Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

Development and evaluation of enzyme-linked immunosorbent assay based on recombinant inorganic pyrophosphatase gene antigen for the detection of Mycoplasma suis antibodies

The recombinant ppa protein of Mycoplasma suis migrated to 21 kDa. Using this antigen, an ELISA system to detect the antibody against M. suis infection in swine was established. The rELISA demonstrated 98.5% specificities among negative samples and 96.9% sensitivity among positive samples with M. suis infection. A comparison of this ELISA system with an indirect hemagglutination assay (IHA) test using 132 swine samples revealed that the positive rate was 34.0% in ELISA and 28.0% in IHA. Compared with IHA, the present rELISA system using recombinant ppa antigen significantly improves the specificity, sensitivity, and stability for serodiagnosis of M. suis infection in swine.     

猪附红细胞体感染对仔猪猪瘟免疫效果的影响

为探讨猪附红细胞体对仔猪猪瘟免疫效果的影响,本试验利用猪瘟抗体检测ELISA试剂盒,对已注射猪瘟疫苗的69头感染猪附红细胞体的仔猪和31头无猪附红细胞体感染的健康仔猪进行了猪瘟抗体检测。结果表明,感染猪附红细胞体仔猪的猪瘟抗体水平低下,其猪瘟疫苗整体免疫合格率(49·2%)明显低于健康仔猪(93·5%),且显性感染仔猪的免疫合格率(41·6%)明显低于隐性感染仔猪(53·5%)。说明猪附红细胞体严重干扰了猪瘟疫苗的免疫效果,且干扰程度与猪附红细胞体的感染程度呈正相关。     

猪附红细胞体对仔猪猪瘟免疫效果的影响

为探讨猪附红细胞体对仔猪猪瘟免疫效果的影响，本试验利用猪瘟抗体检测ELISA试剂盒，对已注射猪瘟疫苗的69头感染猪附红细胞体的仔猪和31头无猪附红细胞体感染的健康仔猪进行了猪瘟抗体检测．结果表明，感染猪附红细胞体仔猪的猪瘟抗体水平低下，其猪瘟疫苗整体免疫合格率（49．2％）明显低于健康仔猪（93．5％），且显性感染仔猪的免疫合格率（41．6％）明显低于隐性感染仔猪（53．5％）、说明，猪附红细胞体严重干扰了猪瘟疫苗的免疫效果，且干扰程度随着猪附红细胞体感染程度的加深而更为明显。     

Comparison of monoclonal antibody-based competitive and indirect enzyme-linked immunosorbent assays for the diagnosis of swine trichinosis

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of swine trichinosis has been developed using a biotinylated monoclonal antibody and an avidin-enzyme conjugate. The assay is based on competitive binding between swine serum antibodies and a monoclonal antibody specific for an antigenic determinant present on proteins from Trichinella spiralis excretory-secretory products with molecular weights of 45,000, 49,000, and 53,000. The competitive ELISA reliably detected pigs infected experimentally with T. spiralis and eliminated false-positive reactions in pigs infected with other swine nematodes, particularly Trichurus suis. When the competitive ELISA and an indirect ELISA using affinity-isolated antigen were compared using serum from pigs with naturally-acquired infections of T. spiralis, both tests were highly effective in detecting infected animals.     

Serologic profile of a cohort of pigs and antibody response to an autogenous vaccine for Actinobacillus suis

