C., Robinson, C., Wright, N.L., Steward, K.F., Anderson, E., Webb, K., Holden, M.T., Efstratiou, A., Broughton, K., Jolley, K.A., Priestnall, S.L., Marotti Campi, M.C., Hughes, M.A., Radford, A., Erles, K., Waller, A.S., 2010a. Identification of three novel superantigenencoding genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP. Infect. Immun. 78, 4817–4827.
S.B. Lindahl et al. / Veterinary Microbiology 166 (2013) 281–285 Paillot, R., Robinson, C., Steward, K., Wright, N., Jourdan, T., Butcher, N., Heather, Z., Waller, A.S., 2010b. Contribution of each of four Superantigens to Streptococcus equi-induced mitogenicity, gamma interferon synthesis, and immunity. Infect. Immun. 78, 1728–1739. Quinn, P.J., Carter, M.E., Markey, B., Carter, G.R., 1994. The Streptococci and related cocci. In: Clinical Veterinary Microbiology. Wolfe Publishing, Mosby Year Book Europe Ltd, London, pp. 127–136. Robinson, C., Steward, K.F., Potts, N., Barker, C., Hammond, T.A., Pierce, K., Gunnarsson, E., Svansson, V., Slater, J., Newton, J.R., Waller, A.S., 2013.
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Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure. Vet. J., http://dx.doi.org/10.1016/j.tvjl.2013.01.033. Timoney, J.F., 2004. The pathogenic equine streptococci. Vet. Res. 35, 397– 409. Webb, K., Jolley, K.A., Mitchell, Z., Robinson, C., Newton, J.R., Maiden, M.C., Waller, A., 2008. Development of an unambiguous and discriminatory multilocus sequence typing scheme for the Streptococcus zooepidemicus group. Microbiology 154, 3016–3024.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Outbreak of upper respiratory disease in horses caused by Streptococcus equi subsp. zooepidemicus ST-24 Susanne B. Lindahl a,b,*, Anna Aspa´n a, Viveca Ba˚verud a, Romain Paillot c, John Pringle b, Nicola L. Rash c, Robert So¨derlund a, Andrew S. Waller c a
Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, PO Box 7054, SE-750 07 Uppsala, Sweden c Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, United Kingdom b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 March 2013 Received in revised form 17 May 2013 Accepted 18 May 2013
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is generally considered a commensal and an opportunistic pathogen of the upper airways in horses. Establishing whether certain strains of S. zooepidemicus can cause upper respiratory disease as a hostspecific pathogen of horses, and if there are certain genogroups of S. zooepidemicus that are more virulent than others is of major clinical importance. In this study, we describe an outbreak of upper respiratory disease in horses that was associated with S. zooepidemicus. Upper respiratory samples were cultured, analyzed by real-time PCR for S. zooepidemicus and S. equi, and genetically differentiated by sequencing of the SzP protein gene and multilocus sequence typing (MLST). Serum samples were analyzed for antibodies against S. equi and common viral respiratory pathogens. The ST-24 strain of S. zooepidemicus was isolated from all horses with clinical signs of disease, while the healthy horses carried other strains of S. zooepidemicus. Bacteriological, molecular and serological analyses strongly suggest that a single strain (ST-24) was responsible for the disease outbreak, and that certain strains of this presumed commensal may be more virulent than others. ß 2013 Elsevier B.V. All rights reserved.
Keywords: S. equi subsp. zooepidemicus MLST SzP Respiratory disease Equine
1. Introduction Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is considered an opportunistic pathogen of the upper airways in horses (Anzai et al., 2000; Timoney, 2004; Webb et al., 2008), unlike the closely related biovar S. equi subsp. equi (S. equi), an obligate pathogen that causes the important equine specific respiratory disease strangles (Timoney, 2004). Since S. zooepidemicus is generally regarded an opportunistic pathogen, the importance of this bacterium in disease outbreaks may be neglected. S. zooepidemicus is genetically diverse (Barquero et al., 2010;
* Corresponding author at: Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden. Tel.: +46 18 67 40 00; fax: +46 18 67 40 93. E-mail address: [email protected] (S.B. Lindahl). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.05.006
Chanter et al., 1997; Holden et al., 2009; Webb et al., 2008), and the importance of particular evolutionary events to the pathogenicity of different strains is currently being investigated (Newton et al., 2008; Paillot et al., 2010a; Webb et al., 2008). 2. Materials and methods 2.1. Study population An outbreak of upper respiratory disease occurred in a herd of 17 Icelandic horses in a loose housing system outdoors in Sweden in late September 2009 and lasted approximately six weeks. Clinical signs were: normal to slightly elevated temperature (37.5–38.5 8C), serous to purulent nasal discharge, lymphadenopathy in the head and neck region, cough and depression. The initial clinical assessment was that the signs were consistent with an
S.B. Lindahl et al. / Veterinary Microbiology 166 (2013) 281–285
282
Fig. 1. Timeline for collection of bacteriological samples and blood samples during an outbreak of respiratory disease (Samplings 0 and I), and after the horses had recovered (Samplings II and III). NP = nasopharyngeal. *sampling performed by the practicing clinician. **one horse, not included in the bacteriological sampling group of 12 horses, was not available for blood collection at the second blood sampling occasion.
