Pathogenic potential to humans of bovine Escherichia coli O26, Scotland

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin-producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx(2) in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26-associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx(2) phage acquisition.     

Causes of pneumonia epizootics among bighorn sheep, Western United States, 2008-2010

Epizootic pneumonia of bighorn sheep is a devastating disease of uncertain etiology. To help clarify the etiology, we used culture and culture-independent methods to compare the prevalence of the bacterial respiratory pathogens Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae in lung tissue from 44 bighorn sheep from herds affected by 8 outbreaks in the western United States. M. ovipneumoniae, the only agent detected at significantly higher prevalence in animals from outbreaks (95%) than in animals from unaffected healthy populations (0%), was the most consistently detected agent and the only agent that exhibited single strain types within each outbreak. The other respiratory pathogens were frequently but inconsistently detected, as were several obligate anaerobic bacterial species, all of which might represent secondary or opportunistic infections that could contribute to disease severity. These data provide evidence that M. ovipneumoniae plays a primary role in the etiology of epizootic pneumonia of bighorn sheep.     

Colour Doppler ultrasound imaging findings in paediatric periocular and orbital haemangiomas

Purpose: To evaluate the combined grey‐scale ultrasonography (US) and colour Doppler imaging (CDI) as the first and primary imaging modalities in diagnosing paediatric orbital haemangiomas. Methods: The charts of 20 consecutive children with a periorbital mass echographically diagnosed as a haemangioma between January 2004 and June 2009 in the Tel‐Aviv Sourasky Medical Center were reviewed. Data on demographic details, clinical findings, US and CDI characteristics, treatment and outcome were retrieved. Results: Twelve (60%) haemangiomas were located on the upper eyelid, five in the lower eyelid (25%) and three in the medial cantus (15%). The tumour resolved completely in 10 children (50%) and in 10 children (50%) partial resolution was documented. Seven (35%) patients underwent treatment (intralesional or oral steroids or propranolol). Grey‐scale US depicted a solid‐tissue mass with low internal echogenicity. Mean haemangioma volume was 1.33 cm³. Colour Doppler imaging demonstrated intralesional flow with a mean peak systolic velocity of 15.2 cm per second and a mean resistance index of 0.51. All US and CDI examinations were carried out on alert children and no sedation or general anaesthesia was needed. During mean follow‐up time of 23 months, no child required any additional imaging or diagnostic procedures to confirm the diagnosis. Conclusion: Combined US and CDI are suggested as the first imaging modalities in cases with a suspected diagnosis of periocular and orbital capillary haemangioma.     

Molecular Typing in Public Health Laboratories: From an Academic Indulgence to an Infection Control Imperative

Using three Austrian case studies, the variegated applications of molecular typing in today''s public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.     

羊膜腔穿刺与经皮脐血取样在重型α-地中海贫血产前诊断的对比分析

目的:探讨羊膜腔穿刺与经皮脐血取样在重型α-地中海贫血产前诊断中的手术指征特点、检出情况、诊断所需时间以及妊娠结局等方面的差异,为早诊断及早干预提供客观依据。方法:以夫妇双方诊断为α-地贫1缺失突变和孕中晚期胎儿超声异常为指征,在超声引导下行羊膜腔穿刺(72例次)或经皮脐血取样(14例次)进行α-地贫基因检测,主要检测东南亚缺失--SEA和罕见的泰国型缺失--THAI,脐血还送血红蛋白电泳检测。结果:羊水组穿刺平均孕周早于脐血组,差异有统计学意义(P<0.01);重型α-地贫羊水组17例,检出率23.61%,脐血组7例,检出率50.00%,两组差异有统计学意义(P<0.05),其中脐血组有1例为罕见的泰国缺失与东南亚缺失双重杂合子(--THAI/--SEA);母体合并症并发症发生率羊水组17.65%,脐血组为86.17%,两组差异有统计学意义(P<0.01)。两组检出的重型α-地贫胎儿均给予终止妊娠引产。结论:羊水组穿刺孕周及终止妊娠孕周明显早于脐血组,相对诊断时间早,母体发生合并症并发症少;而脐血组对重型α-地贫胎儿检出率明显高于羊水组,且脐血血红蛋白电泳可快速诊断重型α-地贫胎儿,是一个补救的措施。     

Immunity to Staphylococcus aureus secreted proteins protects rabbits from serious illnesses

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. This study investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50% of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (β-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins β-toxin and γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.     

Therapeutic vaccination with Salmonella-delivered codon-optimized outer inflammatory protein DNA vaccine enhances protection in Helicobacter pylori infected mice

Vaccination had demonstrated as an alternative way to combat Helicobacter pylori challenge. In the present study, codon-optimized outer inflammatory protein gene (oipA) for Mus species codon usage, the inclusion of optimal Kozak sequence, and modified of GC content was applied to construct a novel DNA construct. The Salmonella-delivered wild type oipA construct (SL7207/poipA) and the Salmonella-delivered codon-optimized oipA construct (SL7207/poipA-opt) were prepared and their therapeutic efficacy was evaluated in H. pylori-infected mice. The codon-optimized oipA construct (poipA-opt) expressed almost six-fold higher protein than that of wild type construct (poipA) as normalized to the β-actin expression in AGS cells. Oral therapeutic immunization with SL7207/poipA-opt significantly eliminated H. pylori colonization in the stomach; and protection was related to a robust Th1/Th2 immune response. Therefore, our results suggested that fine therapeutic efficacy was related to sufficient expression of the antigen. It is supposed that codon-optimized oipA gene improves protein expression and consequently enhances the immunogenicity of DNA vaccine, which resulted in a significant reduction of bacterial loads in H. pylori infected mice. The Salmonella-delivered codon-optimized DNA construct could be a candidate vaccine against H. pylori for the clinical application.     

