IVF-20培养液用于大鼠体外受精的实验研究

目的调查分析小鼠嗜肺巴氏杆菌感染情况。方法对某实验动物设施的空气、排风口、门把手、小鼠咽、饮水、饲料、垫料、粪便采样,进行嗜肺巴氏杆菌检测。结果所采集的201份样品,经细菌分离培养和菌落形态、革兰氏染色、生化试验、血清玻片凝集试验的进一步鉴定,从小鼠咽、饮水、饲料中检出了嗜肺巴氏杆菌。结论该设施饲养的小鼠携带嗜肺巴氏杆菌,饮水和饲料也有嗜肺巴氏杆菌污染,该污染可能与动物饲养密度过高有关。     

嗜肺巴斯德杆菌选择性培养基研制及应用

目的　研制一种对嗜肺巴斯德杆菌表现出强选择作用的选择性培养基,用于该菌的常规检测。方法　药敏试验及抗生素最小抑菌浓度测定。结果　研制了嗜肺巴氏杆菌选择性培养基(PPSM培养基)及嗜肺巴斯德杆菌增菌液(PP肉汤)。嗜肺巴斯德杆菌在PPSM培养基上,37℃48h培养,形成1mm左右,凸起、湿润、灰黑色并有金属光泽的特殊菌落;对表皮葡萄球菌和大肠埃氏菌的抑制率为100%,对变形杆菌的抑制率为76%,并能抑制其迁徙生长;通过PP肉汤增菌培养,PPSM培养基使SPF小鼠粪便中嗜肺巴斯德杆菌检出率从0增至67.2%;用小鼠咽拭子接种该培养基,其初代培养物几乎为纯培养物。结论　该培养基对嗜肺巴氏杆菌具有较强的选择作用,使用该培养基对嗜肺巴氏杆菌进行检测可以简化检测程序、防止漏检、在不处死动物的情况下对嗜肺巴斯德杆菌进行常规监测。     

嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性研究

目的观察不同来源的嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性.方法取源于野鼠、实验大鼠和小鼠的嗜肺巴氏杆菌3株,对30只受试大鼠和小鼠进行交叉人工感染,并于感染后不同时期取咽拭子分离培养,对感染前后菌株,应用RAPD-PCR、SDS-PAGE和Western blot进行基因型、蛋白和抗原成份比较,以及生物学特性的比较.结果受试实验动物对3株嗜肺巴氏杆菌均易感,被接种的动物能稳定携带嗜肺巴氏杆菌直到试验结束,重新分离的嗜肺巴氏杆菌在生物学特性、蛋白成份、抗原性和基因型方面无明显改变.结论同一株嗜肺巴氏杆菌能在实验大鼠和小鼠中相互传染.     

嗜肺巴氏杆菌L型的诱导及其流行病学意义初探

目的 探讨L型嗜肺巴氏杆菌在诊断学及流行病学上的意义。方法 青霉素液体法诱导，并对嗜肺巴氏杆菌L型的生物学特性进行系统观察。结果 嗜肺巴氏杆菌L型具有典型的“煎蛋状”（L型）和“丝状”（F 型）菌落，扫描电镜观察，L型菌体呈球状、杆状、长丝状；革兰染色呈阴性、缠绕的长丝体，并具有圆球体及巨型体，细胞壁染色显示细胞壁缺失；对紫外线、新洁尔灭抵抗力较原菌增强5~10倍；自然干燥环境中，原菌2 d死亡，而L型可存活12 d；能透过0.45μm滤膜；回复的最初几代，其菌体形态较原菌大数倍。结论 L型嗜肺巴氏杆菌在形态学方面有较大变化，对理化因素及外界环境抵抗力增强，有可能透过胎盘屏障垂直传播。本研究提示L型嗜肺巴氏杆菌在该菌诊断学及流行病学上有重要意义。     

