Characterization of a Streptococcus equi ssp. equi Isolate From a Strangles Outbreak in Thailand

Strangles, which is caused by Streptococcus equi ssp. equi, is one of the major infectious respiratory diseases in horses. Knowledge of isolates from different areas of the world is important for investigating the different strains of the disease. In contrast to many other countries, currently little is known about S. equi ssp. equi isolates in Thailand. In 2014, a farm in Thailand imported 20 horses from Europe. Approximately 1 month after arrival, 50% of the horses had developed pyrexia, mucopurulent nasal discharge, and abscesses of the mandibular lymph nodes. Nasal swabs of mucopurulent discharge were sent to a diagnostic laboratory, and two isolates of S. equi ssp. equi were identified. One of the isolates was further characterized using seM gene polymerase chain reaction and sequence analysis. The seM sequence was then compared to the database of PubMLST-seM. It was found to contain SeM allele 48, an allele isolated from horses in the United Kingdom in 2006 and 2010. This result demonstrates the usefulness of SeM allele identification as a tool for investigating the source of related strains and for the epidemiologic study of strangles. To the best of the authors' knowledge, this is the first report of the identification of an SeM allele of the S. equi ssp. equi isolate in Thailand.     

Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India

Present study was undertaken to study the prevalence of β-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.     

Etiologic and epidemiologic analysis of bacterial infectious upper respiratory disease in Thoroughbred horses at the Seoul Race Park

Infectious upper respiratory disease (IURD) of Thoroughbred racehorses has been a frequent problem (29.6% of incidence) at the Seoul Race Park (Korea). Risk factors for IURD include the season with a high transfer rate (summer and fall), the stabling period (≤ 3 months), and age (2 to 3 years old), suggesting that the movement and new environment may have depressed the immune system of the horses and decreased their ability to respond properly to pathogens. The bacterial strains (n = 98) isolated from IURD horses included Pseudomonas spp., Escherichia coli, Staphylococcus spp., Streptococcus equi subsp. equi and zooepidemicus.     

A Multiplex Polymerase Chain Reaction Assay for Direct Detection and Differentiation of β-Hemolytic Streptococci in Clinical Samples from Horses

Streptococcus equi subspecies equi, S equi subspecies zooepidemicus, and S dysgalactiae subspecies equisimilis are β-hemolytic Streptococci, often isolated from horses with respiratory or genital diseases. The aim of this study was (i) defining and validating a multiplex polymerase chain reaction (PCR) protocol for identifying these Streptococci in bacterial cultures and for detecting them directly in equine clinical specimens, and (ii) defining and validating a cheap DNA extraction protocol for clinical specimens. When respiratory and genital samples from symptomatic and asymptomatic horses were tested by bacterial culture and by multiplex PCR, all the 150 samples culture-positive for S equi, S zooepidemicus, or S equisimilis were also positive by PCR. Of 150 culture-negative samples, 143 were negative by PCR. Seven samples were positive by PCR but negative by bacteriology. The multiplex PCR protocol described in this study is proven suitable for a sensitive, specific, and rapid detection and identification of S equi, S zooepidemicus, and S equisimilis in cultured bacterial colonies, as well as in clinical specimens from symptomatic or asymptomatic horses. The inclusion of internal control primers in the PCR protocol excludes false-negative results. A cheap DNA extraction method has been also validated for swabs, tracheal aspirates, bronchoalveolar lavage, and guttural pouches lavage samples.     

First Report of Molecular Characterization of Argentine Isolates of Streptococcus equi subsp. equi by Pulsed-Field Gel Electrophoresis

Strangles is one of the most frequently diagnosed equine respiratory infectious diseases in the world. It is caused by Streptococcus equi subsp. equi (S. equi), and it is an acute infection characterized by pyrexia, nasal discharge, pharyngitis, and abscessation of lymph nodes. Frequently, healthy horses might continue to harbor S. equi after clinical recovery. Although the genetic distance between S. equi isolates is short, strains can be differentiated by pulsed-field gel electrophoresis (PFGE) and single locus sequence typing for epidemiological studies. The aim of this study was to characterize by PFGE Argentine isolates of S. equi obtained from horses with acute strangles and those that had recovered. Bacterial isolation and identification of 80 S. equi isolates by phenotypic and genotypic tests were performed using samples from 29 horses with acute strangles and 95 from healthy animals. Also, the isolates were characterized by PFGE using Bsp120I and SmaI. Visual comparison of macrorestriction patterns generated with both enzymes revealed three different DNA fragment profiles with variations of one or two bands. Interestingly, an identical profile was found in isolates from the same horse and from horses that were infected at the same time, and the horses recovered from strangles continue to carry the same strain. Some vaccinated horses have been mild infected for a different strain from that of carriers suggesting other source of infection. This is the first molecular characterization of Argentine isolates of S. equi, which shows the presence of three strains between 2010 and 2013 in Buenos Aires.     

Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares

Equine babesiosis, a tick transmitted haemoprotozoan disease caused by Theileria equi is globally distributed and responsible for heavy economic losses to the equine husbandry. Equids reared in endemic areas usually pick up infection at an early age and become immune tolerant throughout their life span. We studied the level of passively transferred antibodies in neonate foals born from pre-immuned mares. Latently T. equi infected pre-immuned pony and donkey mares (three each) were selected and T. equi antibody titres in neonates was monitored till 90 days post foaling (DPF) by applying Dot-ELISA on sequentially collected serum samples from foals and their dams. A very high antibody titre was observed in pre-immuned pony and donkey mares. The maximum antibody of 1:60 to 1:80 was observed in pony's and donkey's foal on 2–16 and 2–10 DPF, respectively and thereafter it declined to less than 1:20 on 63–77 and 56–63 DPF. Simultaneously parasite carrying status in neonate foals and their dam was also monitored by applying PCR on blood samples. We could demonstrate PCR amplification in dam's blood samples while no amplification was recorded in neonate's blood samples. This study indicated that new-born foals were born naïve and passively transferred immunity was transitory which wanes after 63–77 DPF.     

Sequelae and complications of Streptococcus equi subspecies equi infections in the horse

Streptococcus equi ssp. equi infection in the horse, or strangles, commonly results in abscessation of the submandibular, submaxillary or retropharyngeal lymph nodes. Although this classical presentation of strangles is associated with a low mortality rate, complications and sequlae may worsen the prognosis and increase mortality rates. This article reviews sequelae and complictions of S. equi ssp. equi infection in the horse, including guttural pouch empyema, bastard strangles and immune mediated diseases such as purpura haemorrhagica, myopathies and myocarditis.     

Identification of a Streptococcus equi Strain Responsible for Four Outbreaks of Strangles in Colorado

The objective of this study was to use a new subtyping technique to determine the identity of five Streptococcus equi isolates from four outbreaks of strangles in Colorado during 2005−2006. All five of the isolates from the four strangles outbreaks in Colorado contained SeM allele 28. This SeM allele is typical of American isolates of S. equi and has previously been linked with strangles outbreaks in Minnesota in 1994 and Kentucky in 1995. Use of the new S. equi strain subtyping method allowed for characterization of recent S. equi isolates associated with outbreaks of strangles in Colorado. To our knowledge, this is the first report of typing of more recent isolates from North America. This approach, however, has been used in situations in the United Kingdom to differentiate the vaccine strain of S. equi from that of the wild type of the bacteria.     

Isolation of Streptococcus agalactiae and an aquatic birnavirus from doctor fish Garra rufa L

Background

The doctor fish, Garra rufa, has become increasingly popular as a treatment for skin disorders and for pedicures in recent years. Despite this there is very little information available regarding the welfare of these fish and the range of potential pathogens they may carry. In this study, a group of fish suffering from post-transport mortalities were examined and the isolated pathogens identified.

Findings

Group B Streptococcus agalactiae was isolated from kidney swabs of the fish and found to be resistant to a number of antibiotics. In addition to this, a fish virus belonging to the aquabirnavirus group, serogroup C was isolated for the first time in Ireland. However, no clinical signs of disease typical of bacterial or viral infections were observed in any fish examined.

Conclusions

As no clinical signs of disease attributable to either of the pathogens identified were found it was concluded that the mortalities were most likely due to transport related stress exacerbated by the presence of the pathogens. Further work is required to assess the suitability of current transport strategies and to examine the potential risk associated with the transport of live ornamental fish.     

Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa

A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; ∼1600 bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.     

Antimicrobial resistant bacteria in wild mammals and birds: a coincidence or cause for concern?

Background

The emergence and dissemination of antimicrobial resistance (AMR) is a growing concern to public and animal health. The contribution attributable to wildlife remains unclear. In this study two unrelated wildlife species herring gulls (Larus argentatus) and a hybrid deer (Cervus elaphus x Cervus nippon) were investigated for the presence of Escherichia coli expressing an AMR phenotype.

