N. Chanter

Genomic evidence for the evolution of Streptococcus equi : host restriction, increased virulence, and genetic exchange with human pathogens

The continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A2 toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci.

Occurrence of Clostridium perfringens β2-toxin amongst animals, determined using genotyping and subtyping PCR assays

Clostridium perfringens isolates are currently classified into one of five biotypes on the basis of the differential production of alpha-, beta-, epsilon- and iota-toxins. Different biotypes are associated with different diseases of man and animals. In this study a multiple PCR assay was developed to detect the genes encoding these toxins. In addition, detection of the genes encoding the C. perfringens enterotoxin and beta2-toxin allowed subtyping of the bacteria. C. perfringens isolates taken from a variety of animals, including foals, piglets or lambs, were genotyped using this assay. Most of the isolates were found to be genotype A and the gene encoding beta2-toxin [corrected] was present in 50% of the isolates genotyped. A significant association between C. perfringens possessing the beta2-toxin gene and diarrhoea in piglets was identified, suggesting that beta2-toxin may play a key role in the pathogenesis of the disease.

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, ∼40% of the ∼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 ∼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.

The molecular basis of Streptococcus equi infection and disease

Streptococcus equi is the aetiological agent of strangles, one of the most prevalent diseases of the horse. The animal suffering and economic burden associated with this disease necessitate effective treatment. Current antibiotic therapy is often ineffective and thus recent attention has focused on vaccine development. A systematic understanding of S. equi virulence, leading to the identification of targets to which protective immunity can be directed, is a prerequisite of the development of such a vaccine. Here, the virulence factors of S. equi are reviewed.

Association between Respiratory Disease and Bacterial and Viral Infections in British Racehorses

ABSTRACT Respiratory disease is important in horses, particularly in young Thoroughbred racehorses, and inflammation that is detected in the trachea and bronchi (termed inflammatory airway disease [IAD]) is more significant in this population in terms of impact and frequency than other presentations of respiratory disease. IAD, which is characterized by neutrophilic inflammation, mild clinical signs, and accumulation of mucus in the trachea, may be multifactorial, possibly involving infections and environmental and immunological factors, and its etiology remains unclear. This 3-year longitudinal study of young Thoroughbred racehorses was undertaken to characterize the associations of IAD and nasal discharge with viral and bacterial infections. IAD was statistically associated with tracheal infection with Streptococcus pneumoniae (capsule type 3), Streptococcus zooepidemicus, Actinobacillus spp., and Mycoplasma equirhinis and equine herpesvirus 1 and 4 infections, after adjustment for variation between training yards, seasons, and age groups. The association with S. pneumoniae and S. zooepidemicus was independent of prior viral infection and, critically, was dependent on the numbers of organisms isolated. S. pneumoniae was significant only in horses that were 2 years old or younger. The prevalence and incidence of IAD, S. zooepidemicus, and S. pneumoniae decreased in parallel with age, consistent with increased disease resistance, perhaps by the acquisition of immunity. The study provided evidence for S. zooepidemicus and S. pneumoniae playing an important etiological role in the pathogenesis of IAD in young horses.

Foal diarrhoea between 1991 and 1994 in the United Kingdom associated with Clostridium perfringens, rotavirus, Strongyloides westeri and Cryptosporidium spp.

A case control study of foal diarrhoea in the United Kingdom was carried out over a 3-year period. Clostridium perfringens was significantly associated with foal diarrhoea (Odds Ratio (OR) = 3.0), being isolated from 57% of 421 animals with diarrhoea but from only 27% of 223 healthy foals. Also, C. perfringens was significantly associated with fatal diarrhoea (OR = 4.5). About half of diarrhoea with a fatal outcome was attributable to this organism. The other pathogens significantly associated with diarrhoea were rotavirus (OR = 5.6), Cryptosporidium spp. (OR = 3.2) and the nematode Strongyloides westeri, which was significant only when present in large numbers (> 2000 eggs/g of faeces: OR = 6.1). Salmonella spp. (OR = 14.2) and Cryptosporidium spp. (OR = 3.0) were the only other pathogens associated with fatal illness. Overall, C. perfringens, rotavirus, and large numbers of Cryptosporidium spp. or S. westeri were isolated from 80% of foals with diarrhoea. Thermophilic Campylobacter spp., Yersinia enterocolitica, Escherichia coli and other parasites were not associated with diarrhoea. Carriage of C. perfringens, rotavirus and Cryptosporidium spp. was significantly greater in healthy foals in contact with cases of diarrhoea than in foals that were not in contact with diarrhoea (P < 0.05). There were no statistical interactions between any of the pathogens associated with diarrhoea although separate cases from one location often involved more than one pathogen.

