'I want to be the sort of owner that he wants me to be': Rationales for biosecurity implementation among British horse owners.

BACKGROUND: Horse owners play a critical role in mitigating the risk of pathogen spread between horses. However, little is known about how they view biosecurity and whether they experience barriers to the uptake of preventive measures. OBJECTIVES: To explore horse owners' attitudes, perceptions, and experiences of biosecurity and identify how these factors shape horse owners' decisions for biosecurity implementation. STUDY DESIGN: Qualitative study using semi-structured interviews. METHODS: Interviews were conducted with 23 horse owners across Great Britain. Participants were purposively selected to include those in different geographic regions, with different management arrangements, and varied length of horse ownership experience. Interviews were audio-recorded, transcribed verbatim, and analysed using a critical realist thematic analysis. RESULTS: Participants felt a moral obligation to prioritise their horse's happiness, which became a challenge when certain biosecurity measures (e.g., quarantine) were perceived as compromising their horse's happiness or comfort (Theme 1). A lack of biosecurity was the social norm among shared yards and competition venues (Theme 2), which made it difficult for participants to implement biosecurity measures effectively on their own. Combined with the sense of moral obligation participants felt towards their horse, this meant that participants had to 'care double' (i.e., be more vigilant than they would otherwise) to compensate for collective inaction (Theme 3). MAIN LIMITATIONS: Participants may have been more interested in and/or knowledgeable about biosecurity than the general horse owning population. CONCLUSIONS: The findings highlight several challenges that could be addressed to improve biosecurity implementation among horse owners. Efforts to encourage improved uptake of biosecurity measures should focus on communicating how reducing the risk of disease aligns with horse care. Further research on social norms in the horse industry is needed, in addition to identifying strategies to encourage a collective adoption of biosecurity measures.

Challenges to exotic disease preparedness in Great Britain: The frontline veterinarian's perspective.

BACKGROUND: Exotic diseases pose a significant risk to horse health and welfare. Several stakeholder groups, including primary care veterinarians, share responsibility for maintaining freedom from pathogens that cause exotic diseases. However, little is known about the current state of exotic disease preparedness within the British horse industry. OBJECTIVES: The aim of this study was to explore equine veterinarians' experiences of, and attitudes towards, exotic disease preparedness in Great Britain. STUDY DESIGN: This is a qualitative interview-based study. METHODS: Semistructured interviews were conducted with 14 primary care equine veterinarians in Great Britain. Participants were purposively selected to include perspectives across varying levels of experience, clientele and location. All interviews were recorded, transcribed verbatim and analysed using thematic analysis. RESULTS: Three themes were developed: (a) the reactive generalist, relating to participants' self-concept of their role as primary care practitioners; (b) working within the bounds of influence, encompassing participants' perceived inability to influence their clients' knowledge and behaviours and (c) a fragmented horse industry, illustrating the wider culture in which participants worked, characterised by a lack of cohesion amongst its members. MAIN LIMITATIONS: Only veterinarians' perspectives have been captured, so viewpoints from other stakeholders, such as horse owners and government officials, should be used to triangulate these findings. CONCLUSIONS: The findings suggest that improvements are required before an optimal level of exotic disease preparedness can be achieved. Additional support provided to frontline veterinarians, such as skills-based training (ie, clinical reasoning and collaborative relationship building), accessible and trusted emergency support networks and clear expectations and responsibilities during an outbreak are recommended to optimise exotic disease preparedness.

Surveillance of strangles in UK horses between 2015 and 2019 based on laboratory detection of Streptococcus equi.