RNAi suppression of rice endogenous storage proteins enhances the production of rice-based Botulinum neutrotoxin type A vaccine

Mucosal vaccines based on rice (MucoRice) offer a highly practical and cost-effective strategy for vaccinating large populations against mucosal infections. However, the limitation of low expression and yield of vaccine antigens with high molecular weight remains to be overcome. Here, we introduced RNAi technology to advance the MucoRice system by co-introducing antisense sequences specific for genes encoding endogenous rice storage proteins to minimize storage protein production and allow more space for the accumulation of vaccine antigen in rice seed. When we used RNAi suppression of a combination of major rice endogenous storage proteins, 13 kDa prolamin and glutelin A in a T-DNA vector, we could highly express a vaccine comprising the 45 kDa C-terminal half of the heavy chain of botulinum type A neurotoxin (BoHc), at an average of 100 μg per seed (MucoRice-BoHc). The MucoRice-Hc was water soluble, and was expressed in the cytoplasm but not in protein body I or II of rice seeds. Thus, our adaptation of the RNAi system improved the yield of a vaccine antigen with a high molecular weight. When the mucosal immunogenicity of the purified MucoRice-BoHc was examined, the vaccine induced protective immunity against a challenge with botulinum type A neurotoxin in mice. These findings demonstrate the efficiency and utility of the advanced MucoRice system as an innovative vaccine production system for generating highly immunogenic mucosal vaccines of high-molecular-weight antigens.     

Bovine lactoferrin digested with human gastrointestinal enzymes inhibits replication of human echovirus 5 in cell culture

Many infant formulas are enriched with lactoferrin (Lf) because of its claimed beneficial effects on health. Native bovine Lf (bLf) is known to inhibit in vitro replication of human enteroviruses, a group of pathogenic viruses that replicate in the gut as their primary infection site. On the basis of a model digestion and human gastrointestinal enzymes, we hypothesized that bLf could retain its antiviral properties against enterovirus in the gastrointestinal tract, either as an intact protein or through bioactive peptide fragments released by digestive enzymes. To test our hypothesis, bLf was digested with human gastric juice and duodenal juice in a 2-step in vitro digestion model. Two gastric pH levels and reduction conditions were used to simulate physiological conditions in adults and infants. The antiviral activity of native bLf and of the digested fractions was studied on echovirus 5 in vitro, using various assay conditions, addressing several mechanisms for replication inhibition. Both native and digested bLf fractions revealed a significant inhibitory effect, when added before or simultaneously with the virus onto the cells. Furthermore, a significant stronger sustained antiviral effect was observed when bLf was fully digested in the gastric phase with fast pH reduction to 2.5, compared with native bLf, suggesting the release of antiviral peptides from bLf during the human digestion process. In conclusion, this study demonstrates that bLf may have a role in the prevention of human gastrointestinal virus infection under physiological conditions and that food containing bLf may protect against infection in vivo.     

突触蛋白质组学分析中双向电泳技术的优化

背景：双向电泳是突触蛋白质组学分析中最流行最通用的蛋白质分离方法之一.但文献中突触蛋白双向电泳对线性固相pH梯度（immobilized pH gradients,IPG）胶条和十二烷基磺酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electroph-oresis,SDS-PAGE）浓度的选择很多,尚未见统一标准.目的：对突触蛋白双向电泳的IPG胶条和SDS-PAGE凝胶浓度进行优化,以获得高质量的突触蛋白双向电泳图谱.方法：以大鼠海马突触蛋白为试材,比较pH 5.0-8.0与pH 3.0-10.0线性IPG胶条,线性与非线性pH 3.0-10.0IPG胶条,以及单一浓度10％与12％ SDS-PAGE对双向电泳的影响.此外,还使用计算机检索了1989至2013年中国知网及PubMed数据库中关于突触蛋白双向电泳的文献,对文献中选择的IPG胶条、SDS-PAGE凝胶浓度进行了统计和评价,并总结了突触蛋白在不同pH值IPG胶条和不同浓度SDS-PAGE凝胶的双向电泳图谱上的分布特征.结果与结论：结果表明,使用pH 3.0-10.0的非线性胶条及单一浓度10％ SDS-PAGE凝胶进行突触蛋白双向电泳较为适宜,电泳图谱质量好,实验操作便利;同时还推荐合并使用pH 4.0-7.0和pH 6.0-11.0的IPG胶条,以及线性梯度浓度9％-16％ SDS-PAGE凝胶,也适用于突触蛋白双向电泳分析.