Actinobacillus suis is a commensal opportunistic pathogen in swine. However, in recent years, an increasing prevalence of clinical signs associated with A. suis has been observed in high health status herds in North America. The objectives of the study were to assess the kinetics of antibodies to A. suis in pigs from a herd showing clinical signs of A. suis infection and, to evaluate the antibody response in gilts following vaccination with an autogenous vaccine. An enzyme-linked immunosorbent assay (ELISA) using a saline extract of boiled-formalinized whole cells of a field strain as the coating antigen was standardized. This ELISA was used as a tool for monitoring, in a comparative way, the variations in A. suis antibody levels. The herd selected for the serologic profile was negative for Actinobacillus pleuropneumoniae infection and showed clinical signs of A. suis infection in 16 to 19-week-old pigs. A cohort of 20 pigs was blood sampled at 5, 8, 12, and 16 weeks of age. The lowest level of serum antibodies was observed between weeks 8 and 12, this probably corresponding to a decrease in maternal immunity. A marked increase in the antibody response was seen at 16-week of age, at the approximate time of onset of A. suis clinical signs in the herd. The evaluation of serum antibody responses to an autogenous vaccine revealed that the humoral immunity of gilts further increased following vaccination although the level of antibodies was already high prior to vaccination. The magnitude of the response to vaccination was higher when the level of antibodies was low prior to the first injection. The ELISA test seems to detect antibodies against the O-chain LPS.     

Development of an enzyme-linked immunosorbent assay for detecting antibodies in sera of Brucella suis-infected swine.

An enzyme-linked immunosorbent assay was developed using a heat-killed Brucella suis antigen for detecting antibodies in the sera of swine from which B. suis was isolated. Optimal enzyme-linked immunosorbent assay reactions were obtained using heat-killed B. suis antigen at a concentration comparable to McFarland Standard No. 1. Statistically significant differences were observed in the enzyme-linked immunosorbent assay results of 40 animals from which B. suis was isolated and the results for 48 noninfected swine at serum dilutions of 1:25 and 1:50 (P < 0.0001). The enzyme-linked immunosorbent assay is a rapid reproducible test which can be readily automated that appears to have practical value for screening large numbers of breeding and slaughter swine for brucellosis.     

Detection of Eperythrozoon suis DNA from swine blood by whole organism DNA hybridizations

A procedure is described for isolating Eperythrozoon suis DNA of sufficient quantity and purity to serve as a probe in whole-organism DNA hybridizations for detecting parasitized swine. The E. suis organisms were isolated from the blood of infected swine; the DNA was recovered and digested with restriction endonucleases and resolved on agarose gels. In DNA hybridizations using recovered E. suis DNA, blood samples from parasitized swine could be differentiated from uninfected, control samples. A high salt lysate recovery technique was used in sampling swine whole blood for E. suis DNA and found to offer many advantages in the collection and recovery process.     

猪附红细胞体病间接ELISA诊断试剂盒的研制及初步应用

以纯化的猪附红细胞体特异性抗原为包被抗原,在建立猪附红细胞体病间接ELISA检测方法的基础上对各反应参数进行了优化,并初步组装成试剂盒。组装的试剂盒抗原最佳包被质量浓度为30 mg/L,被检血清稀释度为1∶100,酶标二抗的最适工作滴度为1∶4 000,底物TMB最适反应条件为室温15 min;检测血清的判定标准为D450值≥0.13定为阳性反应,D450值≤0.11定为阴性反应,0.11~0.13定为疑似反应;该试剂盒不与猪大肠杆菌病、猪弓形虫病及猪瘟等阳性血清发生交叉反应,具有较好的特异性;在不同时间对阴阳性样品重复检测8次,阴阳性血清的D450值变异系数均未超过10%,说明该试剂盒具有良好的重复性;通过对组装试剂盒中各组分的稳定性和保存时间的测定结果证实,该试剂盒的保质期暂定为6个月;应用保存6个月的试剂盒通过对吉林省延边地区的60份猪血清样本检测,并与间接ELISA比较,其阳性符合率达100%,说明该试剂盒具有较好的稳定性。     

Immunization of pigs against Streptococcus suis serotype 2 infection using a live avirulent strain.

