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New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
Prevalence of Streptococcus dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in a sample of healthy dogs, cats and horses. a
b
a
a
bd
ae
c
E Acke , AC Midwinter , K Lawrence , SJG Gordon , S Moore , I Rasiah , K Steward , N b
c
French & A Waller a
Animal and Biomedical Sciences, Massey University Veterinary Teaching Hospital, Institute of Veterinary, Massey University, Palmerston North, New Zealand b
Animal and Biomedical Sciences, Infectious Disease Research Centre, Institute of Veterinary, Massey University, Palmerston North, New Zealand c
Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, United Kingdom
Click for updates
d
Current address: William Stephen Road, RD1, Te Anau, New Zealand
e
Current address: Asteron Centre, 55 Featherston Street, Wellington, New Zealand Accepted author version posted online: 19 Feb 2015.
To cite this article: E Acke, AC Midwinter, K Lawrence, SJG Gordon, S Moore, I Rasiah, K Steward, N French & A Waller (2015): Prevalence of Streptococcus dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in a sample of healthy dogs, cats and horses., New Zealand Veterinary Journal, DOI: 10.1080/00480169.2015.1016133 To link to this article: http://dx.doi.org/10.1080/00480169.2015.1016133
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & New Zealand Veterinary Association Journal: New Zealand Veterinary Journal DOI: 10.1080/00480169.2015.1016133
Scientific Article
Prevalence of Streptococcus dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in a sample of healthy dogs, cats and horses.
rip
t
E Acke*§, AC Midwinter†, K Lawrence*, SJG Gordon*, S Moore†#, I Rasiah*¥, K Steward‡, N French† and A Waller‡
M an
us c
Massey University Veterinary Teaching Hospital, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand † Infectious Disease Research Centre, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand ‡ Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, United Kingdom # Current address: William Stephen Road, RD1, Te Anau, New Zealand ¥ Current address: Asteron Centre, 55 Featherston Street, Wellington, New Zealand § Author for correspondence. Email: [email protected]
pt e
d
Abstract
AIMS: To estimate the prevalence of β-haemolytic Lancefield group C streptococci in healthy dogs, cats and horses; to determine if frequent contact with horses was associated with isolation of these
ce
species from dogs and cats; and to characterise recovered S. equi subsp. zooepidemicus isolates by multilocus sequence typing (MLST). METHODS: Oropharyngeal swabs were collected from 197 dogs and 72 cats, and nasopharyngeal
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
*
swabs from 93 horses. Sampling was carried out at the Massey University Veterinary Teaching Hospital, on sheep and beef farms or on premises where horses were present. All animals were
healthy and were categorised as Urban dogs and cats (minimal contact with horses or farm livestock), Farm dogs (minimal contact with horses) and Stable dogs and cats (frequent contact with horses). Swabs were cultured for β-haemolytic Streptococcus spp. and Lancefield group C streptococcal subspecies were confirmed by phenotypic and molecular techniques. RESULTS: Of the 197 dogs sampled, 21 (10.7 (95% CI= 4.0–25.4)%) tested positive for S. dysgalactiae subsp. equisimilis and 4 (2.0 (95% CI=0.7–5.5)%) tested positive for S. equi subsp.
zooepidemicus. All these isolates, except for one S. dysgalactiae subsp. equisimilis isolate in an Urban dog, were from Stable dogs. S. dysgalactiae subsp. equisimilis was isolated from one Stable cat. Of the 93 horses, 22 (23.7 (95% CI=12.3–40.6)%) and 6 (6.5 (95% CI=2.8–14.1)%) had confirmed S. dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus isolation respectively. Isolation of S. dysgalactiae subsp. equisimilis from dogs was statistically associated with frequent contact with horses (OR=9.8 (95% CI=2.6–72.8)). Three different MLST profiles of S. equi subsp. zooepidemicus that have not been previously reported were recovered from healthy dogs.
rip
t
CONCLUSIONS AND CLINICAL RELEVANCE: Subclinical infection or colonisation by S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis occurs in dogs and further research on
us c
needed. Complete speciation of β-haemolytic streptococci should be recommended in clinical cases and the possible exposure to horses and their environment should be considered in epidemiological
M an
investigations.
