鸡传染性支气管炎ELISA抗体检测试剂盒的研制

采用加蔗糖垫离心纯化鸡传染性支气管炎病毒(IBV)制备抗原，用提纯的IBV抗原包被微量板，建立了检测鸡传染性支气管炎(IB)抗体的ELISA试剂盒。抗原、被检血清和酶标结合物的最佳工作浓度分别为10ug／ml、1：200和1：3200。与IDEXX试剂盒相比，其敏感性、重复性、特异性均接近国外同类产品水平。对SPF鸡血清、实验免疫与攻毒的SPF鸡血清进行检测，结果表明所建立的ELISA特异性为95．6％，与IDEXX试剂盒符合率为95．6％。用于检测抗IBV特异性IgG抗体发现在免疫接种IB弱毒苗后，第4天即可检测到IgG抗体，峰值在第3周。试验鸡在通过滴鼻、点眼途径人工感染IBV强毒后，第5天抗体滴度明显上升。我们认为，该法是目前我国SPF鸡质量监测、养鸡生产中进行IB监测较好的方法。     

Single dilution ELISAs using soluble piroplasm, cellular schizont and soluble schizont antigens for the detection of antibodies against Theileria annulata

Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log10 antibody titres by multi-dilution ELISA. The value of "r" was the highest at the dilution of 1:400. From the log10 antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a + bX, where Y = predicted log10 titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 + 1.35X for soluble piroplasm, Y = 2.67 + 0.547X for cellular schizont and Y = 1.817 + 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multidilution ELISAs showed non-significant differences (P < 0.01) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.     

Use of a constant-virus diluting-serum microneutralization technique for the detection and quantification of infectious bronchitis virus antibodies

A constant-virus diluting-serum microneutralization test (CVMNT) for avian infectious bronchits virus (IBV) was evaluated for both reliability and repeatability. The virus used in the assay was a chick kidney (CK) cell-adapted strain, the Beaudette strain (IBV 42). Sera tested were from 24-week-old broiler-breeder chickens that had been vaccinated 3 times from a combination vaccine of Newcastle disease virus (NDV) and IBV. Test results were not repeatable or comparable when the same sera were tested on different days, but test results were repeatable and comparable when the sera were tested on the same day. Differences in virus titer at the different times that tests were performed appeared to cause the variation in test results. A comparison was made between the CVMNT and a constant-serum diluting-virus microneutralization test (CSMNT). The CVMNT was able to detect differences in flock antibody titers that the CSMNT could not.     

Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite

Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi disease-positive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey/horse sera obtained by serial and single dilution ELISA using the WM antigen did not show any significant difference. Since the single dilution ELISA was found to be more economical, convenient, sensitive, specific than the serial dilution ELISA and has a high predictive value, it is suitable for use in sero-epidemiological studies on B. equi infections in the field.     

鸡传染性支气管炎病毒血凝抑制试验抗原的研制

为应用血凝抑制(HI)试验检验鸡传染性支气管炎疫苗免疫效力(血清、卵黄抗体水平),建立了鸡传染性支气管炎病毒HI试验抗原的制备方法。该方法是通过选取抗原谱最广的鸡传染性支气管炎病毒(IBV)M41株,经SPF鸡胚增殖培养36h后无菌收取鸡胚尿囊液,4℃、12 000r/min离心10min,上清用聚乙二醇(PEG)20000浓缩100倍;兔源A型产气荚膜梭菌中国标准株(C57-1株)37℃增殖培养18h,4℃、12 000r/min离心10min取上清,经PEG 20000透析袋浓缩5倍后通过0.20μm滤膜过滤除菌,然后将IBV液和A型产气荚膜梭菌菌液(含α毒素)二者按一定比例混合后经37℃恒温振荡感作2h,4℃经48h后制成。经大量试验表明,制备的IBV HI试验抗原效价高、稳定性好,可替代进口抗原应用于鸡群IBV疫苗免疫后血清抗体及卵黄抗体的HI效价检测。     