Findings

Bacterial isolates resistant to β-lactam compounds were identified in both animal species and the production of functional β-lactamase was confirmed using nitrocefin. The prevalence of resistant isolates was higher in herring gulls (87%) compared to deer (31%). Resistance to this class of antibiotic was found only in non-pathogenic E. coli in herring gulls and in both pathogenic and non-pathogenic E. coli strains in deer.

Conclusions

The presence of AMR in wildlife has implications for public health, food safety and potable water source protection among others.     

Detection of Streptococcus equi subspecies equi using a triplex qPCR assay

Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2 h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

Spread of extended-spectrum beta-lactamase producing Escherichia coli isolates in Swedish broilers mediated by an incI plasmid carrying blaCTX-M-1

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla_CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla_CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla_CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla_CTX-M-1.     

Prevalence of thermophilic Campylobacter species in Swedish dogs and characterization of C. jejuni isolates

Background

The aims of this study were to investigate the prevalence of Campylobacter species in Swedish dogs, to identify the species of the Campylobacter isolates and to genotype the C. jejuni isolates. Young and healthy dogs were targeted and the sampling was performed at 11 veterinary clinics throughout Sweden from October 2011 to October 2012. Faecal swab samples were collected and sent to the laboratory at the National Veterinary Institute (SVA) for isolation of Campylobacter, speciation and genotyping.

Results

Campylobacter spp. were isolated from 67 of the 180 sampled dogs which yields an overall prevalence of 37%. The most prevalent species of Campylobacter among the participating dogs was C. upsaliensis with 52 of the 67 identified isolates. A lower prevalence was observed for C. jejuni with seven identified isolates and one isolate was identified as C. helveticus. Multi-locus sequence typing (MLST) was carried out on the seven C. jejuni isolates and all sequence types that were found are also commonly found in humans. The dogs were divided into three age groups; 1) under 12 months, 2) 12 to 23 months and 3) 24 months and older. The highest prevalence was found in the two younger age groups. Dogs shedding C. jejuni were between 3–12 months of age while dogs shedding C. upsaliensis were found in all ages.

Conclusions

The present investigation finds that Campylobacter spp. known to cause campylobacteriosis in humans are present in Swedish dogs. The results suggest an age predisposition where dogs under 2 years of age are more likely to shed Campylobacter spp. than older dogs. The most commonly isolated species was C. upsaliensis followed by C. jejuni, which was only detected in dogs up to 12 months of age. All C. jejuni isolates identified in the present study were of the same MLST types that have previously been described both in humans and in animals. The awareness of the Campylobacter risk of healthy young dogs may be an important way to reduce the transmission from dogs to infants, young children and immunocompromised adults.     

Genetic basis of penicillin resistance of S. aureus isolated in bovine mastitis

Background

The blaZ gene encoding penicillin resistance can be located either chromosomally or on plasmids. The aim of this study was to investigate the genetic relationships and to determine the location of the blaZ gene in S. aureus isolated in bovine mastitis in Finland and Sweden.

Methods

Seventy-eight β-lactamase positive S. aureus isolates from bovine mastitis (34 from Finland and 44 from Sweden) were included in the study. The localization of blaZ gene was determined by Southern blotting. The blaZ genes of the isolates were sequenced and the sequences were translated to beta-lactamase proteins and further grouped as different protein signatures. The isolates and, as control, 33 Swedish and 36 Finnish beta-lactamase negative isolates were typed with pulsed-field gel electrophoresis (PFGE).

Results

In 26 out of 34 Finnish isolates (76.5%) and in 25 out of 44 Swedish isolates (56.8%) the blaZ gene was localized on a plasmid. Six different protein signatures were found. One signature was found only in four Swedish isolates, but all other signatures were found both in Finnish and Swedish isolates. The PFGE results revealed a diversity of S. aureus clones. The protein signatures were not clearly associated with certain pulsotypes.

Conclusions

The plasmid location of the blaZ gene was not statistically significantly more common in Finland than in Sweden, and hence does not explain the higher proportion of penicillin-resistant isolates of S. aureus causing bovine mastitis in Finland compared to Sweden.     

