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James A Leigh

Publications and source records attributed to James A Leigh.

12 recordsLinked to original sources
PubMed2006

Multilocus-sequence typing analysis reveals similar populations of Streptococcus uberis are responsible for bovine intramammary infections of short and long duration.

Multilocus-sequence typing (MLST) was used to analyse Streptococcus uberis isolates from a single herd associated with long duration (50-260 days) and rapidly cleared (less than 1 month) bovine intramammary infections to determine whether the bacterial type had any impact on the duration of infection. Most chronic infections (24 of 33) were due to continuous infection of the mammary quarter with the same sequence type, and infections were found to persist for many months. The remaining quarters were re-infected with a different sequence type within a single lactation. No particular sequence type or clonal complex (lineage) was associated with persisting infections, indicating that the outcome of intramammary infections with S. uberis is more likely to be dependent on host factors than on inter-strain differences. Analysis of these strains alongside others obtained from the same herd at a later date revealed the shift in the predominant genotypes with time.

Alleles↗
PubMed2006

First insights into the evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology.

Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis throughout the world. Several procedures to differentiate S. uberis isolates have been proposed. However, all are prone to interlaboratory variation, and none is suitable for the description of the population structure. We describe here the development of a multilocus sequence typing (MLST) scheme for S. uberis to help address these issues. The sequences of seven housekeeping gene fragments from each of 160 United Kingdom milk isolates of S. uberis were determined. Between 5 and 17 alleles were obtained per locus, giving the potential to discriminate between 1.3 x 10(7) sequence types. In this study, 57 sequence types (STs) were identified. Statistical comparisons between the maximum-likelihood trees constructed by using the seven housekeeping gene fragments showed that the congruence was no better than that between each tree and trees of random topology, indicating there had been significant recombination within these loci. The population contained one major lineage (designated the ST-5 complex). This dominated the population, containing 24 STs and representing 112 isolates. The other 33 STs were not assigned to any clonal complex. All of the isolates in the ST-5 lineage carried hasA, a gene that is essential for capsule production. There was no clear association between ST or clonal complex and disease. The S. uberis MLST system offers researchers a valuable tool that allows further investigation of the population biology of this organism and insights into the epidemiology of this disease on a global scale.

Animals↗
PubMed2006

Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom.

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.

Alleles↗
PubMed2005

Identification of multiple linear epitopes of the plasminogen activator A (PauA) of Streptococcus uberis with murine monoclonal antibodies.

Streptococcus (S.) uberis is a common cause of mastitis in cattle. A protein (PauA) secreted by this bacterium is capable of activating plasminogen from sheep and cattle. The PauA first binds to bovine plasminogen (b-plg) to form a PauA-plasminogen complex that subsequently binds to and activates b-plg to form plasmin. We have identified several linear epitopes of PauA that are recognized by murine monoclonal antibodies to PauA. Two of the monoclonal antibodies which neutralized the enzymatic activity of PauA, EC3 and 2.22, recognized common linear peptide sequences with similar charge and spacing patterns. These neutralization epitopes are located in the predicted alpha-domain of the PauA molecule. Further, these same epitopes are in critical structure/function domains identified in other studies. These characterizations may facilitate the design of an efficacious vaccine for streptococcal mastitis in the dairy cow.

Amino Acid Sequence↗
PubMed2004

Complex interactions between bovine plasminogen and streptococcal plasminogen activator PauA.

The interactions between bovine plasminogen and the streptococcal plasminogen activator PauA that culminate in the generation of plasmin are not fully understood. Formation of an equimolar activation complex comprising PauA and plasminogen by non-proteolytic means is a prerequisite to the recruitment of substrate plasminogen; however the determinants that facilitate these interactions have yet to be defined. A mutagenesis strategy comprising nested deletions and random point substitutions indicated roles for both amino and carboxyl-terminal regions of PauA and identified further essential residues within the alpha domain of the plasminogen activator. A critical region within the alpha domain was identified using non-overlapping PauA peptides to block the interaction between PauA and bovine plasminogen, preventing formation of the activation complex. Homology modelling of the activation complex based upon the known structures of streptokinase complexed with human plasmin supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.

Amino Acid Sequence↗
PubMed2004

The exploitation of the genome in the search for determinants of virulence in Streptococcus uberis.

Despite much success in the control of mastitis in dairy cattle, intramammary infection with Streptococcus uberis remains a threat to herd health. This organism is a frequent cause of mastitis worldwide. Recent advances in the ability to genetically manipulate this bacterium, coupled to the determination of a representative genome sequence have already enabled the investigation of certain aspects of disease pathogenesis. Further use of such technology coupled to reliable models of disease and post-genomic analysis will permit the elucidation of further interactions between pathogen and host. This additional information can be usefully targeted at identification of candidates for inclusion in effective vaccines. This communication reviews the current, reported progress using this technology for S. uberis.

Animals↗
PubMed2004

Genetic analysis of Streptococcus uberis plasminogen activators.

