Playback experiment to test maternal responses of Japanese macaques (Macaca fuscata) to their own infant's call when the infants were four to six months old

Calls emitted by infants when the infant loses sight of its parents are useful to estimate the infant's requirement for parental care. When an Old World monkey infant loses sight of its mother it emits whistles. Therefore, it would be interesting to determine whether mothers could distinguish their own infant's whistles from the whistles of other infants. The response of each of seven Japanese macaque (Macaca fuscata) mothers to her own infant's whistle was compared with their responses to another infant's whistle. Matched control playback experiments were performed when the infants were four to six months old. The results showed that each mother could distinguish her own infant's whistle from that of another infant when the infants were four to six months old. Although a stricter experimental plan is required to further examine the issue, we found that the dominance rank of the infant's mother was another important factor in the female response to the infant's call. The finding suggests that females can associate a call emitted by an infant with its mother's rank, even before the infant begins to wander far from its mother.     

Testing of the Biocan B inj. ad us. vet. vaccine and development of the new recombinant vaccine against canine borreliosis

Verification of the efficacy of Biocan B inj. ad us. vet. (Bioveta, a.s.) was done by challenge testing. Ticks collected in the nature were used as natural vectors of the infection. Six beagles and two control ones were used in the test. Formation of outer surface protein A specific antibodies (OspA antibodies) and borrelia specific immonoglobulins (IgG) was measured by Western blot and EIA in the sera samples. The tissue samples were used for detection of borreliae by cultivation method and dark field microscopy (DFM). Formation of IgG antibodies and OspA antibodies after vaccination was observed. The maximum titer level of antibodies was reached between 21. and 49. day after vaccination and then slowly decreased. Presence of borreliae was detected only in skin biopsies of non-vaccinated dogs. The post mortem tissue samples showed presence of borreliae in all of the samples of the non-vaccinated dogs. The tissues of the vaccinated dogs were not infected with borreliae, except for two samples of dog with low titer levels of OspA antibodies. The development of the new vaccine is based on preparation of recombinant outer surface proteins (e.g. rOspA and rOspC) of B. afzelii, B. burgdorferi and B. garinii origin. Chosen recombinant proteins were successfully expressed in E. coli. The obtained purified proteins are currently being tested on laboratory BALB/c mice. Formation of specific antibodies against some recombinant proteins has been confirmed. These proteins are suitable candidates for preparation of a vaccine prototype and they will be subsequently used in challenge tests.     

艰难梭菌毒素B羧基端的表达与卵黄抗体制备简

目的：在大肠杆菌中可溶性表达艰难梭菌毒素B羧基端（TcdB-e）,免疫产蛋鸡,获得针对TcdB-c的卵黄抗体（IgY）。方法：人工合成TcdB-c的基因,将其克隆至pET32b（＋）载体中,转化大肠杆菌BL21（DE3）,诱导表达产物经金属螯合层析纯化,凝血酶酶切后得到目的蛋白TcdB-c;利用兔红细胞凝集和兔肠袢实验检测目的蛋白活性,用TcdB-c免疫产蛋鸡制备鸡卵黄抗体,分离纯化卵黄抗体并经ELISA测定抗体效价,用兔肠袢实验检测抗体的中和活性。结果：构建了TcdB-c的重组表达载体,诱导表达的融合蛋白相对分子质量约为79000,经凝血酶酶切后的相对分子质量约65000;目的蛋白免疫产蛋鸡后获得效价为1：20000的抗TcdB-C卵黄抗体,且该抗体可以中和TcdB-c对兔小肠的毒性作用。结论：获得了具有生物学活性的TcdB-C,并制备了针对TcdB-c的鸡卵黄抗体,为利用基因工程方法防治艰难梭菌感染打下了基础。     

Effect of exogenous interferon on the transplantation reactions of mice]

The authors studied the influence of the serum obtained at various periods after the administration of interferon inductors (New castle disease virus, amino ethylisothiouronium, E. coli endotoxin) on the rate of rejection of the skin or cell transplant of mice C3H and CBA, and also CC57Br. The allogenous skin transplant perished more rapidly; there was also an acceleration of elimination of allogenous lymphoid cells, suppression of colony formation by the cells of allogenous bone marrow in the spleen of the irradiated recipient in administration of the serum obtained at the period of maximal content of interferon induced by the Newcastle disease virus and by amino ethylisothiouronium. The cytotoxic activity of lymphocytes of mice CC57Br against the allogenous target cells rose in the presence of these sera. The serum containing interferon induced with E. coli endotoxin failed to influence the rate of the allotransplant rejection and did not increase the cytotoxic activity of lymphocytes.     

Comparison of serological specificity of anti-endotoxin sera directed against whole bacterial cells and core oligosaccharide of Escherichia coli J5-tetanus toxoid conjugate

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.     