outbreak of strangles (S. equi) and the farm was quarantined. Two horses, of which one also had a ruptured submandibular abscess, were sampled for bacteriological analysis by a nasal swab and an abscess sample respectively (sampling point 0, Fig. 1). Twelve horses (including the two index cases), of which ten horses displayed clinical signs of respiratory disease and two horses were clinically healthy, were included in the study (Table 1). 2.2. Clinical samples The horses were sampled for bacteriological analysis on three occasions: sampling I during the outbreak was performed according to Lindahl et al. (2013) by using two types of nasal swabs, a nasopharyngeal swab and a nasopharyngeal wash. Samplings II and III (four and eight months after the outbreak) were performed using two nasal ESwabs (Copan, Brescia, Italy) and a nasopharyngeal
wash. At sampling points II and III, none of the horses showed any clinical signs of respiratory disease. Blood samples were collected from all 17 horses in the herd three and seven weeks after the onset of the outbreak (Fig. 1). 2.3. Culture, biochemical fermentation and real-time PCR Swabs and wash samples were cultured on Colistin Oxalinic Acid Blood Agar and b-hemolytic colonies were subcultured on 5% horse blood agar (Lindahl et al., 2013). Isolates from the index cases were recovered from frozen stocks (70 8C) and cultured on 5% horse blood agar. DNA was extracted from cultures and directly from swabs/wash samples as previously described (Lindahl et al., 2013). Real-time PCR was performed, using sodA and seeI as target genes (Baverud et al., 2007), on all samples from sampling I, and on culture of the abscess strain from the index case. For samples collected at samplings II and III, b-hemolytic
Table 1 Twelve horses (#1–12) sampled in an outbreak of upper respiratory disease. The results for bacteriologic analysis (Sez = S. zooepidemicus), sequencing of the szP gene and multi-locus sequence typing (MLST) are given for sampling points I-III. szP sequences are labeled according to their GenBank accession numbers. MLST sequence types (ST) were assigned using http://pubmlst.org/szooepidemicus/. Sampling I was performed during the outbreak. Two horses (#11 and 12) were clinically normal. Samplings II and III were performed after the outbreak had resolved; none of the horses showed any clinical signs of upper airway disease at these sampling points. Horse ID
Age (year)
Sampling I October 16th, 2009
Sampling II February 24th, 2010
Culture
szP
MLST
Culture
Diseased horses 1 1 2 2
Sez Sez
AF519488 (n = 5) AF519488 (n = 3)
ST-24 ST-24
NS NG
3 4 5 6 7 8 9 10
1 1 15 8 9 3 2 3
Sez Sez Sez Sez Sez Sez Sez Sez
AF519488 AF519488 AF519488 AF519488 AF519488 AF519488 AF519488 AF519488
(n = 4) (n = 5) n = 2) (n = 6) n = 1) (n = 5) (n = 4) (n = 4)
ST-24 ST-24 ST-24 ST-24 ST-24 ST-24 ST-24 ST-24
Sez NG NG NG NG NG NG NG
Healthy horses 11 2 12 13
Sez Sez
AF519478 (n = 4) AF519475 (n = 4)
ST-70 ST-39
NG Sez
szP
Sampling III June 16th, 2010 MLST
Culture NG Sez
AF519475 (n = 4)
AF519475 (n = 5)
ST-39
ST-39
NG NG Sez NS NS Sez Sez NS
szP
MLST
AF519488 (n = 1) AF519474 (n = 1)
ST-24 ST-238
AF519478 (n = 2)
ST-43
AF519478 (n = 2) AF519474 (n = 2)
ST-70 ST-238
NS NS
n = number of strains investigated. NS = not sampled, the horse had moved to another stable or was unavailable for sampling due to other reasons. NG = no growth of S. zooepidemicus on bacteriological culture.