BACKGROUND & OBJECTIVES: Streptococci produce a diverse range of secreted plasminogen activators capable of converting mammalian plasminogen to plasmin in a species-specific manner. In all examples to date, the host animal's plasminogen and that of a number of additional species have been shown to interact with these molecules leading to the conclusion that the pathogenesis of streptococci is in some way dependent upon activation of host plasminogen. PauA was the first plasminogen activator described from Streptococcus uberis, a pathogen frequently isolated from cases of bovine mastitis. Recently, a second S. uberis plasminogen activator (PauB) was identified from a Danish mastitis isolate. Interestingly, the pauB open reading frame occupied the locus normally filled by pauA. In the present study a genetic screen of streptococcal and field isolates frequently associated with mastitis was undertaken to assess the distribution, chromosomal location and sequence variation of these putative virulence factors. METHODS: Southern analysis of a diverse panel of streptococci and additional bacterial isolates frequently associated with bovine mastitis was performed using pauA and pauB probes. Sequence variation of PauA was assessed at the protein level following nucleotide sequence analysis of pauA alleles amplified from isolates picked from different geographical locations. RESULTS: We observed plasminogen activators to be universally distributed amongst S. uberis. A pauA allele was identified in all but one strain of S. uberis. This strain had a pauB allele substituted for pauA at the same locus. The remarkably low level of sequence variation demonstrated by PauA was further restricted to a limited number of residues within the molecule. INTERPRETATION & CONCLUSION: The high prevalence of PauA alleles in field isolates of S.uberis supported the observation that plasminogen activators are likely to confer an advantage with respect to colonization and growth. The findings of the present study support the theory that PauA plays a critical role in the pathogenesis of S. uberis.

Base Sequence↗
PubMed2003

The hyaluronic acid capsule of Streptococcus uberis is not required for the development of infection and clinical mastitis.

The frequency at which the genes responsible for capsule biosynthesis occurred in field isolates of Streptococcus uberis was determined. Of the two genotypes detected (hasABC and hasC), the capsular genotype (hasABC) was more common. This genotype was present at a higher frequency in a population isolated from mastitis cases than in a population isolated from cattle bedding. The virulence of a mutant strain of S. uberis (TRF0-6) that lacked the ability to produce a hyaluronic acid capsule due to an insertion within its single copy of hasA (P. N. Ward, T. R. Field, W. G. F. Ditcham, E. Maguin, and J. A. Leigh, Infect. Immun. 69:392-399, 2001) was compared to that of the capsular parental strain (0140J). Strains TRF0-6 and 0140J infected all mammary gland quarters following experimental challenge. The wild type and the mutant induced overt signs of disease in four out of four and in six out of eight mammary gland quarters, respectively. Both the wild type and the hasA mutant were resistant to killing by bovine neutrophils following cultivation in bovine milk. The ability to withstand the bactericidal action of neutrophils following growth in milk was therefore independent of the capsule and coincided with the ability of supernatants from such cultures to prevent the bactericidal action of neutrophils. This investigation revealed that, in the absence of the capsule, S. uberis is able to withstand the bactericidal effect of bovine neutrophils and induce mastitis in dairy cows.

Animals↗
PubMed2003

MtuA, a lipoprotein receptor antigen from Streptococcus uberis, is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland.

A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.

Animals↗
PubMed2002

Characterization of PauB, a novel broad-spectrum plasminogen activator from Streptococcus uberis.

A bovine plasminogen activator of atypical molecular mass ( approximately 45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072-1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.

Animals↗
PubMed2002

Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis: identification of virulent clones and potential capsular serotype exchange.

Streptococcus suis is an important pathogen of pigs and occasionally causes serious human disease. However, little is known about the S. suis population structure, the clonal relationships between strains, the potential of particular clones to cause disease, and the relevance of serotype as a marker for epidemiology. Here we describe a multilocus sequence typing (MLST) scheme for S. suis developed in order to begin to address these issues. Seven housekeeping gene fragments from each of 294 S. suis isolates obtained from various S. suis diseases and from asymptomatic carriage representing 28 serotypes and nine distinct countries of origin were sequenced. Between 32 and 46 alleles per locus were identified, giving the ability to distinguish >1.6 x 10(11) sequence types (STs). However only 92 STs were identified in this study. Of the 92 STs 18 contained multiple isolates, the most common of which, ST1, was identified on 141 occasions from six countries. Assignment of the STs to lineages resulted in 37 being identified as unique and unrelated STs while the remaining 55 were assigned to 10 complexes. ST complexes ST1, ST27, and ST87 dominate the population; while the ST1 complex was strongly associated with isolates from septicemia, meningitis, and arthritis, the ST87 and ST27 complexes were found to contain significantly higher numbers of lung isolates. In agreement with the observed distribution of disease-causing isolates of S. suis, most isolates previously characterized as of high virulence in porcine infection models belong to ST1, while isolates belonging to other STs appear to be less virulent in general. Finally nine STs were found to contain isolates of multiple serotypes, and many isolates belonging to the same serotypes were found to have very disparate genetic backgrounds. As well as highlighting that the serotype can often be a poor indicator of genetic relatedness between S. suis isolates, these findings suggest that capsular genes may be moving horizontally through the S. suis population.

Animals↗