Pathogenicities of two CAR bacillus strains in mice and rats

Two strains of CAR bacillus from a mouse (CB-M) and a rat (CB-R) which were passaged 11th in embryonated chicken eggs via the allantoic route were inoculated intranasally in ICR mice and Wistar rats. The histopathological changes and the localization of the CAR bacillus in the tracheas and lungs of these animals were investigated microscopically 2, 4 and 8 weeks postinoculation (PI). Histopathological changes similar to those in natural cases of CAR bacillus infection, showing severe peribronchial lymphoid cuffing, were first recognized 4 weeks PI. CAR bacillus was also found on the cilia of the respiratory epithelium. These histopathological changes were more remarkable in mice inoculated with CB-M and in rats inoculated with CB-R.     

Antigenic analyses of cilia-associated respiratory (CAR) bacillus isolates by use of monoclonal antibodies

Mouse monoclonal antibodies (MAbs) developed to a rat isolate (R-3) of cilia-associated respiratory (CAR) bacillus were used to assess antigenic relationships among three rat and five rabbit CAR bacillus isolates. Evaluation of MAbs by enzyme-linked immunosorbent assays (ELISAs) indicated that 87 of 241 hybridomas secreted CAR bacillus-reactive antibodies that could be grouped into four major groups. Group-I MAbs reacted with epitopes expressed by all CAR bacillus isolates and at least two or more nonrelated species of bacteria. Group-II, -III, and -IV MAbs reacted with only one or more of the rat CAR bacillus isolates; no MAbs reacted only with rat and rabbit CAR bacillus isolates. Western blot analyses indicated that 41-, 50-, and 105-kDa peptides of rat CAR bacillus isolates expressed rat CAR bacillus group- and isolate-specific epitopes. Hyperimmune anti-CAR bacillus antiserum and serum specimens from a CAR bacillus histologically positive mouse and rat also reacted with the 41-, 50-, and 105-kDa peptides. Sera from CAR bacillus histologically negative rats did not react with these peptides. These results suggest that the 41-, 50-, and 105-kDa peptides may represent suitable antigens for development of a specific ELISA for detection of rodent CAR bacillus infections. Furthermore, these data indicate that use of crude CAR bacillus preparations for either rat or rabbit CAR bacillus ELISAs is inappropriate.     

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.     

Circulating autoantibodies to mammalian tissue kallikreins

Autoantibodies to tissue kallikrein (EC 3.4.21.35) were discovered in normal human, rat, mouse, and guinea pig sera. Three independent methods--binding of iodolabeled antigen, enzyme-linked immunosorbent assay (ELISA), and immunoblotting--were used to demonstrate these kallikrein autoantibodies. Autoantibodies from rat and human sera were purified, using rat and human tissue kallikrein-affinity chromatography, respectively. Purified rat kallikrein autoantibody bound 50% of 125I-labeled rat urinary kallikrein upon incubation of antibody at 2.5 X 10(-10) M. The subtypes of rat and human kallikrein autoantibodies were determined by an ELISA, using antisera to immunoglobulin subclasses. In both species, autoantibody was predominantly IgG (approximately 80%) and some IgM (approximately 20%). Purified autoantibodies from rat and human sera were separated on sodium deodecyl sulfate-polyacrylamide gels, and their subunits were identified by Western blot analyses, using anti-rat and anti-human IgG antibodies, respectively. When primary cultures of mouse spleen cells were incubated for 1 to 5 days with lipopolysaccharide (1 to 5 micrograms/ml), the anti-kallikrein antibodies in the media increased up to seven-fold. We have demonstrated circulating autoantibodies that recognize and bind both autologous and heterologous kallikrein; however, their significance to the function of the tissue kallikrein-kinin system in normal and disease states remains to be explored.     