嗜肺巴氏杆菌靛基质试验方法探讨

在生长繁殖过程中产生靛基质是嗜肺巴氏杆菌重要的生物学特性之一,但在常用的手册及标准方法中,仅介绍了常规的靛基质试验方法,按此方法一般不易检测出嗜肺巴氏杆菌产生的较微量的靛基质。为此,我们就嗜肺巴氏杆菌靛基质试验时细菌的接种量,结果观察时靛基质萃取物二甲苯的使用两方面进行了探索,报告如下。     

北京地区实验动物中嗜肺巴斯德杆菌的表型分析

目的对北京地区实验动物中的嗜肺巴斯德杆菌分离株进行表型分析,提高检测的准确性。方法通过生化鉴定和16S rDNA测序,对306株嗜肺巴斯德杆菌可疑菌株进行鉴定。结合分离株在血琼脂平皿上的菌落特征和生化特性,组成53个特征数据谱,进行表型特征的聚类分析。结果 306株分离株中,BD自动细菌鉴定系统和16S rDNA测序鉴定出嗜肺巴斯德杆菌的阳性率分别为164/306和227/306。227株嗜肺巴斯德杆菌真阳性的表型特征共有140种,Heyl型106种,Jawetz型23种。结论北京地区实验动物中嗜肺巴斯德杆菌主要为Heyl型感染,Jawetz型较少,表型特征多样,分布广泛。     

中国灰仓鼠气管和回盲部细菌分离鉴定及药敏试验

目的 对新疆灰仓鼠气管和回盲部携带的细菌种群分布情况及耐药性进行调查分析.方法 对新疆灰仓鼠采样进行细菌学分离和生化鉴定,并选择主要细菌进行药敏试验.结果 共分离到22株细菌.结果 表明,中国灰仓鼠气管和回盲部携带的细菌种群主要有埃希菌属(大肠埃希氏菌)、假单胞菌属(铜绿假单胞菌)、葡萄球菌属(松鼠葡萄球菌)、巴斯德氏菌属(嗜肺巴斯德氏菌、产气巴斯德氏菌、多杀巴斯德氏菌)和芽孢杆菌属(短小芽孢杆菌、蜂房芽孢杆菌、地衣芽孢杆菌、蜡样芽孢杆菌)等,这些菌株对四环素、左氧沙星等药物有很高的敏感性,而对氨苄西林、青霉素G等药物不敏感.结论 中国灰仓鼠携带的细菌种群具有多样性,研究结果为中国灰仓鼠的细菌学检测以及质量标准的制定提供了科学依据.     

嗜肺巴氏杆菌外膜蛋白和脂多糖抗原在血清学诊断中的意义

目的评价嗜肺巴氏杆菌外膜蛋白(OMP)和脂多糖(LPs)作为血清学诊断抗原的敏感性和特异性.方法用OMP、LPS和全菌(WC)作为Western blot和ELISA的诊断抗原检测自然感染和实验感染嗜肺巴氏杆菌小鼠相应的IgG抗体滴度,同时测定3种抗原与实验动物常见致病菌的交叉反应.结果与嗜肺巴氏杆菌自然感染和实验感染小鼠血清的ELISA反应中,不同时期,LPS作为诊断抗原时血清抗体阳性率最高,WC次之,OMP最低.自然感染小鼠群中,出生4周LPS抗体阳性率即可达80%,而同期的WC和OMP仅为25%和20%,故LPS敏感性最高.与实验动物常见致病菌免疫血清和阴性种鼠血清的ELISA反应中,WC抗原表现出较高的吸光度(A)值,经Western blot证实,其反应为非特异性反应,LPS抗原特异性最强,OMP抗原次之.结论混合多株具有型或种特异性的OMP或LPS作为ELISA的诊断抗原,无论从特异性和敏感性上均高于全菌抗原.     