S.B. Lindahl et al. / Veterinary Microbiology 166 (2013) 281–285
streptococci were subtyped by biochemical fermentation (Quinn et al., 1994).
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analyzed using Quansoft software (Techne). Mitogenic activities of S. zooepidemicus culture supernatants were measured as previously described (Paillot et al., 2010a,b).
2.4. Serological analyses Serum was separated from the blood samples and stored at 70 8C until further analysis by iELISA (Animal Health Trust, Newmarket, UK) for the presence of antibodies against S. equi: Ninety-six well Immulon 2HB Flat bottom Microtitre plates were coated overnight at 4 8C with 200 ng of antigen A (Robinson et al., 2013) or antigen B per well. Plates were washed with phosphate buffered saline containing 0.05% Tween 20 (PBST) and blocked with PBST containing 1% non-fat milk (PBSTN) for 1 h at 37 8C. Plates were washed with PBST and 100 mL of test or control serum diluted 1/800 in PBSTN was added to the appropriate well for 1 h at 37 8C. Assays were run in duplicate. Plates were washed with PBST and 100 mL of peroxidaseconjugated goat anti-horse IgG (Kirkegaard and Perry Laboratories) diluted 1/600 in PBSTN was added to each well and the plates incubated for 1 h at 37 8C. Plates were washed with PBST, 100 mL of SureBlue reserve peroxidase substrate 3,30 ,5,50 -tetramethylbenzidine (Kirkegaard and Perry Laboratories) was added and the plate incubated for 10 min at 21 8C. 100 mL of 0.18 M sulphuric acid was added and the optical density at 450 nm (OD450 nm) measured. An OD450 nm of 0.5 was considered to be a positive result for antigen A and an OD450 nm of 1.0 was considered to be a positive result for antigen B iELISAs and for the combined assay a sample was considered to be positive if either assay generated a positive result. Serum samples from the 12 horses included in the bacteriological sampling were also analyzed for the presence of antibodies against equine herpes viruses type 1 and 4 (EHV-1/-4) by complement fixation test, equine arteritis virus (EAV) by serum neutralization test, and equine influenza virus A (equine influenza) by hemagglutination inhibition assay (National Veterinary Institute, Uppsala, Sweden). 2.5. Molecular characterization of S. zooepidemicus isolates Molecular characterization of the isolates was performed by sequencing part of the szP gene (Anzai et al., 2002; Baverud et al., 2007) and by multi-locus sequence typing (MLST) (Webb et al., 2008). Between one and six colonies per horse and sampling occasion were selected for sequencing of the szP gene. MLST was performed on one isolate from each horse, except for horse #2 where two isolates were analyzed (Table 1). MLST and szP sequences were analyzed using BioNumerics 6.5 (Applied Maths, Sint-Martens-Latem, Belgium). Sequence types (STs) were determined using the PubMLST database http://pubmlst.org/szooepidemicus/ (Jolley et al., 2004). Superantigens (sAgs) are believed to be of importance in streptococcal disease (Cole et al., 2011; Commons et al., 2008). Therefore, 20 isolated strains from samplings I-III, including the abscess sample from the index case, were analyzed for presence of S. zooepidemicus superantigens szeF, szeN and szeP, and S. equi superantigens seeH, seeI, seeL and seeM according to Paillot et al. (2010a,b). Data were
3. Results 3.1. Bacteriology All 12 horses sampled during the outbreak (samplings 0 and I) were positive for S. zooepidemicus by real-time PCR and biochemical fermentation. The colonies were mucoid and had a wide zone of b-hemolysis. All isolates fermented sorbitol and lactose, but not trehalose, except for the abscess isolate that only fermented lactose. At sampling point II, two of 11 sampled horses were positive for S. zooepidemicus (sorbitol ve, lactose +ve, trehalose ve), while at sampling point III four of seven sampled horses were positive for S. zooepidemicus (sorbitol +ve, lactose +ve, trehalose ve) (Table 1). 