Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two specie

Gender Differences in Early Reading Strategies: a Comparison of Synthetic Phonics Only with a Mixed Approach to Teaching Reading to 4-5 Year-Old Children

A survey of primary schools in England found that girls outperform boys in English across all phases (Ofsted in Moving English forward. Ofsted, Manchester, 2012). The gender gap remains an on-going issue in England, especially for reading attainment. This paper presents evidence of gender differences in learning to read that emerged during the development of a reading scheme for 4- and 5-year-old children in which 372 children from Reception classes in sixteen schools participated in 12-month trials. There were three arms per trial: Intervention non-PD (non-phonically decodable text with mixed methods teaching); Intervention PD (phonically decodable text with mixed methods teaching); and a ‘business as usual’ control condition SP (synthetic phonics and decodable text). Assignment to Intervention condition was randomised. Standardised measures of word reading and comprehension were used. The research provides statistically significant evidence suggesting that boys learn more easily using a mix of whole-word and synthetic phonics approaches. In addition, the evidence indicates that boys learn to read more easily using the natural-style language of ‘real’ books including vocabulary which goes beyond their assumed decoding ability. At post-test, boys using the nonphonically decodable text with mixed methods (Intervention A) were 8 months ahead in reading comprehension compared to boys using a wholly synthetic phonics approach

Analysis of the novel surface protein P159 and the ribosomal protein L7/L12 of mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae colonise swine ciliated respiratory epithelia, leading to the development of porcine enzootic pneumonia. Heparin and other glycosaminoglycans are known to block adherence of this pathogen to porcine tracheal cilia, however adhesins with heparin-binding capacity have not been identified. Previous studies have implicated a 97 kDa surface protein of M. hyopneumoniae, P97 that appeared to be involved in the interaction of M. hyopneumoniae with swine cilia. This study aimed at identifying other potential adhesins of M. hyopneumoniae. In this study, immuno-electron microscopy and trypsin degradation analyses demonstrated that a 159 kDa protein (P159) resides on the surface of M. hyopneumoniae. Furthermore, proteomic analyses indicate that this molecule is post-translationally cleaved. In vitro, these proteins are found to be cleaved and highly expressed at all stages during the growth cycle of M. hyopneumoniae. This molecule was also shown to be immunogenic due to its reactivity with sera from pigs naturally infected with M. hyopneumoniae. Recombinant expression of P159 domains was undertaken (F1, F2, F3, and F4). Two of these domains (F3 and F4) were found to bind heparin in a dosedependent, saturable and specific manner. The Kd for this interaction was 142.37 + 22.01 nM for F3 and 75.37 + 7.34 nM for F4. Some pathogenic bacteria have been shown to bind heparin which can then bind to further components on the surface of cells, thereby acting as a bridging molecule in adhesion to host cells. Non-labeled heparin was shown to competitively inhibit this interaction with an IC50 value of 52.92 + 1.03 μg/ml for F3 and 66.63 + 1.02 μg/ml for F4. Fucoidan was also shown to competitively inhibit the binding of heparin to F3 (IC50 96.28 + 1.19 μg/ml) and F4 (IC50 36.23 + 1.14 μg/ml). Fluorescent and electron microscopic studies employing latex beads coated with P159 domains revealed that F2, F3 and F4 promoted adherence to the porcine epithelial-like cell line PK15. Additionally, F2 and F4 also mediated the uptake of the latex beads into PK15 cells. Collectively this data suggests that P159 is a good candidate for an adhesin of M. hyopneumoniae and thus may play a role in the colonization of the respiratory tract of swine. Ribosomal protein L7/L12 has been shown to play a role in the pathogenesis of a number of bacterial pathogens in vitro, and has been found to be expressed on the cell surface of some pathogenic bacteria. While work in this study revealed that the L7/L12 molecule is cytoplasmically expressed in M.hyopneumoniae, an immunoreaction with convalescent pig serum indicated the protein has immunogenic properties. The immunogenicity of the M. hyopneumoniae L7/L12 protein may make it a potential target for vaccine development

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species