Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens

A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl--d-glucosaminide, isolated and shown to encode an endo--N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.     

Effects of fungicides applied to seed potato tubers at harvest or after grading on fungal storage diseases and plant development

An assessment was made over 3 years of the efficacy of thiabendazole, 2–aminobutane and a formulation of a mixture of thiabendazole and 2–aminobutane in controlling gangrene, skin spot and dry rot when applied either at harvest or after grading at different dates during the storage season.
After harvest applications, the incidence of gangrene was least on tubers treated with the mixture and generally greatest with thiabendazole. When applied after grading the efficacy of the fungicides was not affected by date of grading. Although 2–aminobutane applications at harvest or after grading were equally effective in controlling gangrene, thiabendazole and the mixture were less effective after grading where gangrene was prevalent in a stock. Thiabendazole and the mixture gave good control of dry rot when applied at harvest but were less effective when applied after grading, particularly late in the storage season. Dry rot was sometimes more prevalent on tubers fumigated with 2–aminobutane, especially those treated after grading. Skin spot was more effectively controlled by the mixture and 2–aminobutane than by thiabendazole but, with applications after grading, the efficacy of all fungicides declined the later the date of grading.
Plants grown from tubers treated with the mixture or its 2–aminobutane glycollate constituent exhibited characteristics associated with genetic variation.     

Contamination of seed potato tubers grown from stem cutting or microplant-derived tubers by Phoma foveata and Polyscytalum pustulans in Scotland: the influence of site and fungicide treatment at harvest

A survey was made over 5 years of the incidence of contamination by Phoma foveata and Polyscytalum pustulans on 3-year-old virus-tested stem cutting (VTSC) stocks assessed on three occasions during the storage season, and on their daughter tubers at five farms. The contamination occurring on plants grown in small plots from stem-cutting/microplant tubers was also assessed at the farms.
Fungicide treatment soon after harvest reduced the incidence of both pathogens, especially P. pustulans . Tuber contamination was similar for samples collected after fungicide treatment and before planting. The incidence of contamination on daughter tubers, relative to that on the untreated mother tubers, was less with P. foveata but was similar or greater with P. pustulans . Contamination by both pathogens varied between farms and was reflected in the degree of contamination occurring on daughter tubers from stem-cutting/microplant tubers.
The incidence of P. foveata on stems, soil and tubers of seed stocks classified at Foundation Stock grade from various regions of Scotland was assessed over 2 years. The incidence of tuber contamination was correlated ( P < 0.05) with the amount of tuber-borne soil contamination for all comparisons, but was correlated with stem inoculum in only 7 out of 13 comparisons. The largest amounts of stem inoculum of P. foveata were found in stocks in Aberdeenshire.
These results are discussed in relation to the spread of both pathogens to healthy VTSC stocks.     

坏疽病原体快速检测方法的临床应用

目的:利用荧光定量PCR技术,建立一种快速、敏感、特异的检测产气荚膜梭菌的方法,及时用于指导临床治疗。方法:以产气荚膜梭菌16srRNA基因作为靶序列,设计一对特异引物和探针,以伤口分泌物和脓液提取的核酸作为模板,利用已经优化的引物和探针进行PCR反应;同时与细菌培养作比较,验证此方法的快速性、敏感性及特异性。结果:建立的反应体系在上游引物浓度为0.45μmol/L,下游引物浓度为0.15μmol/L,探针浓度为0.3μmol/L时,具有很好的敏感性,与21种其他细菌均无交叉反应,其敏感性为9cfu/反应体系。荧光定量PCR检测结果与细菌培养结果完全一致。结论:所建立的荧光定量PCR方法特异、灵敏、快速,能对产气荚膜梭菌感染做出准确的检测报告,具有对战时高发疾病气性坏疽进行快速和定量检测潜质。     

A quantitative electrochemiluminescence assay for Clostridium perfringens alpha toxin

Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.     

Genome mapping ofClostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,, , and, in addition to the enterotoxin and-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins, and, were missing.     

四妙勇安汤加减治疗下肢脱疽的临床观察

目的总结中西医结合治疗下肢脱疽病的临床经验。方法应用四妙勇安汤加减内服,红萝卜加马齿苋水煎浸泡外洗患处;蚕食法清除坏死组织,外敷呋喃西林纱布保护伤口的综合治疗方法。结果36例中治愈27例,好转6例,无效3例。结论以四妙勇安汤加减内服为主的综合疗法是一种有效治疗方法,值得推广应用。     

Comparison of two surgical techniques for mastectomy of goats

Two different techniques for mastectomy were carried out on 14 goats with gangrenous mastitis. The animals were randomly assigned to one of two groups containing seven goats each. The first group was operated via a classical surgical mastectomy technique (either bilateral (n=5) or unilateral (n=2)). The second group was operated via vascular ligation of the external pudendal blood vessels and milk vein and amputation of the affected teat (either bilateral (n=3) or unilateral (n=4)). Comparison between the two groups was carried out. Vascular ligation and teat amputation proved to be an effective, quick, safe, and less expensive technique for mastectomy in goats. Ligation of udder vasculature was less traumatic than surgical amputation and the stress on the patient was minimal.