BACKGROUND: Previously national surveillance data for monitoring strangles (Streptococcus equi infection) in UK horses was limited. Improved awareness and knowledge of positive diagnoses would permit the optimisation of biosecurity protocols, decreasing the prevalence of strangles. METHODS: Seven UK laboratories reported positive strangles diagnoses between 1 January 2015 and 31 December 2019 based on identifying Streptococcus equi via agent detection assays from field-based practitioner-submitted samples. Associated clinical history and animal signalment were collected where provided, and descriptive analysis undertaken. RESULTS: Within the study period, 1617 laboratory-confirmed diagnoses occurred from samples submitted by 315 veterinary practices. Of these, 51.6% were swabs and 44.0% guttural pouch lavages. Diagnoses were primarily based on qPCR alone (59.6%), qPCR and culture (35.8%), or culture alone (4.6%). A total of 1791 clinical signs were reported for 713 diagnoses, where nasal discharge (31.3%) and pyrexia (20.5%) were most frequently reported. Regions with the highest number of diagnoses included North Yorkshire (n = 75, 4.6%), Staffordshire (n = 71, 4.4%) and West Sussex (North East) (n = 63, 3.9%). CONCLUSION: This study presents important insights into the diagnosis and clinical features of strangles in UK horses, even though limited and/or missing clinical history and signalment on laboratory submission forms restricts the completeness of the data.

Preliminary insight into horse owners' perceptions of, and attitudes towards, exotic diseases in the United Kingdom.

BACKGROUND: The potential for an exotic disease incursion is a significant concern for the United Kingdom (UK) equine industry. Horse owners' perceptions of, and attitudes towards, exotic diseases can influence decisions to adopt disease preparedness strategies. The objectives of this study were to describe horse owners' 1) perceptions of the term 'exotic disease', and 2) attitudes towards their risk of being affected by an exotic disease. In order to address these objectives, qualitative content analysis was undertaken on data collected using two open-ended survey questions. RESULTS: Horse owners (n = 423) perceived exotic diseases as 1) belonging somewhere else, and 2) a dangerous threat to their horse(s). The term 'exotic' was associated with being foreign, non-native, and out-of-place in the UK. Attitudes towards exotic disease risk were summarised into four categories: 1) responsible horse owners prevent disease, 2) horse owners need support to stop disease spread, 3) risk depends on proximity to the 'risky' horse, and 4) some risk is inevitable. A 'responsible' owner was aware of health hazards and took actions to protect their horse from disease. Reliance on others, including stakeholders, to uphold disease prevention in the community led to feeling vulnerable to disease threats. When evaluating risk, horse owners considered which horses were the 'riskiest' to their horse's health (horses that travelled, participated in competitions, or were simply unfamiliar) and avoided situations where they could interact. Despite undertaking disease prevention measures, the perceived uncontrollable nature of exotic diseases led some owners to feel an incursion was inevitable. CONCLUSIONS: Without accounting for horse owners' perceptions of, and attitudes towards, exotic diseases, recommendations to increase preparedness may be ineffective. Improved communication among stakeholders in the industry may assist in clarifying expectations for exotic disease-specific prevention measures. A collaborative approach among horse owners and stakeholders is recommended to improve disease preparedness within the industry.

A cross-sectional study of horse owners' awareness and perceived risk of exotic diseases in the United Kingdom.

The international nature of the equine industry provides opportunities for the spread of infectious diseases between countries. While incursions of exotic diseases into the United Kingdom (UK) equine population have been rare, the potential socioeconomic and welfare impacts are a significant concern. However, little is known about leisure horse owners' ability or willingness to prepare for an exotic disease incursion. The objectives of this study were to describe UK leisure horse owners' awareness and perceptions of exotic diseases, and to identify clusters of horse owners characterised by their awareness and perceived risk of exotic diseases. A cross-sectional study of leisure horse owners in the UK was conducted between April and July 2018. Participants (n = 403) completed an online questionnaire with questions pertaining to demographics, experiences with endemic diseases, and awareness and perceptions of exotic diseases. Hierarchical cluster analysis was used to identify groups of participants that were similar in regard to their awareness and perceived risk of exotic diseases. Participants identified a median of 3 (IQR 2-4) exotic diseases, with the most recognised exotic diseases being African horse sickness and West Nile virus. The most frequently mentioned clinical signs that participants thought were associated with exotic diseases included high temperature (57.2%), discharge (46.5%), and lack of energy (41.2%). Hierarchical cluster analysis identified three clusters of participants: 1) those who were aware of exotic diseases and perceived a high amount of risk (n = 78); 2) those who were aware of exotic diseases but perceived a low amount of risk (n = 111); and 3) those who were less aware of exotic diseases and perceived a low amount of risk (n = 214). Efforts to communicate the relevance and consequences of exotic diseases to horse owners should consider the potential difference in receptiveness among horse owners in each cluster. Further investigations are required to determine the implications of horse owners' perceived risk on exotic disease preparedness.