Molecular characterisation of 'strangles' outbreaks in the UK: the use of M-protein typing of Streptococcus equi ssp. equi

Reasons for performing study: Strangles is the most commonly diagnosed and important infectious disease of horses worldwide. Very little is known about the temporo‐spatial and molecular epidemiology of strangles. The disease is not notifiable in the UK and there are few published data on the geographical locations of outbreaks. Objective: To investigate whether typing of a surface protein (SeM) of Streptococcus equi ssp. equi (S. equi), the causative agent of strangles, is a useful epidemiological tool. Methods: The variable region of the SeM gene was amplified from 145 isolates of S. equi by PCR and sequenced. Different SeM gene alleles were assigned based on the SeM database, grouped into phylogenetic clusters using split decomposition analysis and plotted against the submitting veterinary practices. Results: In this study 21 S. equi SeM alleles were found, including 9 previously unidentified alleles and representing 4 phylogenetic groups. S. equi containing SeM alleles 9 and 7 were the most commonly isolated and there was a high number of low frequency alleles. The occurrence of an outbreak cluster in the north‐west of the UK is also reported. Conclusions: Strangles outbreaks can be differentiated on the basis of their SeM allele sequences. The data provide further evidence of SeM mutation leading to the emergence of novel, but related SeM alleles that are geographically linked. Sequencing of the SeM gene is a useful tool for the elucidation of strangles epidemiology at a regional and a national level. Potential relevance: This technique may allow differentiation or linkage of strangles outbreaks and as such may be an effective tool for local as well as national and international disease surveillance.     

Frequency of Rhodococcus equi in Feces of Mares in Central Kentucky

Rhodococcus equi (R equi) pneumonia is an important cause of disease and death in foals. Feces from mares can contain R equi, including virulent R equi, and thus may act as a source of the bacteria at horse breeding farms. A previous report documented that every mare at a farm in central Kentucky shed virulent R equi in at least one of four fecal samples collected serially during the periparturient period. The objective of this study was to assess the extent to which this high prevalence of fecal shedding could be replicated at other horse breeding farms. The frequency of detection of R equi and virulent R equi in fecal samples was studied among 131 mares from 24 farms in central Kentucky. The proportions of fecal samples from mares containing R equi and virulent R equi were 95% and 76%, respectively. These findings indicate that R equi and virulent R equi may be isolated with high frequency from feces of mares at breeding farms in central Kentucky, and that mares are a source of virulent R equi for the environment of their foals.     

Changes in Serum Antibody Levels after Vaccination for Strangles and after Intranasal Challenge with Streptococcus equi subsp. equi in Horses

In this study, to evaluate the influence of strangles vaccination on serological test results, we investigated the changes in strangles serum antibody levels in horses after vaccination and subsequent intranasal challenge with S. equi. The horses were vaccinated for strangles with either a component vaccine (Group C) or a live vaccine (Group L). We measured changes in strangles serum antibody levels weekly for 20 weeks after vaccinating horses twice for strangles over a 3-week interval, and for 7 weeks after intranasal challenge with S. equi in the same horses. Serum antibody responses to the proline-glutamic acid-proline-lysine (PEPK) antigen with five repetitions (PEPK-5R) were higher at all times (up to 2.4-fold) following vaccination in Group C than in Group L, and the value peaked at 2.9-fold above the initial value after the second vaccination in Group C horses. However, the value was lower than that in horses infected with S. equi, and it gradually decreased, reaching the initial (week 0) value by the 15th week. Serum antibody responses to PEPK-5R after challenge with S. equi increased in both groups of horses, but the value tended to be lower than that reported for unvaccinated horses. In addition, the average value in Group C was 2.6-fold higher than that of Group L. These results suggest the serum antibody responses of horses infected with S. equi varies according to the type of vaccine with which they have been vaccinated. Although the serological diagnostic test for strangles in which PEPK-5R is used as an antigen is effective for the investigation of serum antibodies to strangles in vaccinated horses, the present data suggest it is necessary to consider the vaccination history when interpreting the results.     

Septic funiculitis caused by Streptococcus equi subspecies equi infection with associated immune‐mediated haemolytic anaemia

A 2‐year‐old Quarter Horse gelding presented for anaemia, icterus, depression and intermittent colic 2 weeks after routine castration. Bilateral septic funiculitis with Streptoccocus equi ssp. equi with secondary immune‐mediated haemolytic anaemia were diagnosed. A blood transfusion was required to facilitate general anaesthesia for surgical excision of the septic funiculitis. Antibiotic therapy was provided initially with chloramphenicol and later enrofloxacin. Immunosuppressive therapy was provided with dexamethasone and later azathioprine. The horse responded well to treatment and was discharged 8 weeks after presentation. Streptococcus equi ssp. equi should be considered in cases with septic funiculitis and the potential for a secondary immune‐mediated haemolytic anaemia exists with this bacterial species.