不同鸡新城疫疫苗免疫鸡血清HI抗体的测定

将试验鸡分成3个试验组和1个对照组。A组鸡接种鸡新城疫系疫苗,B组鸡接种油乳剂灭活苗,C组鸡接种鸡新城疫系疫苗,并在接种疫苗后第3、4、5、6、7、9、11、13、15、20、25d采取各组鸡血并分离血清,检测HI抗体。结果表明,接种系疫苗的组,HI抗体效价均值从4.67log2上升到10log2,接种后第5d开始上升,接种后第11d达到峰值,持续6d保持高滴度抗体水平。接种系疫苗的组,HI抗体效价均值从4.67log2上升到7log2,接种后第4d开始上升,接种后第9d达到峰值。接种油乳剂灭活苗的组,HI抗体效价均值从4.67log2上升到9.33log2,接种后第5d开始上升,接种后第11d达到峰值,持续16d保持高滴度抗体水平。系疫苗HI抗体效价上升快,效价高,较适合于紧急接种,油乳剂灭活苗HI抗体效价可在高水平维持较长时间,较适合于预防接种。     

Effect of four adjuvants on immune response to F4 fimbriae in chickens

IgG antibody response in chickens immunized with F4 fimbriae extracted from local enterotoxigenic Escherichia coli (ETEC) strain was studied during a 98-day immunization period for comparing the efficacy of four adjuvants: Freund' adjuvant, Quil A (QA), propolis and extract from Cochinchina momordica seed (ECMS). For this purpose, chickens were immunized with F4 fimbriae alone or combined with one of the above adjuvants on days 1 and 21. IgG antibody levels in serum and egg yolk (by ELISA) were measured on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 70, 84 and 98. The egg production of each group was also determined during days 1-7 and the following four weeks. The results showed that QA could enhance antibody titre, as good or almost as good as Freund's adjuvant, whereas the titres of ECMS and propolis groups were relatively lower, with the overall order: Freund's adjuvant>QA>ECMS>propolis both in serum and egg yolk. However, the significant decrease of egg production was merely observed in the Freund's adjuvant group. It is concluded that the four adjuvants tested can stimulate immune response to F4 fimbriae in chickens, with Freund's adjuvant giving the best results, followed by QA.     

Effect of feed restriction early in life on humoral and cellular immunity of two commercial broiler strains under heat stress conditions

1. An experiment was carried out to evaluate the effect of early feed restriction (FR) on immunocompetence of Ross and Arian chickens with separated sexes under heat stress (HS) conditions. 2. Chickens consumed feed ad libitum (AL) or were restricted on alternate days from 11 to 20 d of age. From 35 to 41 d of age, the HS groups were exposed to a high ambient temperature of 39 +/- 1 degreesC for 7 h each day, while the thermoneutral groups (TN) were at 33 degrees C. 3. At 21 and 42d of age, the percentage of CD4+ (helper T cells) and CD8+ (cytotoxic T cells) were determined by flow cytometry. Antibody response to sheep red blood cells (SRBC) and heterophil/lymphocyte (H/L) ratio were determined on d 21 and 42. 4. On d 21, FR elevated the CD4+, antibody titre and H/L ratio, but it decreased the CD8+ T cells. On d 42, HS decreased CD4+, CD8+, and antibody titre, but it increased H/L ratio. Under TN conditions, FR chickens had higher CD4+ than AL chickens. On d 42, FR/HS chickens had higher CD4+ and antibody titre, but they had lower CD8+ and H/L ratio than AL/HS chickens. 5. On d 42, the TN-Ross strain had lower CD4+, but they had higher CD8+ and antibody titres than the TN-Arian strain. On d 42, the HS-Arian strain had higher antibody titres and a lower H/L ratio than the HS-Ross strain. 6. Male chickens had higher CD4+, CD8+, antibody titres and H/L ratios 25 in all treatment groups. 7. In conclusion, FR early in life reduced some of the negative effects of the heat stress on the immune system of broiler chickens when exposed to high environmental temperatures later in life.     

A standardized enzyme-linked immunosorbent assay for infectious bronchitis virus: comparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody levels in chickens

An enzyme-linked immunosorbent assay (ELISA) was developed to measure antibodies to infectious bronchitis virus (IBV) in chickens. The results are reported in IBV standard ELISA values calculated by comparing antibody levels in test sera with antibody levels in a series of standard reference sera. The IBV standard ELISA values were good indicators of responses to vaccination and the immune status of experimentally challenged birds. Although the assay was not serotype-specific, the sensitivity makes it ideally suited for determining the immune status of poultry flocks. The assay results compared favorably with other laboratory results, including virus-neutralization titers, hemagglutination-inhibition levels in sera, virus isolation from vaccinated/challenged birds, and the tracheal ring test results.     