3.2. Multi-locus sequence typing (MLST) and szP sequencing The szP gene was amplified from all isolates of S. zooepidemicus. Isolates from sampling I (outbreak) showed identical szP sequences (GenBank acc. no. AF519488) in all b-hemolytic colonies (n = 39) recovered from the ten horses with signs of clinical disease. The S. zooepidemicus isolates from healthy horses displayed sequences of szP (AF519475 and AF519478) that differed from the sequence of the isolates from diseased horses. The abscess strain (sampling 0) also displayed an AF519475 sequence; however, the isolates from the nasopharyngeal sampling of this horse (#2) at sampling point I displayed an AF519488 sequence (Table 1). MLST analysis identified a single sequence type (ST-24) in all isolates from diseased horses. The isolates recovered from the two healthy horses were identified as ST-70 and ST-39 (Table 1). The abscess isolate was also identified as ST-39. At sampling points II and III (recovered/healthy horses), only two and four horses respectively were positive for S. zooepidemicus. All, except horse #2, displayed STs and szP types not found in isolates from diseased horses during the outbreak. Horse # 2 was the sole animal in this study to have two different strains of S. zooepidemicus (sampling III) (Table 1). MLST STs and szP-types generally corresponded, except one in horse, #5, at sampling III (Table 1). 3.3. Superantigens and mitogenic activity S. zooepidemicus sAgs szeF, szeN and szeP were not present in the ST-24 isolates, nor were the S. equi sAgs seeH, seeI, seeL and seeM. The ST-39 isolate from the lymph node abscess did not possess any sAgs or mitogenic activity in vitro. Mitogenic activity was detected in ST-70 and ST-238 strains, which agreed with the presence of sAgs szeN and szeP in these isolates. 3.4. Serology One horse (#4) showed serologic evidence of previous exposure to S. equi; this horse was re-sampled after two
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months and tested negative on the iELISA. The remaining horses tested negative for antibodies against S. equi on paired samples. None of the 12 horses in the sampling group were positive for antibodies against EHV-1/-4 or EAV. Ten of the 12 horses were positive for antibodies against equine influenza. However, there was no evidence of seroconversion and nine of the ten positive horses had been vaccinated against equine influenza the previous year. The remaining seropositive horse (#1) was unvaccinated, but had a very low antibody titer (1:8 on both samples). 4. Discussion In this outbreak a single clone (ST-24) of S. zooepidemicus, as determined by sequencing of the szP gene and MLST, appeared to have selective pathogenic potential. There are 13 isolates with ST-24 previously described in the PubMLST database. Eleven of these isolates, as well as all four isolates in the database that are single locus variants of ST-24 (ST-79, ST-84, ST-161) were recovered from the respiratory tract of horses, suggesting that the ST24 group of S. zooepidemicus may be more adapted to infect this niche. An ST-24 strain was also isolated from horse #2 eight months after the outbreak. This suggests that either the ST-24 strain may persist in the respiratory tract of convalescent horses facilitating transmission to naı¨ve animals, or a separate incursion of an ST-24 strain had occurred. No superantigen genes or mitogenic activity were detected in any of the ST-24 isolates. This result is supported by a recent prevalence study that highlighted the absence of association between superantigen genes and upper-respiratory infection (N. Rash, personal communication). Although our evidence points to the pathogenic potential of this strain of S. zooepidemicus, more studies are needed to determine what mechanisms influence the pathogenicity of this strain. The discrepancy between the lymph node abscess isolate and the ST of the nasal samples from cases of respiratory disease highlights that these two conditions were not linked and were due to different strains of S. zooepidemicus. Usually isolates of S. zooepidemicus recovered from non-strangles lymph node abscesses contain coding sequences for superantigens (p = 0.0006) or have mitogenic activity in vitro (p = 0.003) (Paillot et al., 2010a) and so the abscess isolate (ST-39) is unusual, but not unique, in that it did not. As the majority of the ill horses displayed moderate clinical signs similar to strangles and one horse had a ruptured abscess, the clinical diagnosis of strangles could not be ruled out at the time of the outbreak. Significantly, involvement of S. equi in the clinical course of these horses can be fully ruled out on the basis of the uniformly negative bacteriological results from multiple samplings of each horse, and all but one horse (which had a declining titer) showed seronegativity to S. equi. Furthermore, the seronegativity to EAV, EHV-1 and EHV-4, along with the vaccinal associated seropositivity to influenza virus, strongly suggests that the S. zooepidemicus strain (ST-24/ szP type AF519488), identified solely in horses with clinical signs, was the primary pathogen.