Streptococcus suis capsular type 2 is still an important cause of economic losses in the swine industry. At the present time, vaccination of pigs against this infection is generally carried out with autogenous bacterins and results are equivocal. In this study, the protective effect of a live avirulent S. suis type 2 strain (#1330) which had induced a good protection in mice, was evaluated in swine. The experiment was performed in triplicate using 4 week-old piglets. A total of 15 piglets were vaccinated 3 times, 15 others were vaccinated 2 times, and 15 piglets were injected 3 times with sterile Todd-Hewitt broth. Using an indirect ELISA, an increase in the IgG response to S. suis antigens was noted in 27 of the 30 vaccinated piglets. On day 21 post-vaccination, all animals were challenged intravenously with a virulent S. suis type 2 strain (#999). In the 2 vaccinated groups, 26 animals were fully protected. Only 1 out of the 15 piglets vaccinated 3 times developed mild clinical signs. In the group vaccinated twice, 3 piglets showed clinical signs and 1 of them died after the challenge. In the control group, 7 animals died out of the 11 with clinical signs of infection. In conclusion, a protective immunity was observed in swine when using strain 1330. However, more studies are needed to assess the use of a live S. suis strain in a vaccine for pigs.     

猪戊型肝炎病毒ORF2主要抗原表位区间接ELISA方法的初步建立

应用原核表达系统表达猪戊型肝炎病毒ORF2蛋白C端和N端的主要抗原表位区,重组蛋白命名为ORF2-C和ORF2-N,初步建立间接ELISA诊断方法。用重组蛋白ORF2-C和ORF2-N作为诊断抗原,对反应条件进行优化,初步建立ELISA诊断方法。抗原最适包被浓度ORF2-C为8 μg/mL、ORF2-N为12 μg/mL;血清最适稀释度均为1∶50,ORF2-C作用时间为50 min、ORF2-N作用时间为90 min;酶标抗体最适稀释度为1∶5000;ORF2-C判定标准：D450 nm值≥0.348为阳性,D450 nm值<0.348为阴性;ORF2-N判定标准：D450 nm值≥0.397为阳性,D450 nm值<0.397为阴性。与戊型肝炎病毒诊断试剂盒检测结果相比,阳性符合率分别为93.3%、86.7%。利用重组蛋白建立的ELISA方法特异性、敏感性和重复性均较好,且ORF2-C的检测效率明显高于ORF2-N,该方法的初步建立为进一步完善猪戊型肝炎病毒诊断方法奠定基础。     

Recombinant DNA probe detecting Eperythrozoon suis in swine blood

A genomic library to Eperythrozoon suis DNA was constructed in lambda gt11, and from this library, E suis clone KSU-2 was identified as a potential diagnostic probe. In hybridization experiments that used 100-microliters samples of blood collected in chaotropic salt solutions, the KSU-2 probe hybridized strongly with purified E suis organisms and blood samples from splenectomized swine that were parasitized with E suis. However, the probe under stringent conditions did not give radiographic indications of hybridizing with equine blood DNA, bovine blood DNA infected with Anaplasma marginale, canine blood DNA infected with Ehrlichia canis, feline blood DNA infected with Haemobartonella felis, or uninfected swine blood DNA.     

Trichuris suis excretory secretory products (ESP) elicit interleukin-6 (IL-6) and IL-10 secretion from intestinal epithelial cells (IPEC-1)

Immune responses to gastrointestinal helminth infections have received increasing attention due to similarities to allergen-induced responses. In fact, the whipworm parasite of swine, Trichuris suis, has been used in beginning clinical trials as an antidote to inflammatory bowel disease. This strategy was based on this similarity and the recognition that other worms have been documented to induce anti-inflammatory responses in the host. In an effort to understand the basis for this response, we hypothesized that the proteins and peptides secreted by T. suis stimulate local intestinal epithelial cells to produce anti-inflammatory cytokines. To test this hypothesis in a correlate system of the natural swine host, T. suis excretory secretory products (ESP) were used to treat both differentiated and undifferentiated intestinal pig epithelial cells (IPEC-1) in vitro as a model for the effect on villus tip and crypt epithelial cells in the vicinity of the worms. IPEC-1 were exposed to low-level doses (0.3mg/ml) of T. suis ESP, and IL-4, IL-6 and IL-10 cytokine responses were measured by an enzyme-linked immunosorbant assay (ELISA). IL-6 was the predominant cytokine produced, accompanied by moderate IL-10 secretion from both differentiated and undifferentiated cells. As expected, IL-4 was not produced by IPEC-1. Additionally, IL-6 and IL-10 cytokines were produced within 24h, suggesting that these two cytokines form part of the primary host response to T. suis infections. These data suggest that T. suis ESP could enhance host immune responses and modulation through the induction of enteric IL-6 and IL-10.     