小鼠嗜肺巴氏杆菌病诊断及带菌状况调查

本文对一起罕见的清洁级昆明小鼠群中散发的嗜肺巴氏杆菌病进行了临床病理观察和病原学诊断，并对该动物群进行了带菌状况调查。该鼠群中嗜肺巴氏杆菌病发病率为3．8％，发病小鼠全部为三同龄左右．全身状况良好，仅表现出结膜炎，剖检除眼眶有米粒大脓肿外全身各脏器均未见异常；从脓肿中仅分离到嗜肺巴氏杆菌，本次流行株病原菌生物学特性显示部分菌株可有溶血性，吲哚试验阴性；其带菌率为：种鼠80％、5～6周龄90％，3～4同龄100％，提示：幼龄鼠对该菌的易感性高于成年鼠。     

嗜肺巴氏杆菌外膜蛋白和脂多糖提取及其活性的初步研究

目的　提取嗜肺巴氏杆菌外膜蛋白 (OMP)和脂多糖 (LPS) ,测定其稳定性和抗原性 ,探讨其作为诊断抗原的可能性。方法　选取具有代表性的嗜肺巴氏杆菌 15株及参考株共 16株 ,提取OMP、LPS ,通过SDS -PAGE、Westernblot、ELISA等方法在稳定性、抗原性等方面与全菌抗原 (WC)进行比较。结果　 16株嗜肺巴氏杆菌OMP的SDS-PAGE图谱基本一致 ,不同培养基成分、培养时间和不同体外传代次数的提取物均显示 5条带 ,相对分子质量大约是 (17、2 6、31、37、4 1)× 10 3。其中 5株嗜肺巴氏杆菌的LPS为光滑型和粗糙型LPS的混合物 ,主带相对分子质量在 14 .4× 10 3左右。外膜蛋白和脂寡糖提取物保持了其抗原反应性和免疫原性。OMP和LPS具有种和型的特异性。OMP的 5条条带抗原性存在差异 ,相对分子质量 (2 6、37)× 10 3的蛋白不具有抗原性 ,相对分子质量 17× 10 3的蛋白抗原为受试菌株的共同抗原 ,(31、4 1)× 10 3抗原具有大鼠株和小鼠株的特异性。LPS表现出较强的型的特异性。结论　本研究提取的OMP和LPS活性稳定 ,并保持了抗原性和免疫原性。     

Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odisha,India

Objective

To screen and isolate an eco-friendly, a thermophilic and potent L-asparaginase producing bacterium, with novel immunological properties that may obviates hypersensitivity reactions.

Methods

In the present study bacterial strain isolated for extracellular L-asparaginase production from hotspring, identified by morphological, biochemical and physiological tests followed by 16S rDNA technology and the L-asparaginase production ability was tested by both semi quantitative and quantitative enzymatic assay.

Result

The bacterial strain was identified as Bacillus subtilis strain hswx88 (GenBank Accession Number: {"type":"entrez-nucleotide","attrs":{"text":"JQ237656.1","term_id":"374341559","term_text":"JQ237656.1"}}JQ237656.1). The extracellular enzyme yielding capacity isolate Bacillus subtilis strain hswx88 (23.8 IU/mL) was found to be 1.7 and 14.5 times higher than the reference organism Pectobacterium carotovorum MTCC 1428 (14.2 IU/mL) and Bacillus sp. BCCS 034 (1.64 IU/mL).

Conclusion

The isolate is eco-friendly and useful to produce bulk quantity of extracellular, thermophilic L-asparaginase for the treatment of various tumor cases and for preparation of acrylamide free fry food preparation.     

Molecular analysis of Pasteurella multocida strains isolated from fowl cholera infection in backyard chickens

Objective

To characterize Pasteurella isolated from backyard chickens using whole cell protein lysate profiles and random amplified polymorphic DNA (RAPD) techniques to show their genetic relationship because Pasteurella multocida (P. multocida) is an important cause of fatal infections in backyard chickens.