Asia I口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia I口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法。用纯化的Asia I型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠, 将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合, 采用间接ELISA和有限稀释法筛选杂交瘤细胞。分别用ELISA、Western blotting检测mAb腹水的效价及其特异性。筛选到杂交瘤细胞2株, 腹水效价均在100×29以上; 以纯化后的Asia I型口蹄疫病毒vp2重组蛋白作为抗原, 利用Asia I型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia I型口蹄疫抗体。临床应用表明, 该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%, 和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%。     

Protective effect of rabbit immune serum administered orally to rats infected by a human pathogenic strain of E. coli.

1-week-old rats were inoculated orally with a strain of E. coli (serotype 078) isolated from the blood of a newborn baby who had died of septicemia. During the 3 weeks following inoculation, approximately 50% of the animals died of septicemia and 60% of the surviving rats had pathogenic bacteria in their rectum. Some of the surviving rats were severely impaired in their development. Autopsy showed evidence of active intestinal infection localized mainly in the ileum and cecum. A rabbit anti-E. coli (strain 23) serum (agglutinating titer: 1/2,500) afforded 100% protection when as little as 0.03 mg of serum protein per gram of rat body weight was orally administered in a single dose. The immune serum had an effect both on the mortality rate and on the growth of the rats. However, it never affected the survival of pathogenic bacteria in the rectum, even when administered at a daily dose of 1.5 mg of serum protein per gram of rat body weight on 4 consecutive days. The immune rabbit serum had only a weak bactericidal effect in vitro. The hemagglutination test showed the presence in the immune serum of antibodies against the fimbriae of the pathogenic E. coli strain (titer: 1/1,000). The role of antibody in inhibiting the adherence of bacteria to epithelial cells and/or their progression across the mucous layer are discussed as possible immune mechanisms in the intestinal lumen.     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies

A recombinant fusion protein consisting of native Escherichia coli heat-stable enterotoxin (STa) and a dimer of a synthetic IgG-binding fragment (ZZ), derived from Staphylococcus aureus protein A was produced in E. coli. The fusion protein (ZZSTa) was secreted in large quantities into the growth medium and recovered by affinity chromatography on IgG-Sepharose. Rabbits immunized with the fusion protein responded by producing high serum levels of anti-STa antibodies that also effectively neutralized STa toxicity in infant mice. The fusion peptide ZZSTa had a substantially decreased toxicity as compared with native STa. A polymeric form of ZZSTa separated by size fractionation was about 100 times less toxic than the monomeric fusion protein, yet both forms had the same capacity to induce neutralizing antibodies. This suggests that modified non-toxic forms of ZZSTa with retained immunogenicity may be produced and tested for their usefulness as functional components in a vaccine against diarrhoea caused by enterotoxigenic E. coli.     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters.

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

甘蔗花叶病毒CP基因的高效表达和抗血清制备

将甘蔗花叶病毒的CP基因克隆到表达载体pET22b( ),并在大肠杆菌BL21(DE3)中得到高效表达,表达蛋白约占总蛋白的15％。用此蛋白制备了效价高专化性强的抗体。此方法可以解决制备马铃薯Y病毒属病毒的血清时遇到的病毒提纯产量低和寄主蛋白影响等问题。     

西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli [pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.     

DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli

Abstract In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli , E. coli DH5α cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized components) on the transformant outer surface.     

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579-36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg)+/-LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>Eagg, Eagg:LBP (E/LBP approximately 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

Specificity and Sensitivity of the Streptozyme Test for the Detection of Streptococcal Antibodies

A comparison between the results of the streptozyme hemagglutination test and serological titers for anti-streptolysin O (ASO), anti-hyaluronidase (AH), anti-deoxyribonuclease B (ADN-B), and anti-nicotinamide adenine dinucleotidase (ANAD) was made in two groups of human sera. In one group, serological titers for all the four antibodies were lower than the threshold of sensitization reported by the producing firm. In the second group, the titer of at least one of the four antibodies was equal to or higher than the threshold. False-positive and false-negative reactions occur with those sera when one or more antibody titer is at or near the threshold of the test as described by the manufacturer. The test was positive for all sera where either the ASO was greater than 166 or the ANAD was greater than 270, and for 98% of the sera with ADN-B greater than 360. It is, therefore, concluded that the streptozyme test can be used as an adjunct to the clinical diagnosis of streptococcal infections and their nonsuppurative sequelae. It is less useful to assess the levels of antibodies in sera from general population surveys. For such sera, the relative specificity and sensitivity of the test might yield misleading results. Until more experience is gained with the test, caution should be used in its application to infant and older adult age groups, where significant streptococcal antibody titers are frequently near the threshold of the test.