大通县城奶牛乳样中葡萄球菌的分离与鉴定

通过细菌分离培养法对大通县城奶牛乳汁样品36份进行金黄色葡萄球菌的分离，并采用金黄色葡萄球菌常规鉴定方法血浆凝固酶试验、甘露醇发酵试验和接触酶试验进行了鉴定，36份乳样中检出金黄色葡萄球菌阳性7份，阳性率为19．7％(7／36)。     

Study on Expression and Preliminary Immunocompetence of CIC Protein Containing Molecular Adjuvants

To express CTB-IsdBid-Clfais(CIC) protein and evaluate its immunogenicity, CTB (as a molecular adjuvant) could be tandem linked with IsdBid-Clfais gene by the overlapping PCR method, then CTB-IsdBid-Clfais(CIC) was inserted into pET-32a(+) vector to construct recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais. The recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais were transformed into Escherichia coli (E.coli) BL21 to express the CTB-IsdBid-Clfa(CIC) protein, the CIC expression protein and its immune activity were detected by Western blotting and ELISA,respectively. The results showed that the length of CIC gene were 2 072 bp, and CIC was correctly inserted into the pET-32a(+) plasmids, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed. Western blotting result confirmed that the molecular weight of CIC proteins was 95.9 ku, which were correctly expressed by E.coli BL21 with pET-32a (+)-CTB-IsdBid-Clfais plasmids. ELISA results showed that there was no significant difference among the CIC, IsdBid and Clfais protein groups (P>0.05), and there was extremely significant difference between CIC and BSA protein groups (P<0.01). In conclusion, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed, CIC proteins were correctly expressed, and were able to react with serum from mice immunized with IsdBid and Clfais,respectively,therefore, CIC proteins had strong immune activity.     

金黄色葡菌球菌脂蛋白对M1型小鼠骨髓源巨噬细胞免疫作用的影响

本研究旨在阐明金黄色葡萄球菌脂蛋白对M1型小鼠骨髓源巨噬细胞免疫作用的影响，为金黄色葡萄球菌致病性研究提供理论参考。以金黄色葡萄球菌野生株SA113（WT SA113）和SA113 lgt：：ermB脂蛋白表达缺失菌株（SA113Δlgt株）体外感染M1型小鼠骨髓源巨噬细胞，分为3组：空白对照组、WT SA113感染组（MOI：3：1）、SA113Δlgt感染组（MOI：3：1）。采用ELISA法检测M1型小鼠骨髓源巨噬细胞中肿瘤坏死因子-α（TNF-α）、白细胞介素1β（IL-1β）、趋化因子（RANTES）和白细胞介素10（IL-10）的水平，实时荧光定量PCR检测Toll样受体2（TLR2）、Toll样受体4（TLR4）和含NLR家族Pyrin域蛋白3（NLRP3）基因的表达，免疫荧光法检测脂蛋白对M1型小鼠骨髓源巨噬细胞吞噬金黄色葡萄球菌作用的影响。结果显示，与空白对照组相比，WT SA113感染组和SA113Δlgt感染组均可显著上调M1型小鼠骨髓源巨噬细胞中TNF-α、RANTES、IL-10分泌量以及WT SA113感染组TLR2、NLRP3基因表达水平（P<0.05），而TLR4基因表达量显著降低（P<0.05）；与WT SA113感染组相比，SA113Δlgt感染组M1型小鼠骨髓源巨噬细胞中TNF-α、IL-1β、RANTES、IL-10分泌量以及TLR2（12 h除外）、NLRP3基因表达水平显著降低（P<0.05）。免疫荧光结果显示，M1型巨噬细胞对SA113Δlgt株的吞噬作用显著低于对WT SA113株的吞噬作用（P<0.05）。综上，金黄色葡萄球菌的脂蛋白在M1型小鼠骨髓源巨噬细胞中主要通过激活TLR2和NLRP3受体，诱导细胞因子TNF-α、IL-1β、RANTES和IL-10的产生和释放。     

Prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma

Currently, antimicrobial-resistant staphylococci, particularly methicillin-resistant Staphylococcus pseudintermedius (MRSP), are frequently isolated from canine superficial pyoderma in Japan. However, little is known regarding the nasal prevalence of MRSP in pet dogs. Here, we determined the prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma. Of the 125 nares and 108 affected sites of pet dogs with superficial pyoderma, 107 (13 species) and 110 (eight species) staphylococci strains, respectively, were isolated. The isolation rate of S. pseudintermedius from pyoderma sites (82/110 strains, 74.5%) was significantly higher than that from nares (57/107 strains, 53.3%) (P<0.01). Notably, the prevalence of MRSP (18/57 strains, 31.6%) in nares was equivalent to that in pyoderma sites (28/82 strains, 34.1%). Furthermore, the phenotypes and genotypes of antimicrobial resistance in MRSP strains from nares were similar to those from pyoderma sites. Our findings revealed that the prevalence of antimicrobial-resistant staphylococci in the nares of pet dogs with superficial pyoderma is the same level as that in affected sites. Therefore, considerable attention should be paid to the antimicrobial resistance of commensal staphylococci in companion animals.     

新疆某牧场乳房炎奶样的细菌分离鉴定与药物敏感试验

目前,乳房炎仍然是奶牛养殖过程中最常见和最严重的疾病之一,每年给我国奶牛养殖业造成巨大的损失。为了解新疆某规模化牧场奶牛乳房炎主要致病菌,通过实验鉴定和分离其场内乳房炎奶样的致病菌,并进行药物敏感试验,以期为奶牛乳房炎临床治疗以及预防提供有效指导意见。试验结果分离到大肠杆菌、链球菌、金黄色葡萄球菌,表明该牧场乳房炎是由多种致病菌引起的。药敏试验结果显示,头孢吡肟、头孢曲松对上述致病菌抑制效果最佳。     

新疆部分地区生乳中金黄色葡萄球菌的分离鉴定及耐药性分析

【目的】探明新疆部分地区生乳中金黄色葡萄球菌流行情况及耐药性,为该地区生乳中金黄色葡萄球菌的监测及保障生乳质量安全提供理论依据。【方法】试验随机从南北疆规模化奶牛场采集110份生乳样品。采用增菌培养、分离纯化、革兰氏染色、生化鉴定及PCR法对奶样中金黄色葡萄球菌进行分离鉴定,并利用微量肉汤稀释法及PCR方法对金黄色葡萄球菌进行16种抗菌药物的耐药表型及耐药基因分析。【结果】从110份奶样中分离出18株金黄色葡萄球菌,分离株呈浅黄色、光滑凸起的圆形菌落,革兰氏染色镜检呈紫色、短链状排列的革兰氏阳性菌,生化试验结果符合金黄色葡萄球菌生化特点,经16S rDNA及nuc基因PCR扩增鉴定为金黄色葡萄球菌,分离率为16.36%(18/110)。药敏试验结果显示,金黄色葡萄球菌分离株对氨苄西林、青霉素及磺胺异噁唑具有较高的耐药性,耐药率分别为100%、83.33%和77.78%,而对万古霉素、复方新诺明、苯唑西林、头孢噻吩、头孢噻呋、利福昔明及环丙沙星7种抗菌药物高度敏感,敏感率分别为100%、100%、94.44%、94.44%、94.44%、94.44%和94.44%。其中有14株金黄色葡萄球菌为多重耐药菌,多重耐药率为77.78%。耐药基因检测结果显示,大环内酯类耐药基因ermB检出率最高,为50.00%,其次为β-内酰胺类耐药基因mecA,检出率为27.28%,磺胺类耐药基因Sul1的检出率为22.22%。【结论】新疆奶牛场生乳中分离的金黄色葡萄球菌的流行及耐药情况仍较严重,且存在多重耐药现象,因此,对生乳中金黄色葡萄球菌耐药性的监测有重要意义。     

