Laura McAuliffe

Differentiation of Mycoplasma species by 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis fingerprinting.

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of a 16S ribosomal DNA PCR product was used to differentiate 32 mycoplasma species of veterinary significance. Twenty-seven (85%) species could be differentiated by DGGE. This method could enable the rapid identification of many mycoplasma species for which there is no specific PCR available and which are currently identified by using culture and serological tests.

Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England

SHEEP and goats suffer from a number of serious diseases caused by Mycoplasma species in mainland Europe, in particular contagious agalactia and pleuropneumonias, which lead to significant economic losses as a result of decreases in milk production, morbidity and, occasionally, mortality (Nicholas 2002). In contrast, only two Mycoplasma species have been shown to cause disease in small ruminants in Britain. They are Mycoplasma conjunctivae, which causes a serious but self-limiting keratoconjunctivitis (Baker and oth-

Molecular epidemiological analysis of Mycoplasma bovis isolates from the United Kingdom shows two genetically distinct clusters.

ABSTRACT Mycoplasma bovis is an important veterinary pathogen causing pneumonia, arthritis, and mastitis in infected cattle. We investigated the genetic diversity of 53 isolates collected in the United Kingdom between 1996 and 2002 with pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) analysis. In addition, the influence of variable surface protein (Vsp) profiles on the profiles generated with molecular typing techniques was studied. Both AFLP and RAPD separated the isolates into two distinct groups, but PFGE showed less congruence with the other techniques. There was no clear relationship between the geographic origin or year of isolation of the isolates and the profiles produced. No correlation between Vsp profiles and any of the molecular typing techniques was observed. We propose that RAPD and AFLP provide valuable tools for molecular typing of M. bovis.

RNase P RNA Gene (rnpB) Phylogeny of Hemoplasmas and Other Mycoplasma Species

ABSTRACT Partial sequences of the RNase P RNA gene (rnpB) were obtained from a number of hemoplasmas and other Mycoplasma species. Phylogenetic analysis of these sequences showed that all hemoplasmas were present within a single clade and were most closely related to Mycoplasma fastidiosum, similar to the results found with 16S rRNA gene phylogeny.

Vaccines for Mycoplasma Diseases in Animals and Man

Vaccines for important mycoplasma diseases, including contagious bovine and caprine pleuropneumonia, have been used for centuries, consisting mainly of infected tissue or fluids which are inoculated into sites at which the risk of severe infection is slight, such as the tail and bridge of the nose. Surprisingly, little progress has been made in developing safe, defined and protective alternatives, the vaccines today still consisting of mildly attenuated strains serially passaged in eggs or in culture. Ill-defined temperature-sensitive mutants are widely used for mycoplasmoses in poultry despite uncertainty about their mode of protection. Inactivated vaccines for enzootic pneumonia appear to have improved pig health worldwide, but disease reduction has been generally modest. Ironically, attempts to develop subunit preparations have often led to exacerbation of disease, particularly in human atypical pneumonia. Promising results have been seen in DNA vaccine technology, which has been applied to the development of mycoplasma vaccines for porcine enzootic pneumonia, but field trials still seem a long way off. No commercial vaccines exist for Mycoplasma bovis, despite evidence that this is a major cause of calf pneumonia, mastitis and arthritis.

Multilocus sequence typing of Mycoplasma agalactiae.

Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

Strain typing of Mycoplasma cynos isolates from dogs with respiratory disease

Abstract The association of Mycoplasma cynos with canine infectious respiratory disease is increasingly being recognised. This study describes the strain typing of 14 M. cynos isolates cultured from trachea and bronchoalveolar lavage samples of six dogs with respiratory disease, from two separate kennels in the United Kingdom. The genetic similarity of the isolates was investigated using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Most of the isolates from four dogs housed at a re-homing kennel were genetically similar and some isolates from different dogs were indistinguishable by both PFGE and RAPD. These isolates were cultured from dogs with non-overlapping stays in the kennel, which may indicate maintenance of some strains within kennels. A small number of isolates showed much greater genetic heterogeneity and were genetically distinct from the main group of M. cynos strains. There was also a high degree of similarity of the M. cynos type strain (isolated from a dog with respiratory disease in Denmark in 1971) to at least one of the United Kingdom isolates using PFGE analysis, which may suggest possible conservation of pathogenic strains of M. cynos.

Biofilm-grown Mycoplasma mycoides subsp. mycoides SC exhibit both phenotypic and genotypic variation compared with planktonic cells

Biofilm formation where bacterial cells adhere to a surface and surround themselves in a polysaccharide matrix is thought to be an important factor in disease initiation and persistence for many bacterial species. We have examined biofilm formation by Mycoplasma mycoides subsp. mycoides small colony using a simple model without an air/liquid interface and have found that adherent Mmm SC was more resistant to many stresses, including heat, osmotic shock and oxidative stress. Biofilms of Mmm SC also exhibited remarkable persistence and were able to survive for up to 20 weeks in stationary phase. Significant variation was seen between Mmm SC strains in their ability to form a biofilm and the morphology of the biofilm produced with some strains unable to produce microcolonies. Proteomic analysis found that a number of proteins linked to adherence were over-expressed in biofilms compared with planktonic cells.

Protein-specific analysis of humoral immune responses in a clinical trial for vaccines against contagious bovine pleuropneumonia.

ABSTRACT Specific humoral immune responses in a clinical trial on cattle for vaccines against contagious bovine pleuropneumonia (CBPP) were investigated. The trial included a subunit vaccine consisting of five recombinant putative variable surface proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) compared to the currently approved attenuated vaccine strain T1/44 and untreated controls. Humoral immune responses to 65 individual recombinant surface proteins of M. mycoides SC were monitored by a recently developed bead-based array assay. Responses to the subunit vaccine components were found to be weak. Animals vaccinated with this vaccine were not protected and had CBPP lesions similar to those of the untreated controls. In correlating protein-specific humoral responses to T1/44-induced immunity, five proteins associated with a protective immune response were identified by statistical evaluation, namely, MSC_1046 (LppQ), MSC_0271, MSC_0136, MSC_0079, and MSC_0431. These five proteins may be important candidates in the development of a novel subunit vaccine against CBPP.

UK epidemic strains of meticillin-resistant Staphylococcus aureus in clinical samples from Malta

Since 1999, the European Antimicrobial Resistance Surveillance System (EARSS) has monitored the rise in infection due to a number of organisms, including meticillin-resistant Staphylococcus aureus (MRSA). The EARSS reported that MRSA infections within intensive care units account for 25-50 % of infections in many central and southern European countries, these included France, Spain, Great Britain, Malta, Greece and Italy. Each country has defined epidemic MRSA (EMRSA) strains; however, the method of spread of these strains from one country to another is unknown. In this current study, DNA profiles of 473 isolates of MRSA collected from the UK and Malta were determined by PFGE. Analysis of the data showed that two countries separated by a large geographical distance had a similar DNA profile pattern. Additionally it was demonstrated that strains of EMRSA normally found in the UK were also found in the Maltese cohort (EMRSA 15 and 16). A distinct DNA profile was found in the Maltese cohort, which may be a local EMRSA, and accounted for 14.4 % of all Maltese isolates. The appearance of the same MRSA and EMRSA profiles in two separate countries suggests that MRSA can be transferred out of their country of origin and potentially establish in a new locality or country.