ELISA of rat sera infected with Paragonimus iloktsuenensis

Enzyme-linked immunosorbent assay (ELISA) of paragonimiasis iloktsuenensis rat sera was performed using crude antigens of Paragonimus iloktsuenensis (PIA), P. westermani (PWA) and Clonorchis sinensis (CSA). Three crude antigens (PIA, PWA, CSA) were prepared to saline homogenated supernatants of whole adult worms. Infected rat sera were obtained biweekly from the albino rats fed 50-80 metacercariae of P. iloktsuenensis through gastric catheter. Experimental groups were divided into 4 groups: GI (controls), GII, GIII and GIV according to 1-7 worms as GII, 10-19 worms as GIII and 22-40 worms as GIV, respectively. In ELISA, the mean OD values of each group for the homologous antigen (PIA) were increased significantly compared to the control sera at the 4th week of infection. With the progress of duration of infection, the mean OD values of infected sera of GII & GIV continuously increased up to the 12th week (last week), but in GIII the mean OD value increased until the 10th week. No significance was noted among the infection dose groups (GII, GIII and GIV), after the 6th week of infection. Also, the OD values of all infected rats did not show any proportional relationships to the number of worms recovered. In brief, the antibody productivity of individual rats were strongly different. The rat sera infected with P. iloktsuenensis cross-reacted with those infected with P. westermani or C. sinensis, as identified by OD values.     

A new approach to the design of hybrid hybridomas based on the use of an actinomycin D-resistant line of murine myeloma]

The hybrid hybridomas (tetradomas) were produced from the fusion of the double mutant actinomycin Dr (ADr)/HATs hybridoma to horseradish peroxidase (HRP) and wild type hybridoma to alpha-endorphin (EP). The double mutant phenotype was constructed using the new strategy, based on the fusion of immune mouse splenocytes with mouse myeloma (X63.Ag8, 653) cell variants, made resistant to 30 ng/ml of AD by stepwise selection. This allowed the direct introduction of the dominant selective marker (ADr) into the hybrid cells. Tetradomas secreted the bispecific monoclonal antibodies (bi Mabs), simultaneously binding to EP and HRP in double antigen ELISA, the ELISA plates covered with EP-bovine serum albumin conjugate. Using rat pituitary the bi Mabs were shown to be effective for immunostaining of EP-producing cells. EP-producing cells.     

鸭瘟病毒gB蛋白N端主要抗原域的表达及间接ELISA检测

在分析鸭瘟病毒gB蛋白抗原性的基础上,设计一对引物克隆gB蛋白N端抗原性较好的抗原域编码基因.将克隆的基因定向插入pET-32a的EcoR Ⅰ和HindⅢ之间,构建了gB蛋白主要抗原域原核表达载体pET-gB1.将pET-gB1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了DPV gB蛋白主要抗原域的高效表达.免疫印迹试验表明获得的表达产物具有良好的反应原性.应用His·Bind亲和层析柱纯化重组DPV gB蛋白,以纯化的重组gB1蛋白作为检测抗原,初步建立了检测鸭瘟病毒抗体的igB1-ELISA.结果表明,抗原的最佳包被浓度为6.5μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490＞0.4,且待检血清OD490/阴性血清OD490＞2.应用igB1-ELISA对鸭血清样品进行检测,结果表明igB1-ELISA与全病毒包被的iDPV-ELISA符合率达到95.6%.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

应用转化细胞分泌的乙型肝炎病毒e抗原检测人血清中的乙型肝炎病毒e抗体

应用乙型肝炎病毒核心抗原基因转化的小鼠L细胞分泌的乙型肝炎病毒e抗原,采用ELISA法与Abbott公司抗-HBe EIA诊断盒平行比较,检测了31份抗-HBe阳性和19份抗-HBe阴性的人血清,结果完全相符。经多次重复试验,本法的OD490nm值的误差不超过8％。OD490nm值与血清稀释度之间呈直线关系。细胞培养液不经纯化即可应用,一般做1:4稀释。细胞分泌的抗原无感染性,价格低廉,不会因结合人血清蛋白而产生非特异性反应。因此比一般诊断盒中所用的人血清HBeAg有很大的优越性。     

Reactivities of 4 murine coronavirus antigens with immunized or naturally infected rat sera by enzyme linked immunosorbent assay

Four murine coronavirus antigens, sialodacryoadenitis virus (SDAV) strain TG, Parker's rat coronavirus (PCV) strain 8190, mouse hepatitis virus (MHV) strains S and NuU, were examined for their reactivities to hyperimmunized and naturally infected rat sera by ELISA. With the immunized sera, SDAV and PCV antigens reacted best with respective homologous sera. MHV antigens reacted with all antisera, anti-SDAV, anti-PCV, and anti-MHV-S at approximately the same level, and MHV-S showed a slightly higher reactivity than MHV-NuU. The reactivities of the sera from various colonies to these antigens were in the order--from high to low--of SDAV, MHV-S, MHV-NuU, and PCV. None of sera negative for SDAV antigen reacted positively to the other antigens. Within the sera positive for SDAV, the positivities were in the order of MHV-S, MHV-NuU, and PCV. These results suggested that, although homologous antigens are best to detect SDAV or PCV infection by ELISA, MHV antigen can be used if highly cross-reactive viral strain is selected.     