Methods

Twenty one P. multocida isolates were recovered previously from clinical cases of fowl cholera belonging to individual owners and phenotypically analyzed using biochemical tests and serotyping were used for the genetic characterization.

Results

Phylogenetic study based on both methods revealed that the recovered population of P. multocida isolated from backyard chickens differs markedly, constituting a well-separated cluster and appearance of 3 distinguishing lineages with greater discrimination shown by RAPD-PCR that resulted in two suclusters in cluster A and three subclusters in cluster B and were related greatly with capsular serogroups for the examined strains. The whole cell protein revealed the presence of dominant protein bands at approximately 41 and 61 kDa in all of the examined isolates that may be a virulent proteins share in the increasing of its pathogenicity. Clear distinctive bands ranged from 123 to 1 554 bp.

Conclusions

Based on the previous findings, there are three spreading clusters that may indicate the association of a small number of P. multocida variants with the majority of cases suggesting that certain clones of P. multocida are able to colonize the examined backyard chickens. Also, the ease and rapidity of RAPD-PCR support the use of this technique as alternative to the more labour-intensive SDS-PAGE system for strain differentiation and epidemiological studies of avian P. multocida. Further application of RAPD technology to the examination of avian cholera outbreaks in commercially available flocks may facilitate more effective management of this disease by providing the potential to investigate correlations of P. multocida genotypes, to identify affiliations between bird types and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission.     

嗜肺巴斯德杆菌基因分型及在分子流行病学中的意义

目的　探讨我国部分地区嗜肺巴斯德杆菌分子流行病学特征。方法　随机多态扩增PCR(RAPD -PCR) ,两条单一随机引物S1和S3 ,对分离来自北京和南京不同实验动物饲养单位的大鼠、小鼠、野鼠的 1 5株嗜肺巴斯德杆菌及参考株共 1 6株菌基因组随机扩增 ,比较DNA多态性图谱。结果　 1 5个流行株可分成 4种基因型 :来自相同饲养单位的小鼠株、大鼠株及野鼠株基因型亦不相同 ;野鼠株与部分小鼠分离株基因型相同。结论　同一地区嗜肺巴斯德杆菌流行株呈明显的多态性 ;野鼠有可能成为实验动物嗜肺巴斯德杆菌的传染源     

嗜肺巴斯德杆菌研究进展

嗜肺巴斯德杆菌(Pasteurella pneumotropica)是一种条件致病菌,人兽共患。主要危害啮齿类动物,特别是免疫缺陷或抑制的动物,可引起炎症和脓肿等症状。该菌是实验动物中感染率最高的病原菌之一,多呈隐性感染,给动物实验带来了极大的干扰。本文针对嗜肺巴斯德杆菌的流行病学、检测和鉴定、分子分型和防治等方面进行综述。     

Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens

Objective

To isolate and identify Bacillus subtilis (B. subtilis) from soil and to characterize and partially purify the bacteriocin. To evaluate the antimicrobial activity against four diabetic foot ulcer bacterial pathogens.

Methods

Genotypic identification was done based on Bergey''s manual of systemic bacteriology. Antimicrobial susceptibility test was done by Kirby-Bauer disc diffusion method. Colonies were identified by colony morphology and biochemical characterization and also compared with MTCC 121 strain. Further identification was done by 16S rRNA sequencing. Inhibitory activities of partially purified bacteriocin on all the DFU isolates were done by agar well diffusion method. The strain was identified to produce bacteriocin by stab overlay assay. Bacteriocin was extracted by organic solvent extraction using chloroform, further purified by HPLC and physical, and chemical characterization was performed.

Results

The four isolates showed high level of resistance to amoxyclav and sensitivity to ciprofloxacin. HPLC purification revealed that the extracts are bacteriocin. The phylogenetic tree analysis results showed that the isolate was 99% related to B. subtilis BSF01. The results reveled activity to all the four isolates and high level of activity was seen in case of Klebsiella sp.