Demonstration of circulatinf antibodies to Eimeria tenella by the indirect immunofluorescent antibody test using sprozoites and second-stage schizonts as antigen

In the indirect immunofluorescent antibody (IFA) test using sporozoites as an antigen, sera from chickens immunized via the natural route were positive in dilutions as high as 1 : 1 048. Serum from a rabbit, immunized only to sporozoites by subcuntaneous injection, was positive in a dilution of 1 : 4 4 096. Non-immunized chicken andn rabbit sera were positive in dilutions varying from 1 : 20 to 1 : 64.Sporozoites within sporocytes were not stained. In frozen sections of infected chorioallantoic membranes and ceca, sporozoites were not traced with the IFA test with immunized chicken or rabbit sera. However, with the rabbit serum specific diffuse intraepithelial and subepithelial fluorescence was observed in the ceca from 4 to 11 h after infection.Fluorescence was never associated with the first-stage schzonts and gamates, but second-stage schizonts were positive with both chicken and rabbit serum. The titres obtained with this antigen were about the same as those obtained with sporozoite smears. The possible presence of common antigens in sporozoites and second stage schizints is discussed.     

Ciliary activity: a criterion for associating resistance to infectious bronchitis virus infection with ELISA antibody titer

Vaccination and challenge experiments using infectious bronchitis virus (IBV) were conducted on groups of specific-pathogen-free chickens. Three weeks post-vaccination with one of the four IBV strains used, chickens were challenged with the homologous immunizing strain of IBV. Subsequently, the chickens were sacrificed, their tracheas were examined for ciliostasis, and the specific IBV antibody content of their sera was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that protection was conferred by primary vaccination, as ciliostasis was not observed in tracheas from groups vaccinated and then challenged. No protection was observed in control groups that received only a challenge exposure, and the virus was readily recovered from their tracheas. Homologous protection was present in chickens that had ELISA titers as low as 624 and neutralization indices as low as 2.9, whereas susceptible controls had titers of less than 100 and less than 1.0, respectively.     

Immunizing efficacy of aromatic-dependent Salmonella dublin in mice and calves

Mice immunized with an aromatic-dependent (aro-) S. dublin strain CS101 by either the intraperitoneal (i.p.) or oral route, were protected against oral challenge with a virulent S. dublin strain CS90, the degree of protection being the greatest when mice had received 3 immunizing doses at weekly intervals. Mice immunized with an aromatic-dependent (aro-) S. typhimurium strain CS332 by the i.p. or oral routes were protected against challenge with virulent S. dublin strain CS90 at 1 or 2 weeks but not at 3 or 4 weeks post-immunization. Mice immunized with 1 dose of aro- S. dublin strain CS101 by the i.p. route developed low levels of lipopolysaccharide (LPS) and flagellin-specific antibody but no delayed-type hypersensitivity (DTH) whereas those immunized with 2 or 3 doses developed significantly higher antibody titres and DTH. In contrast, mice immunized by the oral route developed neither significant antibody response nor DTH. The aro- S. dublin strain CS101 could not be detected beyond day 28 post-inoculation in visceral organs including liver, spleen, mesentery, small intestine, caecum or large intestine of mice inoculated by the i.p. route or in mice inoculated by the oral route with the exception of day 42 post-inoculation. Challenge of mice previously immunized with 3 doses of the aro- S. dublin strain CS101 by the i.p. or oral route with virulent S. dublin strain CS90 resulted in their rapid clearance from the above visceral organs. Calves immunized with the aro- S. dublin strain CS101 by either the intramuscular (i.m.) or oral routes were significantly protected against oral challenge with virulent S. dublin strain CS90. In contrast to the observations in mice, somatic (O) and flagellar (H) antibody titres of calves immunized by either route were negligible as were anti-LPS antibody titres. However, flagellin-specific antibody titres were higher in calves immunized by the i.m. than the oral route. These results indicate that the protection observed in immunized mice or calves against oral challenge with virulent S. dublin was unlikely to have been mediated by humoral salmonella-specific immune mechanism(s).     

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.     