猪源ST9型耐甲氧西林金黄色葡萄球菌中前噬菌体的流行状况与转导分析

本研究旨在分析猪源ST9型耐甲氧西林金黄色葡萄球菌(Staphylococcus aureus,MRSA)中前噬菌体的流行情况、结构特点和转导能力,探究前噬菌体在猪源MRSA流行克隆形成中的作用。基于全基因组信息,分析了近年来从我国多省分离的131株ST9型MRSA中前噬菌体的流行率、分型、亲缘关系和结构特征;选取含不同分型前噬菌体的菌株进行诱导,对诱导获得的噬菌体颗粒进行转导,测定转导子的耐药表型及体外适应性。研究结果显示：猪源ST9型MRSA的前噬菌体携带率为78.6%(103/131株),其中,63株携带完整前噬菌体序列,所有前噬菌体序列均不含耐药基因,仅2.9%(3/103株)的前噬菌体序列含毒力基因;前噬菌体谱型丰富,其整合酶分型主要为Sa2int和Sa4int;各型别前噬菌体结构同源性较高,完整前噬菌体可被诱导为长尾噬菌体;噬菌体颗粒可包装供体菌的aadD、tet(L)耐药基因并转导至受体菌中;转导子可获得卡那霉素、四环素耐药表型,体外生长能力与受体菌株无明显差异(P>0.05)。研究结果表明：猪源ST9型MRSA的前噬菌体携带率较高,谱型丰富,不携带耐药基因,部分噬菌体可包装供体菌的耐药基因转导至受体菌,产生的适应性代价小。     

Characterization of a pothos (Scindapsus aureus) virus with unusual properties

A virus for which the name of pothos latent virus (PoLV) is proposed, was isolated by inoculation of sap from symptomless plants ofScindapsus aureus. PoLV had isometric particlesc. 30 nm in diameter, a monopartite genome consisting of a non polyadenylated, single-stranded RNA moleculec. 4,300 nucleotides in length, constitutingc. 17% of the particle weight, and a single type of coat protein subunit with aM _r ofc. 40,000 Daltons. The biological properties (host range reactions) of PoLV resembled those ofTombusviridae for it infected most of the artificial hosts locally, inducing symptoms recalling those elicited by several species of the above family. Like tombus- and carmoviruses, PoLV had two subgenomic RNAs which, however, differed in size from those of both genera. The dsRNA pattern was also distinctly different. Cytopathological features recalled those of tombusviruses except for the lack of multivesicular inclusion bodies. PoLV was serologically related to, but distinct from twoCarmovirus (i.e., galinsoga mosaic and Ahlum waterborne viruses) and threeTombusvirus species (i.e. eggplant mottled crinkle, Sikte waterborne and Lato river viruses). Thus, PoLV had properties somewhat intermediate between those ofTombusvirus andCarmovirus genera but bridged the two taxa through the serological relationship with some of their species. The taxonomic position of PoLV is still undetermined. It must await the results of molecular investigations now underway.     

奶牛乳房炎金黄色葡萄球菌疫苗研究进展

奶牛乳房炎是影响乳品工业经济效益的重要疾病之一,引起奶牛乳房炎的致病菌约有150多种,但金黄色葡萄球菌是引起奶牛乳房炎的主要病原。由它引起的乳房炎很难治愈,而且对抗生素产生耐药性的菌株已经相当普遍,因此使用预防或治疗性疫苗防治乳房炎就成为首选。然而金黄色葡萄球菌的致病力由许多不同的因子(如黏附素、荚膜等)决定,这使得制备非常有效的疫苗变得很困难。本文就这些致病因子在疫苗研制方面的进展做一简要介绍。     

A linear programming assessment of the profit from strategies to reduce the prevalence of Staphylococcus aureus mastitis

We used a linear programming model to estimate the financial returns to a Staphylococcus aureus testing and control program over a 1-year period for a 100-cow herd, with a 8636-kg rolling-herd average. Six tests, which vary in sensitivity from 0.80 to 0.98 and specificity of 0.99, were examined in simulated herds with 10, 20 and 30% prevalence of S. aureus infection. Sensitivity of these results to a range of assumptions regarding rolling-herd average, milk price, somatic cell-count premium, and cost and cure rate of dry treatment were examined to determine the profits from the program. The profits of a control program are most dependent upon prevalence, cell-count premium, and cost of dry treatment. In our simulation for a 100-cow herd, a testing and control program appears to cost less than US$10 per cow per year, and pays for itself within 1 yr, except under the lowest prevalence and most-adverse conditions (low yield, high cost of dry treatment, or low SCC premium).