Conclusions

Partially purified bacteriocin was found to have antimicrobial activity against the four diabetic foot ulcer bacterial pathogens, which can thus be applied as a better drug molecule on further studies. The strain B. subtilis are found to be safe for use and these antimicrobial peptides can be used as an antimicrobial in humans to treat DFU bacterial pathogens.     

Antioxidant and Anti-inflammatory Capacity of Ferulic Acid Released from Wheat Bran by Solid-state Fermentation of Aspergillus niger

Objective

A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated.

Characterization of cytotoxic compound from marine sediment derived actinomycete Streptomyces avidinii strain SU4

Objective

To investigate the cytotoxic activity of actinomycete isolated from marine sediment.

Methods

In the present study the DNA was isolated and the ITS region of 16s rRNA was amplified by polymerase chain reaction, using two universal bacterial primers, 1492R (5′-GGTTACCTTGTTAC GACTT-3′) and Eubac27F (5′-AGAGTTTGATCCTGGCTC AG-3′). The amplified products were purified using TIANgel mini purification kit, ligated to MD18-T simple vector (TaKaRa), and transformed into competent cells of Escherichia coli DH5α. 16S rRNA gene fragment was sequenced using forward primer M13F (-47) and reverse primer M13R (-48). Blast search sequence similarity was found against the existing non-redundant nucleotide sequence database thus, identified as Streptomyces sp SU, Streptomyces rubralavandulae strain SU1, Streptomyces cacaoi strain SU2, Streptomyces cavourensis strain SU3, Streptomyces avidinii strain SU4, Streptomyces globisporus strain SU5, Streptomyces variabilis strain SU6, Streptomyces coelicolor strain SU 7. Among the eight identified isolates, one actinomycete Streptomyces avidinii strain SU4 was selected for further study.

Results

Crude extract of the actinomycete isolate exhibited IC₅₀ in 64.5 µg against Hep-2 cell line, 250 µg in VERO cell line. This value is very close to the criteria of cytotoxicity activity for the crude extracts, as established by the American National Cancer Institute (NCI) is in IC₅₀ < 30 µg/mL. The GC MS analysis showed that the active principle might be 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (12.17%), isooctyl phthalate (15.29%) with the retention time 15.642 and 21.612, respectively.

Conclusions

This study clearly proves that the marine sediment derived actinomycetes with bioactive metabolites can be expected to provide high quality biological material for high throughout biochemical and anticancer screening programs. These results help us to conclude that the potential of using metabolic engineering and post genomic approaches to isolate more bioactive compounds and make their possible commercial application is not far off.     

Low copulatory activity in selectively bred Sardinian alcohol-nonpreferring (sNP) relative to alcohol-preferring (sP) rats

Background

There is a growing consensus that similar neural mechanisms are involved in the reinforcing properties of natural rewards, like food and sex, and drugs of abuse. Rat lines selectively bred for high and low oral alcohol intake and preference have been useful for understanding factors contributing to excessive alcohol intake and may constitute proper animal models for investigating the neurobiological basis of natural rewarding stimuli.

Methods

The present study evaluated copulatory behavior in alcohol and sexually naïve Sardinian alcohol-preferring (sP) and -nonpreferring (sNP) male rats in three consecutive copulatory behavior tests.

Results

The main finding was that, under the conditions used in this study, sNP rats were sexually inactive relative to sP rats. To gain more information about the sexual behavior in sP rats, Wistar rats were included as an external reference strain. Only minor differences between sP and Wistar rats were revealed.

Conclusions

The reason behind the low copulatory activity of sNP rats remains to be elucidated, but may in part be mediated by innate differences in brain transmitter systems. The comparison between sP and Wistar rats may also suggest that the inherent proclivity to excessive alcohol drinking in sP rats may mainly be dependent on its anxiolytic properties, as previously proposed, and not changes in the reward system.