The first strain of Clostridium perfringens isolated from an avian source has an alpha-toxin with divergent structural and kinetic properties

Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.     

Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature

Toxigenic types of Clostridium perfringens are important causes of enteric disease in domestic animals, although type E is putatively rare, appearing as an uncommon cause of enterotoxemia of lambs, calves, and rabbits. We report here two geographically distinct cases of type E enterotoxemia in calves, and diagnostic findings which suggest that type E may play a significant role in enteritis of neonatal calves. The cases had many similarities, including a history of diarrhea and sudden death, abomasitis, and hemorrhagic enteritis. In both cases, anaerobic cultures of abomasum yielded heavy growth of C. perfringens genotype E. Four percent of > 1000 strains of C. perfringens from cases of enteritis in domestic animals were type E, and all (n=45) were from neonatal calves with hemorrhagic enteritis. Furthermore, type E isolates represented nearly 50% of all isolates submitted from similar clinical cases in calves. Commercial toxoids available in North America have no label claims for efficacy against type E infections. Consideration should be given to type E-associated enteritis when planning for the health care of calves.     

Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana

Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based protein profiling (ABPP) revealed that the activities of papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.     

利用红外相机技术监测道路对野生动物丰富度的影响——以观音山国家级自然保护区道路为例

2014年7月至2018年10月,利用20台红外相机监测陕西观音山国家级自然保护区一条内部道路对兽类和鸟类活动的影响.监测期间获得照片16168张,共鉴定到13种兽类和14种鸟类,包括4种国家Ⅰ级重点保护野生动物和7种国家Ⅱ级重点保护野生动物.结果 显示,距道路的距离越远,出现的物种种类越多.有2种兽类只活动于距离道路...     

Isolations of aerobic bacteria from wild desert bighorn sheep (Ovis canadensis nelsoni and O. c. mexicana) in Arizona

Nasal, pharyngeal, cervical and vaginal swab specimens were obtained from 74 desert bighorn sheep for the purpose of investigating the normal aerobic bacterial flora of wild sheep. A total of 281 isolates was obtained and identified by standard microbiologic tests. One hundred seven of these isolates were gram positive and included Bacillus sp. (36%), Staphylococcus epidermidis (8%), S. aureus (4%), Corynebacterium sp. (diphtheroids, 4%), and Streptococcus sp. (48%). gram negative isolates totaled 174 and included Neisseria sp. (18%), Citrobacter sp. (3%), Enterobacter sp. (2%), Escherichia coli (2%), Proteus sp. (2%) and non-fermentative bacilli (NFB) (73%). Of the NFB isolates, Pseudomonas sp. (25%), Acinetobacter sp. (18%), Moraxella sp. (15%) were identified.     

Autoclavable ultrahigh-efficiency filter for use with flexible film germfree animal isolators

An autoclavable ultrahigh-efficiency filter unit, which accommodated 99.98% efficient respirator filter cartridges, was built for use with flexible film germfree animal isolators.