KEY WORDS: Streptococcus dysgalactiae subsp. equisimilis, S. equi subsp. zooepidemicus,
d
prevalence, horse, dog, epidemiology
Generalised estimated equations
MLST
Multilocus sequence typing
MUVTH
Massey University Veterinary Teaching Hospital
ce
RTD
pt e
GEE
Respiratory tract disease
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
inter-species transmission and the pathogenic potential of these Lancefield group C streptococci is
ST
Sequence type
Introduction Streptococcus equi subsp. equi, S. equi subsp. zooepidemicus, and S. dysgalactiae subsp. equisimilis are β-haemolytic Lancefield group C streptococci that can cause disease in horses but can also occur subclinically (Timoney 2004; Erol et al. 2012; North et al. 2014). S. equi subsp. zooepidemicus is a commonly isolated opportunistic pathogen from the equine skin, respiratory tract, and urogenital tract (Lavoie et al. 1991; Timoney 2004; Newton et al. 2008). The organism is
a multi-host pathogen that has been associated with infections in several animal species (Webb et al. 2008) and its significance appears to be increasing world-wide. Most concerning in companion animal medicine are the increasing number of reports of haemorrhagic pneumonia and more severe presentations of the canine infectious respiratory disease complex associated with S. equi subsp. zooepidemicus infections in intensively housed dogs in several geographical locations including the United States of America (USA; Garnett et al. 1982; Pesavento et al. 2008), Europe (Chalker et al. 2003), and Asia (Kim et al. 2007; Byun et al. 2009). The epidemiology and pathogenesis of S. equi subsp. zooepidemicus infections in dogs and cats remains poorly understood and the source of
t
infection unknown (Priestnall and Erles 2011). S. equi subsp. zooepidemicus is potentially zoonotic
rip
with sporadic reports of meningitis and other infections in humans attributed to close contact with horses (Jovanovic et al. 2008; Brouwer et al. 2010; Eyre et al. 2010). Dog-to-human infection was
us c
al. 2010). S. dysgalactiae subsp. equisimilis has become an important pathogen in horses where it may cause a range of opportunistic infections (Erol et al. 2012), and also in humans (Brandt and
M an
Spellerberg 2009). Comprehensive studies on β-haemolytic streptococci are limited even in horses; S. dysgalactiae subsp. equisimilis is suspected to have a much more important role than previously thought and has been isolated from a broad range of equine tissues, especially from the reproductive tract (Erol et al. 2012). The importance of this organism in dogs and cats remains very poorly documented in the veterinary literature. In addition to this, β-haemolytic Lancefield group C
d
streptococci are a grouping in a state of taxonomic flux (Vandamme et al. 1996; Preziuso et al.
pt e
2014) which complicates interpretation of bacteriology results previously reported in the veterinary literature. S. equi subsp. equi, the causative agent of equine strangles, also a member of βhaemolytic Lancefield group C and very closely related to S. equi subsp. zooepidemicus, is rarely
ce
reported in non-equine hosts (Holden et al. 2009) with one case of S. equi subsp. equi infection in a dog with severe regional lymphadenopathy reported (Ladlow et al. 2006). Infection of humans has also been reported (Elsayed et al. 2003; Popescu et al. 2006).
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
confirmed in one case where a dog residing on a horse farm infected a veterinary nurse (Abbott et
The range of subspecies recovered from healthy cats and dogs and the role of horses in the epidemiology of Lancefield group C streptococci has not been investigated previously. Very few studies have reported complete taxonomic identification of β-haemolytic streptococci isolated from the oral or nasal cavities in dogs and cats. As many commercial diagnostic laboratories do not routinely identify streptococcal isolates to species level in clinical samples from small animals, the importance and pathogenic potential of S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis infections in companion animal medicine may be underestimated. In addition, contact with horses or exposure to a farming environment may be a risk factor for Lancefield C group streptococcal infection in dogs and cats (Acke et al. 2010; Priestnall et al. 2010).
The objectives of this study were first to estimate the prevalence of β-haemolytic Lancefield group C streptococcal species in healthy dogs, cats, and horses sampled in the lower North Island of New Zealand, secondly to determine if frequent contact with horses was associated with isolation of these species from dogs and cats, and finally to document the genetic diversity of S. equi subsp. zooepidemicus isolates by analysis of the multilocus sequence type (MLST) profiles of isolates obtained from horses, dogs and cats.