The study was approved by the Swedish Ethical Committee on Animal Experiments (C199/8). Acknowledgements This work was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS). The authors thank Helena Ljung (National Veterinary Institute, Uppsala, Sweden) for excellent support during sampling of the horses and in the laboratory. Olga Stephansson (National Veterinary Institute) is acknowledged for performing sequencing of the szP gene. Sofia Lindstro¨m (National Veterinary Institute) is acknowledged for performing MLST. Thomas Manske, DVM, PhD, is acknowledged for collecting the blood samples. Dr. Keith Jolley (Department of Zoology, University of Oxford) is acknowledged for maintaining the MLST database. The authors also express their gratitude to the horse owner for cooperation in examination and sampling. References Anzai, T., Timoney, J.E., Kuwamoto, Y., Wada, R., Oikawa, M., Higuchi, T., 2002. Polymerase chain reaction-restriction fragment length polymorphism analysis of the SzP gene of Streptococcus zooepidemicus isolated from the respiratory tract of horses. Am. J. Vet. Res. 63, 1298– 1301. Anzai, T., Walker, J.A., Blair, M.B., Chambers, T.M., Timoney, J.F., 2000. Comparison of the phenotypes of Streptococcus zooepidemicus isolated from tonsils of healthy horses and specimens obtained from foals and donkeys with pneumonia. Am. J. Vet. Res. 61, 162–166. Barquero, N., Chanter, N., Laxton, R., Wood, J.L., Newton, J.R., 2010. Molecular epidemiology of Streptococcus zooepidemicus isolated from the respiratory tracts of Thoroughbred racehorses in training. Vet. J. 183, 348–351. Baverud, V., Johansson, S.K., Aspan, A., 2007. Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Vet. Microbiol. 124, 219–229. Chanter, N., Collin, N., Holmes, N., Binns, M., Mumford, J., 1997. Characterization of the Lancefield group C streptococcus 16S-23S RNA gene intergenic spacer and its potential for identification and subspecific typing. Epidemiol. Infect. 118, 125–135. Cole, J.N., Barnett, T.C., Nizet, V., Walker, M.J., 2011. Molecular insight into invasive group A streptococcal disease. Nat. Rev. Microbiol. 9, 724–736. Commons, R., Rogers, S., Gooding, T., Danchin, M., Carapetis, J., RobinsBrowne, R., Curtis, N., 2008. Superantigen genes in group A streptococcal isolates and their relationship with emm types. J. Med. Microbiol. 57, 1238–1246. Holden, M.T., Heather, Z., Paillot, R., Steward, K.F., Webb, K., Ainslie, F., Jourdan, T., Bason, N.C., Holroyd, N.E., Mungall, K., Quail, M.A., Sanders, M., Simmonds, M., Willey, D., Brooks, K., Aanensen, D.M., Spratt, B.G., Jolley, K.A., Maiden, M.C., Kehoe, M., Chanter, N., Bentley, S.D., Robinson, C., Maskell, D.J., Parkhill, J., Waller, A.S., 2009. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathog. 5, e1000346. Jolley, K.A., Chan, M.S., Maiden, M.C., 2004. mlstdbNet - distributed multilocus sequence typing (MLST) databases. BMC Bioinformatics 5, 86. Lindahl, S., Baverud, V., Egenvall, A., Aspan, A., Pringle, J., 2013. Comparison of sampling sites and laboratory diagnostic tests for S. equi subsp. equi in horses from confirmed strangles outbreaks. J. Vet. Intern. Med. 27, 542–547. Newton, J.R., Laxton, R., Wood, J.L., Chanter, N., 2008. Molecular epidemiology of Streptococcus zooepidemicus infection in naturally occurring equine respiratory disease. Vet. J. 175, 338–345. Paillot, R., Darby, A.C., Robinson, C., Wright, N.L., Steward, K.F., Anderson, E., Webb, K., Holden, M.T., Efstratiou, A., Broughton, K., Jolley, K.A., Priestnall, S.L., Marotti Campi, M.C., Hughes, M.A., Radford, A., Erles, K., Waller, A.S., 2010a. Identification of three novel superantigenencoding genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP. Infect. Immun. 78, 4817–4827.
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