PinR mediates the generation of reversible population diversity in Streptococcus zooepidemicus.

Opportunistic pathogens must adapt to and survive in a wide range of complex ecosystems. Streptococcus zooepidemicus is an opportunistic pathogen of horses and many other animals, including humans. The assembly of different surface architecture phenotypes from one genotype is likely to be crucial to the successful exploitation of such an opportunistic lifestyle. Construction of a series of mutants revealed that a serine recombinase, PinR, inverts 114 bp of the promoter of SZO_08560, which is bordered by GTAGACTTTA and TAAAGTCTAC inverted repeats. Inversion acts as a switch, controlling the transcription of this sortase-processed protein, which may enhance the attachment of S. zooepidemicus to equine trachea. The genome of a recently sequenced strain of S. zooepidemicus, 2329 (Sz2329), was found to contain a disruptive internal inversion of 7 kb of the FimIV pilus locus, which is bordered by TAGAAA and TTTCTA inverted repeats. This strain lacks pinR and this inversion may have become irreversible following the loss of this recombinase. Active inversion of FimIV was detected in three strains of S. zooepidemicus, 1770 (Sz1770), B260863 (SzB260863) and H050840501 (SzH050840501), all of which encoded pinR. A deletion mutant of Sz1770 that lacked pinR was no longer capable of inverting its internal region of FimIV. The data highlight redundancy in the PinR sequence recognition motif around a short TAGA consensus and suggest that PinR can reversibly influence the wider surface architecture of S. zooepidemicus, providing this organism with a bet-hedging solution to survival in fluctuating environments.

Vaccination with a live multi-gene deletion strain protects horses against virulent challenge with Streptococcus equi.

Strangles, caused by Streptococcus equi subspecies equi (S. equi) is one of the most frequently diagnosed infectious diseases of horses and there remains a significant need to develop new preventative vaccines. We generated a live vaccine strain of S. equi containing deletions in six genes: sagA, hasA, aroB, pyrC, seM and recA, which was administered to nine Welsh mountain ponies via the intramuscular route. Four vaccinated ponies developed adverse reactions following the first vaccination from which the live vaccine strain was isolated. Two of these ponies were withdrawn from the study and seven ponies received a second vaccination, one of which then developed an adverse reaction. Nine control ponies injected with culture media alone developed no adverse reactions. Following challenge with a virulent strain of S. equi, none of the seven vaccinated ponies had developed clinical signs of strangles eleven days post-challenge, compared to six of nine control ponies over the same period (P=0.0114). A lymph node abscess was identified in one of the seven vaccinated ponies at post-mortem examination, whilst all nine control ponies had at least one lymph node abscess (P=0.0009). Three of the six vaccinated ponies that were protected from strangles had not developed an adverse reaction following vaccination, suggesting that a better understanding of the pro-inflammatory responses to S. equi could lead to the development of a live attenuated vaccine against strangles that is safe for administration via intramuscular injection.

Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions.

Equine herpesvirus myeloencephalitis (EHM) remains one of the most devastating manifestations of equine herpesvirus type 1 (EHV-1) infection but our understanding of its pathogenesis remains rudimentary, partly because of a lack of adequate experimental models. EHV-1 infection of the ocular vasculature may offer an alternative model as EHV-1-induced chorioretinopathy appears to occur in a significant number of horses, and the pathogenesis of EHM and ocular EHV-1 may be similar. To investigate the potential of ocular EHV-1 as a model for EHM, and to determine the frequency of ocular EHV-1, our goal was to study: (1) Dissemination of virus following acute infection, (2) Development and frequency of ocular lesions following infection, and (3) Utility of a GFP-expressing virus for localization of the virus in vivo. Viral antigen could be detected following acute infection in ocular tissues and the central nervous system (experiment 1). Furthermore, EHV-1 infection resulted in multifocal choroidal lesions in 90% (experiment 2) and 50% (experiment 3) of experimentally infected horses, however ocular lesions did not appear in vivo until between 3 weeks and 3 months post-infection. Taken together, the timing of the appearance of lesions and their ophthalmoscopic features suggest that their pathogenesis may involve ischemic injury to the chorioretina following viremic delivery of virus to the eye, mirroring the vascular events that result in EHM. In summary, we show that the frequency of ocular EHV-1 is 50-90% following experimental infection making this model attractive for testing future vaccines or therapeutics in an immunologically relevant age group.