Materials and Methods
t
This was a prospective cross-sectional study using a convenience sample from horses, dogs and cats
rip
in the lower North Island of New Zealand. All the animals belonged to clients of the Massey
University Veterinary Teaching Hospital (MUVTH) and the study was conducted during 2010 and
us c
that were mutually exclusive in their exposure history to horses and farmed livestock. The first population of animals sampled, referred to as Urban dogs and Urban cats (minimal contact with
M an
horses or farm livestock), were domestic pets presented for routine vaccination, health check, neutering or dental treatment visits at the MUVTH. Only dogs and cats that had no or infrequent contacts with horses or livestock were included in this group and the samples were collected from February to May 2010. Approximately five to 10 animals were sampled weekly depending on owner consent and staff availability. The second population of animals sampled, referred to as Farm
d
dogs (minimal contact with horses), were working farm dogs living and working on sheep or beef
pt e
farms but without frequent contact with horses. The third population of animals sampled, referred to as Stable dogs and Stable cats (frequent contact with horses), were dogs and cats living on premises where horses were present or with frequent contact with horses, defined as being on premises with
ce
horses several times a week. Some livestock could also be present on these premises but the dominant exposure was equine. In this last group samples were also collected from the horses present on the premises to investigate if there was an association between the isolation of S.
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
2011. An a priori decision was made to sample animals from three disparate populations of animals
dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in dogs and cats and exposure to horses. Visits were completed over 1 year and locations were selected haphazardly in the lower North Island. Each dog or cat had one oropharyngeal swab collected and one nasopharyngeal swab was collected from the horses. All animals sampled in this study were presumed healthy and were not receiving any antimicrobial treatment at the time of sample collection. Information was obtained at the time of sampling regarding age, use of the animal, breed, any past illnesses, vaccination history, in contact animals, travel or shelter/boarding kennel history and recent drug treatments. Sample collection for this study was approved by the Massey University Animal Ethics Committee (Palmerston North, NZ).
Swabs were placed in an Amies transport medium with charcoal (Copan, Brescia, Italy) following sample collection (Day 0), refrigerated and cultured within 24 hours. Each swab was cultured onto Columbia Colistin and Nalidixic acid blood agar (Fort Richard, Auckland, NZ), and incubated at 37°C in 5% CO2 on Day 1. On Days 2 and 3, β-haemolytic colonies were subcultured onto Columbia horse blood agar (Fort Richard) at 37°C in 5% CO2. Multiple colonies were subcultured from each sample as indicated based on different morphology of the colonies. On Days 3 and 4, a Gram stain and catalase test were performed on β-haemolytic cultures and Lancefield grouping was performed on all Gram positive, catalase-negative organisms using an Oxoid Streptococcal
t
Grouping Kit (Oxoid, Basingstoke, UK). A suspension of any Lancefield group C cultures was
rip
made in phosphate-buffered saline, and lactose, sucrose, sorbitol, trehalose and ribose sugar
fermentation tests were performed for subspecies confirmation as illustrated in Table 1 (Cowan and
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zooepidemicus isolate as previously described (Webb et al. 2008) and results were compared to those in the Streptococcus zooepidemicus MLST website (http://pubmlst.org/ szooepidemicus/)
M an
developed by Jolley and Maiden (2010). Streptococcus dysgalactiae subsp. equisimilis isolates obtained from horses were confirmed by PCR as described by Preziuso et al.( 2010). PCR products from a subset of S. dysgalactiae subsp. equisimilis isolates from dogs were purified using the Purelink PCR cleanup kit (Invitrogen, Carlsbad, USA) and sequenced using the Sdy519F and 920R primers. The sequences were submitted to the National Centre for Biotechnology Information,
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Statistical Analysis
d
Basic Local Alignment Search Tool search tool for subspecies confirmation.
Data collected during the study were checked for errors against the written records prior to analysis.
ce
For each of the sampled populations estimated apparent prevalences, with 95% CI, were calculated for each of the confirmed isolates of S. dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus. For each species sampled in the Urban dog and Urban cat populations, where each
Ac
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Steel 2003). Multilocus sequence typing (MLST) analysis was performed on each S. equi subsp.