Inflammatory airway disease, nasal discharge and respiratory infections in young British racehorses

REASONS FOR PERFORMING STUDY Respiratory disease is important in young Thoroughbred racehorses, but the variation in the rates of occurrence between different ages and training groups has not been characterised. OBJECTIVES To determine the rates of respiratory disease, particularly inflammatory airway disease (IAD), as well as evidence of infection, and their variation between age and group. METHODS Horses were examined monthly in 7 British flat training yards over a 3 year period. IAD was defined as increased mucus in the trachea with increased proportions of neutrophils in tracheal wash samples. Frequencies of disease outcomes were estimated from the data. RESULTS The prevalence of IAD was 13.8% and the incidence was 8.9 cases/100 horses/month. Rates varied with training and age groups, decreasing in older animals. The prevalence of nasal discharge (ND) was 4.1%. Rates of bacterial isolation were more common than viral infections. The incidence and prevalence of several bacterial species decreased with age. CONCLUSIONS IAD and ND were common in young racehorses, varying significantly between training groups and decreasing with age, consistent with infection playing a role in aetiology. POTENTIAL RELEVANCE The high prevalence of IAD in 2-year-old horses in Britain suggests that routine endoscopic examination may be helpful in providing early diagnosis and appropriate therapy. The transmission of bacteria and viruses within and between groups of young animals and the role of infection, stable environment and factors inherent to each horse, including their genetic make-up, in the multifactorial aetiology of the disease all merit further study.

Evidence for niche adaptation in the genome of the bovine pathogen Streptococcus uberis

BackgroundStreptococcus uberis, a Gram positive bacterial pathogen responsible for a significant proportion of bovine mastitis in commercial dairy herds, colonises multiple body sites of the cow including the gut, genital tract and mammary gland. Comparative analysis of the complete genome sequence of S. uberis strain 0140J was undertaken to help elucidate the biology of this effective bovine pathogen.ResultsThe genome revealed 1,825 predicted coding sequences (CDSs) of which 62 were identified as pseudogenes or gene fragments. Comparisons with related pyogenic streptococci identified a conserved core (40%) of orthologous CDSs. Intriguingly, S. uberis 0140J displayed a lower number of mobile genetic elements when compared with other pyogenic streptococci, however bacteriophage-derived islands and a putative genomic island were identified. Comparative genomics analysis revealed most similarity to the genomes of Streptococcus agalactiae and Streptococcus equi subsp. zooepidemicus. In contrast, streptococcal orthologs were not identified for 11% of the CDSs, indicating either unique retention of ancestral sequence, or acquisition of sequence from alternative sources. Functions including transport, catabolism, regulation and CDSs encoding cell envelope proteins were over-represented in this unique gene set; a limited array of putative virulence CDSs were identified.ConclusionS. uberis utilises nutritional flexibility derived from a diversity of metabolic options to successfully occupy a discrete ecological niche. The features observed in S. uberis are strongly suggestive of an opportunistic pathogen adapted to challenging and changing environmental parameters.

Characterization of the Lancefield group C streptococcus 16S-23S RNA gene intergenic spacer and its potential for identification and sub-specific typing

The 16S-23S RNA gene intergenic spacers of isolates of Streptococcus equi (n = 5), S. zooepidemicus (n = 5), S. equisimilis (n = 3) and S. dysgalactiae (n = 2) were sequenced and compared. There were distinct regions within the spacer, arranged in the order 1-9 for all S. equi and one S. zooepidemicus isolate and 1,2 and 4-9 for the remaining isolates. Region 4 was identical to the tRNA(ala) gene found in the 16S-23S intergenic spacers of other streptococci. Regions 1, 5, 6 and 7 had distinct variations, each conserved in different isolates. However, amongst the intergenic spacers there were different combinations of variant regions, suggesting a role for DNA recombination in their evolution. The intergenic spacer of all isolates of S. equi and one S. zooepidemicus isolate were almost identical. Primers derived from the variant sequences of regions 1 and 5 to 6 were used to group all S. zooepidemicus (n = 17) and S. equi (n = 5) into 1 of 8 types by polymerase chain reaction; three S. zooepidemicus isolates typed the same as S. equi. S. equi and S. zooepidemicus were clearly distinguishable from S. equisimilis and S. dysgalactiae which had shorter regions 5 and 6 and no region 7. Most homology for the group C sequences was found in previously published sequences for the 16S-23S intergenic spacers of S. anginosis, S. constellatus, S. intermedius, S. salivarius and S. agalactiae. A 75-90 nucleotide length shared with S. anginosus and S. intermedius in opposite orientations in the two main variants of region 6 supported the role for DNA recombination in the evolution of the spacer. The 16S-23S intergenic spacers indicate that S. zooepidemicus was the archetypal species for S. equi and that both are genetically more distant from S. equisimilis and S. dysgalactiae. The intergenic spacer can be used to identify specifically the group C streptococci and as an epidemiological marker for S. zooepidemicus.

Actinobacillus and Pasteurella species isolated from horses with lower airway disease

Seventy-three bacterial isolates from 65 horses with and without evidence of lower airway disease were identified to assess whether the association with disease was accounted for by a small or large number of species. Just over half (50.5 per cent were Actinobacillus equuli, 17.8 per cent were A suis-like, 11 per cent were Pasteurella pneumotropica, 8.2 per cent were A lignieresii, 6.8 per cent were P haemolytica and 5.5 per cent were P mairii. These results suggest that a range of Actinobacillus and Pasteurella species can be isolated from the lower airways of horses, with many of the isolates being A equuli. Among the horses examined, lower airway inflammation was significantly associated with A suis-like bacteria and A lignieresii. However, these two species constituted too small a proportion of the undifferentiated Actinobacillus/Pasteurella species to explain the association of this group of bacteria with lower airway disease. Since A equuli constituted just over 50 per cent of the group of bacteria it is possible that it too is playing a significant role in lower airway disease.