猪乙型脑炎病毒囊膜E蛋白间接ELISA诊断方法的建立与初步应用

本研究利用纯化的原核表达乙型脑炎囊膜E蛋白作为包被抗原,建立了乙型脑炎间接ELISA诊断方法。对检测的各种条件进行了优化,优化反应条件后确定的抗原最适包被浓度为2μg/mL,抗原最佳包被条件为37℃包被2 h,血清的最适稀释度为1∶160,酶标抗体最适稀释度为1∶5000,最佳封闭条件为1%BSA,阴阳性临界值判定标准为D492 nm=0.254。该方法不与猪瘟、猪繁殖与呼吸综合征、猪圆环病毒2型、猪伪狂犬病毒阳性血清反应,其D492 nm0.254,说明该方法具有良好的特异性。采用该方法对150份疑似乙型脑炎血清样品进行检测,结果显示,与某猪乙型脑炎试剂盒相比符合率为90.77%,表明建立的间接ELISA方法具有较高的敏感性和特异性,因此,本研究成功建立了能特异性检测抗乙型脑炎血清抗体的ELISA检测方法。     

A sandwich enzyme-linked immunosorbent assay for the detection of Streptococcus suis.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and the identification of Streptococcus suis capsular types 1, 2, 1/2, 3 and 22. The specificity of this test was first evaluated using reference strains of S. suis capsular types 1 to 28 and 1/2 as well as 15 different bacterial species susceptible to be isolated from swine. The ELISA developed was very specific for capsular types 1, 3 and 22 but it could not discriminate between capsular types 2 and 1/2. In a second study, S. suis isolates from 328, 493, 368 and 76 diseased pigs were used to detect capsular types 1, 2 or 1/2, 3 and 22 respectively. The relative specificity and sensitivity varied between 98% and 100%. The ELISA results were in excellent agreement with the standard techniques (biochemical tests, coagglutination and capsular reaction tests) in detecting both positive and negative strains. Kappa values were 0.80, 0.99, 0.97 and 1.00 for detecting S. suis capsular types 1, 2 or 1/2, 3, and 22 respectively. To evaluate the relative-sensitivity of the test, primary cultures from 73 diseased pigs and tissue samples from 67 diseased pigs were used directly for detecting these capsular types. With primary cultures, the relative specificity and sensitivity (95.9% and 91.6% respectively) remained high and the test was very suitable (Kappa = 0.87). The ELISA using tissue samples gave a good specificity (97.6%), a moderate sensitivity (62.5%) and a low agreement with standard tests (Kappa = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)     

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.     

Quantitative and Qualitative Study of Enzyme-linked Immunosorbent Assay to Detect IgG against Japanese Encephalitis Virus in Swine Sera

This study was designed to investigate the application of indirect enzyme-linked immunoassay (ELISA) in detecting IgG against Japanese encephalitis virus in swine sera and the qualitative nature of this test. The attenuated strain SA14-14-2 of Japanese encephalitis virus (JEV) was inoculated into 9-day-old chicken embryos and virus was harvested, purified and suspended in 0.9% saline as JEV antigen. The control antigen was prepared by the same method as for the antigen. In the ELISA, the optimal concentrations of antigen coated and dilution factor were selected using chi2 test. Ninety-two swine sera negative to haemagglutination inhibition (HI) were tested by this assay and the positive threshold was determined. The results of this study indicate that indirect ELISA has high specificity, sensitivity and reproducability. Simultaneous testing of 74 serum samples from nine pig farms was carried out to compare the existing HI test and the indirect ELISA. The coincidence rate of the two assays was 85.1% (63/74) and no significant difference was observed between them (p > 0.05). This ELISA test can detect 46 swine serum samples qualitatively and the titre of eight swine serum samples through endpoint dilution quantitatively within one 96-well plate.     