animal was sampled independently, Wilson's method (Wilson 1927) was used to calculate 95% CI for prevalence. For the Farm dog, the Stable dog and Stable cat and equine population data, where varying numbers of animals were sampled at each premise, the underlying dependency in the data was controlled for by using a generalised estimating equations (GEE) model. An exchangeable correlation structure was used for this analysis based on the working correlation structure which gave the lowest Quasilikelihood under the Independence model Criterion statistic (Pan 2001). For some of the premises data there were no isolates for a particular species so the GEE model could not be used. Instead a range for the upper 95% CI is shown, that was calculated using the binomial proportion where the lower figure corresponds to a sample size equal to the number of
animals tested and the upper figure corresponding to a sample size equal to the number of visits. In these cases the effective sample size cannot be calculated but the upper 95% CI must lie between these two extremes, as the nearer the within cluster correlation is to one the closer the effective sample size will be to the number of clusters (premises in this case) sampled. Given the small sample sizes and the small number of positive samples detected, Bayesian modelling was used to give a more robust estimate of measures of association (Messam et al. 2008). Markov chain Monte Carlo methods were used to sample from a joint posterior distribution formed by combining the prior distribution and the likelihood, given the data, to generate an estimate of the
rip
t
odds of being infected with either S. dysgalactiae subsp. equisimilis or S. equi subsp.
zooepidemicus, for dogs from the Stable dog population compared to dogs from the Urban dog and
us c
beta (1,1) uniform prior for the prevalence of either of the Streptococcus spp. isolated from the respective dog populations. For the Bayesian analyses, the Markov chain Monte Carlo sampler was run for 110,000 iterations and the first 10,000 burn in samples were discarded. Model convergence
M an
was assessed as satisfactory by visual inspection of the trace plots, density plots and summary statistics.
Owing to the small sample sizes, Fisher’s exact test was used to test the null hypothesis that there was no association between isolate status and history of respiratory tract disease (RTD), animal age,
d
gender, kennel cough or cat flu vaccination, past treatments including antibiotics, or travel or
pt e
boarding history. All statistical analyses were completed using either R v2.14.1 (R Development Core Team, 2004; R Foundation for Statistical Computing, Vienna, Austria) or WinBUGS Version
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1.4.
Results
A summary of important descriptive data for the sampled populations is shown in Table 2. For the
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Farm dog populations. This was carried out using WinBUGS (Spiegelhalter et al. 1996) using a
Urban dog and cat populations, age was only categorised as less than or greater than 6 months; 38/53 (71.7%) Urban cats, and 12/47 (25.5%) Urban dogs were
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
Prevalence of Streptococcus dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in a sample of healthy dogs, cats and horses. a
b
a
a
bd
ae
c
E Acke , AC Midwinter , K Lawrence , SJG Gordon , S Moore , I Rasiah , K Steward , N b
c
French & A Waller a
Animal and Biomedical Sciences, Massey University Veterinary Teaching Hospital, Institute of Veterinary, Massey University, Palmerston North, New Zealand b
Animal and Biomedical Sciences, Infectious Disease Research Centre, Institute of Veterinary, Massey University, Palmerston North, New Zealand c
Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, United Kingdom
Click for updates
d
Current address: William Stephen Road, RD1, Te Anau, New Zealand
e
Current address: Asteron Centre, 55 Featherston Street, Wellington, New Zealand Accepted author version posted online: 19 Feb 2015.
To cite this article: E Acke, AC Midwinter, K Lawrence, SJG Gordon, S Moore, I Rasiah, K Steward, N French & A Waller (2015): Prevalence of Streptococcus dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in a sample of healthy dogs, cats and horses., New Zealand Veterinary Journal, DOI: 10.1080/00480169.2015.1016133 To link to this article: http://dx.doi.org/10.1080/00480169.2015.1016133
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & New Zealand Veterinary Association Journal: New Zealand Veterinary Journal DOI: 10.1080/00480169.2015.1016133
Scientific Article
Prevalence of Streptococcus dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in a sample of healthy dogs, cats and horses.