鸡滑液囊支原体活疫苗(MS-H株)同时免疫对新支二联活疫苗免疫效力影响的调查

为了解新支二联活疫苗和鸡滑液囊支原体活疫苗之间有无相互影响,选用70只1日龄SPF鸡随机分为4组进行比对试验,其中:G1组20只,联合免疫新支二联苗活疫苗和鸡滑液囊支原体活疫苗(MS-H株);G2组20只,为新支二联活疫苗单独免疫组;G3组20只,为传染性支气管炎病毒(IBV)攻毒组;G4组10只,为空白对照组.免疫后...     

禽传染性支气管炎病毒单克隆抗体的制备及其B细胞抗原表位的鉴定

为鉴定传染性支气管炎病毒(IBV)的抗原表位,本研究将IBV ck/CH/LDL/091022株免疫6周龄～8周龄BALB/c小鼠,将小鼠脾脏细胞与骨髓瘤细胞SP2/0融合,经过有限稀释法克隆筛选,得到一株针对IBV核衣壳(N)蛋白的单克隆抗体(MAb)2F2,经鉴定其染色体数目为101条,MAb亚类鉴定其重链属于IgG1,轻链属于K链.用肽扫描方法将IBV ck/CH/LDL/091022株N基因截短为具有相互部分重叠的23段,表达带GST标签的重组蛋白,与MAb 2F2进行western blot和ELISA反应后,鉴定其表位为397INWGDSAL404.将表位序列进行Blast结果表明,本研究鉴定的表位在大部分IBV株中均为保守抗原表位,为进一步建立IBV的检测方法奠定了基础.     

Detection of infectious bronchitis virus antigen from experimentally infected chickens by indirect immunofluorescent assay with monoclonal antibody

Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis.     

Pathological and immunological study of an in ovo complex vaccine against infectious bursal disease

The appearance of very virulent strains of infectious bursal disease (IBD) virus at the end of the 1980s made it necessary to develop more effective immunization procedures. To facilitate this, the immunogenicity and the immunosuppressive effect of a mild (G-87), an intermediate (LIBD) and an intermediate-plus (IBDV 2512) IBDV strain were tested after the in ovo inoculation of 18-day-old SPF and broiler chicken embryos. It was established that no noteworthy difference existed between the immunized and the control embryos in hatching rate and hatching weight. The higher the virulence of the vaccine virus strain, the more severe damage it caused to the lymphocytes of the bursa of Fabricius. In SPF chickens, the haemagglutination inhibition (HI) titres induced by a Newcastle disease (ND) vaccine administered at day old decreased in inverse ratio to the virulence of the IBD vaccine strain, while in broiler chickens this was not observed. Despite the decrease of the HI titre, the level of protection did not decline, or did so only after the use of the 'hot' strain. SPF chickens immunized in ovo with a complex vaccine prepared from strain IBDV 2512 and IBD antibody showed the same protection against Newcastle disease as the broilers. In broiler chicken embryos immunized in ovo, only strain IBDV 2512 induced antibody production, and such chickens were protected against IBD at 3 weeks of age. The complex vaccine administered in ovo has been used successfully at farm hatcheries as well.     

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).     

鸡传染性支气管炎病毒HN99株血清学和免疫学特性的研究

在鸡胚肾细胞上对HN₉₉株病毒进行了克隆纯化，经检测无外源病原污染。应用血清交叉中和试验确定了IBV HN₉₉株的血清型，并用免疫保护试验进行了验证，结果证明HN₉₉株与T株属同一血清型，与TJ、SD、湖北等地方分离株有一定的相关性。不同免疫剂量的效力试验结果表明用0.2 ml剂量的HN₉₉灭活苗接种试验鸡，能够对HN₉₉攻毒提供100%的保护，证明HN₉₉株具有良好的免疫原性。     

Rapid serological profiling by enzyme-linked immunosorbent assay. II. Comparison of computational methods for measuring antibody titer in a single serum dilution

An enzyme-linked immunosorbent assay (ELISA) was used to measure specific antibody activity from a single serum dilution in sera of chickens exposed to Newcastle disease virus (NDV). Observed endpoint titers were used to formulate regression equations, and then absorbance data obtained at a single serum dilution were converted directly to antibody titer by three methods: a correction factor method, a subtraction method, and a double-regression method. Each method was evaluated for three criteria: the overall stability of between-test antibody titer for control sera, the linearity of the relationship of the absorbance values at a single working dilution to the observed antibody titers, and the method's accuracy in predicting titers. Although a nearly linear relationship was obtained for all treatment methods examined, the double-regression method provided the best reduction of between-test titer variation and also best predicted titers.