马动脉炎病毒GL蛋白主要抗原域的表达及间接ELISA的初步建立

在分析马动脉炎病毒GL蛋白抗原性的基础上,设计一对引物克隆GL蛋白一段抗原性较好的抗原域编码基因。将克隆的基因插入pET-32a的BamHⅠ和XhoⅠ之间构建了GL蛋白主要抗原域原核表达载体pET-GL1。将pET-GL1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了EAV GL蛋白主要抗原域的高效表达。免疫印迹试验表明获得的表达产物具有良好的反应原性。应用His.Bind亲和层析柱纯化重组EAV-GL1蛋白,以纯化的重组GL1蛋白作为检测抗原,初步建立了检测马动脉炎病毒抗体的iGL1-ELISA。结果表明,抗原的最佳包被浓度为9.65μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490>0.4,且待检血清OD490/阴性血清OD490>2。应用iGL1-ELISA对马血清样品进行检测,结果表明iGL1-ELISA与病毒中和试验的符合率达到94.1%,与国外同类试剂盒的符合率达到95.6%。     

Detection of IgG antibody against Neospora caninum in cattle in Korea

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasma gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only. And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.     

Pathogenesis of CAR bacillus in rabbits, guinea pigs, Syrian hamsters, and mice.

Cilia-associated respiratory (CAR) bacillus isolated from infected mice (designated, CBM) and propagated in embryonated chicken eggs was inoculated intranasally in rabbits (Oryctolagus cuniculus), guinea pigs (Cavia porcellus), hamsters (Mesocricetus auratus) and mice (Mus musculus). Gross and microscopic lesions, localization of CBM antigen in the respiratory tract, development of antibody, and ability to reisolate the CAR bacillus were studied in animals killed at 2-, 4-, or 8-week intervals postinoculation (PI). In rabbits, although no histopathological changes were observed in the respiratory tract, CBM antigen was detected on the ciliated epithelium of the respiratory tract, and serum CBM antibody was also detected 4 and 8 weeks PI. In guinea pigs, no histopathological changes were noted, CBM antigen was detected in the respiratory tract 2 and 4 weeks PI but not 8 weeks PI, and serum CBM antibody was detected 4 and 8 weeks PI. In hamsters, mononuclear cell proliferation in the submucosa of the bronchus and trachea was observed 8 weeks PI. CBM antigen was detected at first in the nasal cavity 2 weeks PI and in the lower respiratory tract 4 and 8 weeks PI and serum CBM antibody was detected 4 and 8 weeks PI. In mice, histopathological changes, CBM antigen and CBM antibody were observed. CBM was reisolated from the tracheal washouts of hamsters and mice 8 weeks PI but not from those of rabbits and guinea pigs. These results confirm and extend previous reports of experimentally-induced CAR bacillus infection in mice, guinea pigs, and rabbits. To this list of susceptible laboratory animals, we now add hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.     

Pathology of rats intranasally inoculated with the cilia-associated respiratory bacillus

Five-week-old Wistar/Ms rats were inoculated intranasally with a lung homogenate containing a strain of cilia-associated respiratory (CAR) bacillus and were examined on days 4, 7, 14, 21, 28 and 56 postinoculation (PI). Some rats showed clinical signs with wheezing and considerable body weight loss from day 21 PI. Gross lesions, including enlargement of lungs with focal atelectasis, bronchiectasis and emphysema, were observed from day 21 PI. Histologically, round cell infiltration was first present in the lamina propria of the nasal respiratory mucosa on day 7 PI. From day 14 PI, colonization of the CAR bacillus (4-8 micron in length), associated with round cell infiltration in the lamina propria and the peripheral regions, was observed in the ciliated mucosa of the bronchioles, bronchi, trachea and nasal cavities. Generally, the lesions progressed and expanded from upper to lower airways with time. Sporadic mucopurulent bronchopneumonia was observed from day 21 PI in some rats. The CAR bacilli (0.2-0.25 micron in diameter) were also demonstrated electron-microscopically in the ciliated epithelium of the intrapulmonary airways. The CAR bacillus antigen was demonstrated on the ciliated mucosa of the affected airways by the indirect immunofluorescence assay technique. Microbiological examination revealed that the rats used in this study were free from other known respiratory pathogens throughout the experimental period. Thus, it is suggested that the CAR bacillus alone can produce a murine respiratory disease. Fourteen days were needed for pathological lesions to develop.     