rip
t
E Acke*§, AC Midwinter†, K Lawrence*, SJG Gordon*, S Moore†#, I Rasiah*¥, K Steward‡, N French† and A Waller‡
M an
us c
Massey University Veterinary Teaching Hospital, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand † Infectious Disease Research Centre, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand ‡ Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, United Kingdom # Current address: William Stephen Road, RD1, Te Anau, New Zealand ¥ Current address: Asteron Centre, 55 Featherston Street, Wellington, New Zealand § Author for correspondence. Email: [email protected]
pt e
d
Abstract
AIMS: To estimate the prevalence of β-haemolytic Lancefield group C streptococci in healthy dogs, cats and horses; to determine if frequent contact with horses was associated with isolation of these
ce
species from dogs and cats; and to characterise recovered S. equi subsp. zooepidemicus isolates by multilocus sequence typing (MLST). METHODS: Oropharyngeal swabs were collected from 197 dogs and 72 cats, and nasopharyngeal
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
*
swabs from 93 horses. Sampling was carried out at the Massey University Veterinary Teaching Hospital, on sheep and beef farms or on premises where horses were present. All animals were
healthy and were categorised as Urban dogs and cats (minimal contact with horses or farm livestock), Farm dogs (minimal contact with horses) and Stable dogs and cats (frequent contact with horses). Swabs were cultured for β-haemolytic Streptococcus spp. and Lancefield group C streptococcal subspecies were confirmed by phenotypic and molecular techniques. RESULTS: Of the 197 dogs sampled, 21 (10.7 (95% CI= 4.0–25.4)%) tested positive for S. dysgalactiae subsp. equisimilis and 4 (2.0 (95% CI=0.7–5.5)%) tested positive for S. equi subsp.
zooepidemicus. All these isolates, except for one S. dysgalactiae subsp. equisimilis isolate in an Urban dog, were from Stable dogs. S. dysgalactiae subsp. equisimilis was isolated from one Stable cat. Of the 93 horses, 22 (23.7 (95% CI=12.3–40.6)%) and 6 (6.5 (95% CI=2.8–14.1)%) had confirmed S. dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus isolation respectively. Isolation of S. dysgalactiae subsp. equisimilis from dogs was statistically associated with frequent contact with horses (OR=9.8 (95% CI=2.6–72.8)). Three different MLST profiles of S. equi subsp. zooepidemicus that have not been previously reported were recovered from healthy dogs.
rip
t
CONCLUSIONS AND CLINICAL RELEVANCE: Subclinical infection or colonisation by S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis occurs in dogs and further research on
us c
needed. Complete speciation of β-haemolytic streptococci should be recommended in clinical cases and the possible exposure to horses and their environment should be considered in epidemiological
M an
investigations.
KEY WORDS: Streptococcus dysgalactiae subsp. equisimilis, S. equi subsp. zooepidemicus,
d
prevalence, horse, dog, epidemiology
Generalised estimated equations
MLST
Multilocus sequence typing
MUVTH
Massey University Veterinary Teaching Hospital
ce
RTD
pt e
GEE
Respiratory tract disease
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
inter-species transmission and the pathogenic potential of these Lancefield group C streptococci is
ST
Sequence type
Introduction Streptococcus equi subsp. equi, S. equi subsp. zooepidemicus, and S. dysgalactiae subsp. equisimilis are β-haemolytic Lancefield group C streptococci that can cause disease in horses but can also occur subclinically (Timoney 2004; Erol et al. 2012; North et al. 2014). S. equi subsp. zooepidemicus is a commonly isolated opportunistic pathogen from the equine skin, respiratory tract, and urogenital tract (Lavoie et al. 1991; Timoney 2004; Newton et al. 2008). The organism is
a multi-host pathogen that has been associated with infections in several animal species (Webb et al. 2008) and its significance appears to be increasing world-wide. Most concerning in companion animal medicine are the increasing number of reports of haemorrhagic pneumonia and more severe presentations of the canine infectious respiratory disease complex associated with S. equi subsp. zooepidemicus infections in intensively housed dogs in several geographical locations including the United States of America (USA; Garnett et al. 1982; Pesavento et al. 2008), Europe (Chalker et al. 2003), and Asia (Kim et al. 2007; Byun et al. 2009). The epidemiology and pathogenesis of S. equi subsp. zooepidemicus infections in dogs and cats remains poorly understood and the source of
t
infection unknown (Priestnall and Erles 2011). S. equi subsp. zooepidemicus is potentially zoonotic
rip
with sporadic reports of meningitis and other infections in humans attributed to close contact with horses (Jovanovic et al. 2008; Brouwer et al. 2010; Eyre et al. 2010). Dog-to-human infection was
us c
al. 2010). S. dysgalactiae subsp. equisimilis has become an important pathogen in horses where it may cause a range of opportunistic infections (Erol et al. 2012), and also in humans (Brandt and
M an
Spellerberg 2009). Comprehensive studies on β-haemolytic streptococci are limited even in horses; S. dysgalactiae subsp. equisimilis is suspected to have a much more important role than previously thought and has been isolated from a broad range of equine tissues, especially from the reproductive tract (Erol et al. 2012). The importance of this organism in dogs and cats remains very poorly documented in the veterinary literature. In addition to this, β-haemolytic Lancefield group C
d
streptococci are a grouping in a state of taxonomic flux (Vandamme et al. 1996; Preziuso et al.