Experimental infection with Tritrichomonas suis in heifers.

Nine heifers were intravaginally challenged with 9.3x10(6) Tritrichomonas suis reference strains. Vaginal mucus and serum samples were collected weekly 4 weeks post-inoculation. Vaginal mucus was cultured for T. suis and sera was tested by ELISA against whole cell antigens for T. suis and Tritrichomonas foetus. All vaginal mucus cultures were T. suis-negative during the experiment. ELISA values for both antigens were similar and differences were not significant (P>0.05). Positive control serum samples from one heifer vaccinated against T. foetus showed anti-T. suis ELISA values. We concluded that T. suis intravaginal inoculation induced a low level of serum immune response in heifers measured by ELISA and both protozoa probably share a common antigen. However, under the experimental conditions of this trial, colonization of the heifers' genital tract was not possible in any of the nine animals.     

Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45

Enteric chlamydial infections of pigs with Chlamydia (C.) suis are frequent and often subclinical. The enteric pathogenicity of C. suis strain S45 was investigated in gnotobiotic piglets. Piglets from three litters (n=31) were inoculated with egg-grown chlamydiae at 2-3 days of age (n=17) or used as controls (n=14). They were observed for clinical signs, killed and necropsied sequentially at 2-13 days postinoculation (DPI). Feces were collected daily and investigated with an ELISA for chlamydial antigen. At necropsy, specimens were collected for histopathology and for immunohistochemical, PCR-based, and serological (complement fixation test, ELISA) detection of chlamydiae. Chlamydial replication and associated symptoms and lesions were observed from 2 to 13 DPI and were particularly pronounced within the first week PI. Clinical symptoms consisted of moderate-to-severe diarrhea, slight and transient anorexia, weakness and body weight loss. Immunohistochemistry and ELISA revealed that chlamydial replication was particularly marked at 2-4 DPI and primarily located in the small intestinal villus enterocytes. Further sites of replication included large intestinal enterocytes, the lamina propria and Tunica submucosa, and the mesenteric lymphnodes. Histopathological changes included moderate-to-severe villus atrophy with flattened enterocytes and focal villus tip erosions, and moderate mucosal inflammatory cell infiltrates and lymphangitis in the small intestine. PCR of spleen tissue and blood was mostly negative for chlamydiae, indicating that they did not substantially disseminate into the host up to 13 DPI. All sera were negative for anti-chlamydial antibodies. In conclusion, C. suis strain S45 elicited significant enteric disease and lesions in gnotobiotic piglets indicating its pathogenic potential for swine.     

In vitro susceptibility of porcine respiratory pathogens to tilmicosin.

Bacterial isolates obtained from swine with various clinical diseases were tested for susceptibility to tilmicosin by minimum inhibitory concentration (MIC) and Kirby-Bauer disk diffusion tests using National Committee on Clinical Laboratory Standards methodology. The tilmicosin MIC90 was < or =0.125 microg/ml for Erysiopelothrix rhusiopathiae, < or = 1 microg/ml for Haemophilus parasuis isolates, 8 microg/ml for Actinobacillus suis and Pasteurella multocida type A, 16 microg/ml for toxigenic and nontoxigenic P. multocida type D, 64 microg/ml for Bordetella bronchiseptica, and >128 microg/ml for Staphylococcus hyicus and Streptococcus suis. The results of disk diffusion testing matched well with the MIC results for each pathogen. This in vitro survey of tilmicosin activity against various swine isolates suggests that further clinical evaluation of tilmicosin in swine may be warranted for disease associated with E. rhusiopathiae, H. parasuis, and A. suis but not B. bronchiseptica, S. suis, or S. hyicus.