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.     

Haemorrhagic fever with renal syndrome: evaluation of ELISA for detection of Puumala-virus-specific IgG and IgM

IgM and IgG ELISA to Puumala virus were evaluated using sera from patients with haemorrhagic fever with renal syndrome (HFRS) from different geographical regions: Sweden, Denmark, Norway, Belgium and the European USSR.IgM ELISA proved useful in the diagnosis of HFRS in patients from all the regions mentioned above. Specific IgM could be detected as early as day 1 post onset of disease, and patients remained IgM-positive for several months. Specific IgG ELISA antibodies were also frequently detected in acute sera, and acute-convalescent serum pairs often failed to show a significant titre rise or increase in optical density (OD) values. This limits the use of IgG ELISA in patient diagnosis. Sera collected 2 years after infection revealed higher IgG ELISA OD readings than convalescent sera, and very high values were still detectable 10 to 20 years postinfection. IgG ELISA is therefore useful for the testing of immunity and in seroepidemiological studies.Acute and convalescent sera from HFRS patients in Korea and the Asian USSR showed no or only very weak reactivity in the Puumala virus IgG and IgM ELISA. These results are consistent with the “one-way” crossing described earlier.     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

牛流行热病毒G1抗原表位基因在大肠杆菌中的表达、纯化及抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G1抗原表位区基因,与表达载体pGEX-4T-1连接,成功构建重组质粒pGEX-G₁。重组质粒转化BL21（DE3）,以IPTG进行诱导,并确定了最佳表达条件的IPTG浓度为0.1mmol/L、反应温度为16℃、诱导时间为18h。可溶性表达的目的蛋白经Glutathione Sepharose TM^4B介质纯化,纯度达80%;以包涵体形式存在的重组蛋白以2%的脱氧胆酸钠洗涤、0.5%的N-十二烷基肌氨酸钠溶解、透析复性、Glutathione Sepharose TM^4B纯化后,纯度达85%以上。Western blot试验表明纯化的目的蛋白有良好的反应原性。经间接ELISA检测,测得牛流行热病毒12份阳性血清的OD490值平均为1.813±0.231,12份阴性血清的OD490值平均为0.359±0.032,差异极显著（P<0.01）。将重组蛋白作为抗原免疫兔子,试验兔均产生了高滴度的抗体,证实该蛋白有免疫原性。将目的蛋白作为包被抗原,测得8份狂犬病病毒阳性血清的OD490值平均为0.324±0.031,与所测12份阴性血清的OD490值接近,说明不存在交叉反应。以上结果均证实纯化后的重组蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA试剂盒。     

Effect of antibodies against seminal vesicle secretion on fertility in the rat.

Fertile female Wistar rats were immunised against rat and mouse seminal vesicle secretion (SVS) to test the production of allo-antibodies and the effect of the antibodies elicited on fertility. Twenty-six per cent of the rat and mouse SVS-immunised females were infertile after the treatment. The sera were titrated by ELISA and used in Western blots to detect the proteins recognised. Although neither the antibody titres nor the proteins recognised by the sera showed a close relation with the degree of fertility, in all females the highest antibody titre in the fluids from the genital tract was found in the oviductal fluid and during the night of oestrus. This fact suggested that the site of action of the antibody could be the oviduct. Similar results were obtained using mouse SVS as immunogen--a fact that can be related to the antigenic similarity between the SVS of the two species. The antibodies react with the spermatozoa but not with eggs or embryos. Analyses performed on embryos collected from sterile females showed that there was a delay in fertilisation and normal embryogenesis. Our results suggest that SVS proteins are antigenic and that these antigens are bound to the spermatozoa and could take part in early pre-fertilisation events such as capacitation or sperm transport.