pt e
2014) which complicates interpretation of bacteriology results previously reported in the veterinary literature. S. equi subsp. equi, the causative agent of equine strangles, also a member of βhaemolytic Lancefield group C and very closely related to S. equi subsp. zooepidemicus, is rarely
ce
reported in non-equine hosts (Holden et al. 2009) with one case of S. equi subsp. equi infection in a dog with severe regional lymphadenopathy reported (Ladlow et al. 2006). Infection of humans has also been reported (Elsayed et al. 2003; Popescu et al. 2006).
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
confirmed in one case where a dog residing on a horse farm infected a veterinary nurse (Abbott et
The range of subspecies recovered from healthy cats and dogs and the role of horses in the epidemiology of Lancefield group C streptococci has not been investigated previously. Very few studies have reported complete taxonomic identification of β-haemolytic streptococci isolated from the oral or nasal cavities in dogs and cats. As many commercial diagnostic laboratories do not routinely identify streptococcal isolates to species level in clinical samples from small animals, the importance and pathogenic potential of S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis infections in companion animal medicine may be underestimated. In addition, contact with horses or exposure to a farming environment may be a risk factor for Lancefield C group streptococcal infection in dogs and cats (Acke et al. 2010; Priestnall et al. 2010).
The objectives of this study were first to estimate the prevalence of β-haemolytic Lancefield group C streptococcal species in healthy dogs, cats, and horses sampled in the lower North Island of New Zealand, secondly to determine if frequent contact with horses was associated with isolation of these species from dogs and cats, and finally to document the genetic diversity of S. equi subsp. zooepidemicus isolates by analysis of the multilocus sequence type (MLST) profiles of isolates obtained from horses, dogs and cats.
Materials and Methods
t
This was a prospective cross-sectional study using a convenience sample from horses, dogs and cats
rip
in the lower North Island of New Zealand. All the animals belonged to clients of the Massey
University Veterinary Teaching Hospital (MUVTH) and the study was conducted during 2010 and
us c
that were mutually exclusive in their exposure history to horses and farmed livestock. The first population of animals sampled, referred to as Urban dogs and Urban cats (minimal contact with
M an
horses or farm livestock), were domestic pets presented for routine vaccination, health check, neutering or dental treatment visits at the MUVTH. Only dogs and cats that had no or infrequent contacts with horses or livestock were included in this group and the samples were collected from February to May 2010. Approximately five to 10 animals were sampled weekly depending on owner consent and staff availability. The second population of animals sampled, referred to as Farm
d
dogs (minimal contact with horses), were working farm dogs living and working on sheep or beef
pt e
farms but without frequent contact with horses. The third population of animals sampled, referred to as Stable dogs and Stable cats (frequent contact with horses), were dogs and cats living on premises where horses were present or with frequent contact with horses, defined as being on premises with
ce
horses several times a week. Some livestock could also be present on these premises but the dominant exposure was equine. In this last group samples were also collected from the horses present on the premises to investigate if there was an association between the isolation of S.
Ac
Downloaded by [Universite Laval] at 23:52 03 March 2015
2011. An a priori decision was made to sample animals from three disparate populations of animals
dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus in dogs and cats and exposure to horses. Visits were completed over 1 year and locations were selected haphazardly in the lower North Island. Each dog or cat had one oropharyngeal swab collected and one nasopharyngeal swab was collected from the horses. All animals sampled in this study were presumed healthy and were not receiving any antimicrobial treatment at the time of sample collection. Information was obtained at the time of sampling regarding age, use of the animal, breed, any past illnesses, vaccination history, in contact animals, travel or shelter/boarding kennel history and recent drug treatments. Sample collection for this study was approved by the Massey University Animal Ethics Committee (Palmerston North, NZ).