From glanders to Hendra virus: 125 years of equine infectious diseases.

Josh Slater looks back at the past 125 years of developments in equine infectious disease, including landmark discoveries in microbiology and genomics, and considers what the future may hold.

Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure.

The detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet - diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi.

Ensuring equine biosecurity at London 2012.

The London 2012 Olympic and Paralympic Equestrian Games were the highest profile event in the 2012 equestrian calendar and were the culmination of four years of detailed and meticulous biosecurity planning to ensure that all horses arrived, competed and returned home safely and in good health. Josh Slater, Anthony Greenleaves and Andy Paterson describe how this was achieved.

The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis.

BACKGROUND: Streptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2) are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor. RESULTS: Two mutants (G6G and M3G) possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757) that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9%) and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%), which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200), motif II (His239), and motif III (Ser568). SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly) at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p < 0.001). CONCLUSION: In summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

Rapid evolution of virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis.

BACKGROUND: Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, approximately 40% of the approximately 2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three approximately 90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. CONCLUSIONS/SIGNIFICANCE: The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.

New putative virulence factors of Streptococcus suis involved in invasion of porcine brain microvascular endothelial cells.

Streptococcus suis serotype 2 is an important pathogen causing a wide range of infections in swine, the most important being meningitis. Few virulence factors have been identified and the pathogenesis of infection is not well understood. Recently, we demonstrated the ability of S. suis to adhere to and invade porcine brain microvascular endothelial cells (PBMEC) forming the blood-brain barrier. In this paper we describe the screening of a mutant library, produced by insertion of transposon Tn917 into the chromosome of S. suis strain P1/7, for mutants that are less able to interact with PBMEC. Both qualitative and quantitative screening assays were used to identify poorly invasive mutants. Tn917 insertion sites from nineteen poorly invasive mutants were sequenced and characterized. Five mutants were selected and their virulence was assessed in a mouse model of infection. Two out of these five mutants were attenuated as measured by decreased colonization of organs, as well as reduced mortality and morbidity. When tested in swine these two attenuated mutants led to decreased bacterial loads in blood, less severe and delayed clinical signs, and lower plasma IL-6 levels than did infection with the wild-type strain. Overall, our results suggest that these two genes may contribute to the virulence of S. suis.

Mutation of the maturase lipoprotein attenuates the virulence of Streptococcus equi to a greater extent than does loss of general lipoprotein lipidation.

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (DeltaprtM(138-213), with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Deltalgt(190-685)). Moreover, mucus production was significantly greater in both wild-type-infected and Deltalgt(190-685)-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Deltalgt(190-685) mutant did still exhibit signs of disease. In contrast, only the DeltaprtM(138-213) mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Deltalgt(190-685) mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

Development of an in vivo Himar1 transposon mutagenesis system for use in Streptococcus equi subsp. equi.

Streptococcus equi subsp. equi is the causative agent of the equine disease strangles. In this study we describe the development of an in vivo Himar1 transposon system for the random mutagenesis of S. equi and, potentially, other Gram-positive bacteria. We demonstrate efficient and random transposition of a modified mini-transposon onto the chromosome by Southern blot analysis and insertion site sequencing. Non-haemolytic mutants were isolated at a frequency of 0.2%, and acapsular mutants at a frequency of 0.04%. Taken together, these data demonstrate that in vivo Himar1 mutagenesis can be used for genomic-scale mutational analysis of S. equi, and is likely to be applicable to the study of other streptococci.