Swabs were placed in an Amies transport medium with charcoal (Copan, Brescia, Italy) following sample collection (Day 0), refrigerated and cultured within 24 hours. Each swab was cultured onto Columbia Colistin and Nalidixic acid blood agar (Fort Richard, Auckland, NZ), and incubated at 37°C in 5% CO2 on Day 1. On Days 2 and 3, β-haemolytic colonies were subcultured onto Columbia horse blood agar (Fort Richard) at 37°C in 5% CO2. Multiple colonies were subcultured from each sample as indicated based on different morphology of the colonies. On Days 3 and 4, a Gram stain and catalase test were performed on β-haemolytic cultures and Lancefield grouping was performed on all Gram positive, catalase-negative organisms using an Oxoid Streptococcal
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Grouping Kit (Oxoid, Basingstoke, UK). A suspension of any Lancefield group C cultures was
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made in phosphate-buffered saline, and lactose, sucrose, sorbitol, trehalose and ribose sugar
fermentation tests were performed for subspecies confirmation as illustrated in Table 1 (Cowan and
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zooepidemicus isolate as previously described (Webb et al. 2008) and results were compared to those in the Streptococcus zooepidemicus MLST website (http://pubmlst.org/ szooepidemicus/)
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developed by Jolley and Maiden (2010). Streptococcus dysgalactiae subsp. equisimilis isolates obtained from horses were confirmed by PCR as described by Preziuso et al.( 2010). PCR products from a subset of S. dysgalactiae subsp. equisimilis isolates from dogs were purified using the Purelink PCR cleanup kit (Invitrogen, Carlsbad, USA) and sequenced using the Sdy519F and 920R primers. The sequences were submitted to the National Centre for Biotechnology Information,
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Statistical Analysis
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Basic Local Alignment Search Tool search tool for subspecies confirmation.
Data collected during the study were checked for errors against the written records prior to analysis.
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For each of the sampled populations estimated apparent prevalences, with 95% CI, were calculated for each of the confirmed isolates of S. dysgalactiae subsp. equisimilis and S. equi subsp. zooepidemicus. For each species sampled in the Urban dog and Urban cat populations, where each
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Steel 2003). Multilocus sequence typing (MLST) analysis was performed on each S. equi subsp.
animal was sampled independently, Wilson's method (Wilson 1927) was used to calculate 95% CI for prevalence. For the Farm dog, the Stable dog and Stable cat and equine population data, where varying numbers of animals were sampled at each premise, the underlying dependency in the data was controlled for by using a generalised estimating equations (GEE) model. An exchangeable correlation structure was used for this analysis based on the working correlation structure which gave the lowest Quasilikelihood under the Independence model Criterion statistic (Pan 2001). For some of the premises data there were no isolates for a particular species so the GEE model could not be used. Instead a range for the upper 95% CI is shown, that was calculated using the binomial proportion where the lower figure corresponds to a sample size equal to the number of
animals tested and the upper figure corresponding to a sample size equal to the number of visits. In these cases the effective sample size cannot be calculated but the upper 95% CI must lie between these two extremes, as the nearer the within cluster correlation is to one the closer the effective sample size will be to the number of clusters (premises in this case) sampled. Given the small sample sizes and the small number of positive samples detected, Bayesian modelling was used to give a more robust estimate of measures of association (Messam et al. 2008). Markov chain Monte Carlo methods were used to sample from a joint posterior distribution formed by combining the prior distribution and the likelihood, given the data, to generate an estimate of the
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odds of being infected with either S. dysgalactiae subsp. equisimilis or S. equi subsp.
zooepidemicus, for dogs from the Stable dog population compared to dogs from the Urban dog and
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beta (1,1) uniform prior for the prevalence of either of the Streptococcus spp. isolated from the respective dog populations. For the Bayesian analyses, the Markov chain Monte Carlo sampler was run for 110,000 iterations and the first 10,000 burn in samples were discarded. Model convergence
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was assessed as satisfactory by visual inspection of the trace plots, density plots and summary statistics.
Owing to the small sample sizes, Fisher’s exact test was used to test the null hypothesis that there was no association between isolate status and history of respiratory tract disease (RTD), animal age,
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gender, kennel cough or cat flu vaccination, past treatments including antibiotics, or travel or
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boarding history. All statistical analyses were completed using either R v2.14.1 (R Development Core Team, 2004; R Foundation for Statistical Computing, Vienna, Austria) or WinBUGS Version
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1.4.
Results
A summary of important descriptive data for the sampled populations is shown in Table 2. For the
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Farm dog populations. This was carried out using WinBUGS (Spiegelhalter et al. 1996) using a
Urban dog and cat populations, age was only categorised as less than or greater than 6 months; 38/53 (71.7%) Urban cats, and 12/47 (25.5%) Urban dogs were
