Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody

The intestinal and systemic antibody response of calves vaccinated and/or challenged with rotavirus was studied employing isotype-specific ELISAs for the detection of IgG1, IgG2, IgM and IgA antibodies to rotavirus. Monoclonal antibodies to bovine immunoglobulin isotypes of proven specificity were used as conjugated or catching antibody. Five days after oral inoculation (dpi) of a 5-day-old gnotobiotic calf with rotavirus, IgM rotavirus antibodies were excreted in faeces, followed 5 days later by IgA rotavirus antibodies. The increase in IgM rotavirus antibody titre coincided with the inability to detect further rotavirus excretion. Faeces IgM and IgA rotavirus antibody titres fell to low levels within 3 weeks post infection. IgG1 and IgG2 rotavirus antibodies were not detected in faecal samples. In serum, antibodies to rotavirus of all four isotypes were detected, starting with IgM at 5 dpi. Two SPF-calves, which were fed colostrum free of rotavirus antibodies, were vaccinated with a modified live rotavirus vaccine and challenged with virulent rotavirus 6 days later. Upon vaccination, the calves showed an antibody response similar to the response of the infected gnotobiotic calf. Intestinal IgM rotavirus antibodies were excreted before or on the day of challenge and appeared to be associated with protection against challenge infection with virulent virus and rotavirus-induced diarrhoea. In 3 control calves, which were challenged only, the antibody patterns also resembled that of the gnotobiotic calf and again the appearance of IgM rotavirus antibodies coincided with the end of the rotavirus detection period. Two other groups of 3 SPF-calves were treated similarly, but the calves were fed colostrum with rotavirus antibodies during the first 48 h of life. These calves excreted passively acquired IgG1 and IgG2 rotavirus antibodies in their faeces from 2 to 6 days after birth. After vaccination, no IgM or IgA antibody activity in serum or faeces was detectable. Upon challenge, all calves developed diarrhoea and excreted rotavirus. Seven to 10 days after challenge low levels of IgM rotavirus antibody were detected for a short period. These data indicate that the intestinal antibody response of young calves to an enteric viral infection is associated with the excretion of IgM antibodies, immediately followed by IgA antibodies. This response is absent or diminished in calves with passively acquired specific antibodies which may explain the failure to induce a protective intestinal immune response by oral vaccination with modified live rotavirus of calves fed colostrum containing rotavirus antibodies.     

网状内皮组织增殖病病毒感染SPF雏鸡后局部体液免疫球蛋白含量变化

试验对ＳＰＦ雏鸡接种网状内皮组织增殖病病毒（ＲＥＶ）后,应用间接酶联免疫吸附分析法检测了泪液、气管液、肠液和胆汁的免疫球蛋白ＩｇＧ、ＩｇＭ、ＩｇＡ含量的动态变化。结果发现,感染ＳＰＦ雏鸡泪液、气管液、肠液和胆汁的ＩｇＧ、ＩｇＭ、ＩｇＡ含量于感染后７～４２ｄ明显降低,表明ＲＥＶ感染鸡的局部体液免疫能力降低     

Distribution of antibodies to rotavirus in serum and lacteal secretions of naturally infected swine and their suckling pigs

Rotavirus antibodies were demonstrated in lacteal secretions and sera of 20 parturient sows and in sera of their newborn by an enzyme-linked immunosorbent assay blocking technique, using bovine rotavirus cell culture antigen and monospecific antibody to bovine rotavirus. Antibodies to rotavirus occur in the 3 immunoglobulin (Ig) classes IgM, IgA, and IgG in lacteal secretions. High and long-persisting antibody activity was mainly associated with the IgA class. The IgM and IgG decreased to undetectable concentrations in most sows during the 14-day investigation period. Serum antibodies of newborn pigs nursing their dams also decreased rapidly during this time. The heterologous enzyme-linked immunosorbent assay blocking technique was a reliable and rapid procedure for the demonstration of rotavirus antibodies.     

Detection of antibodies against African swine fever virus using infected monolayers and monoclonal antibodies

Three monoclonal antibodies, specific for porcine IgG, IgM and IgA, were used to develop isotype-specific immunoperoxidase monolayer assays for the detection of antibodies against African swine fever virus. A mixture of anti-IgM and anti-IgG monoclonal antibodies was used in an assay designed for screening sera. This test was compared with a commercially available ELISA by using experimental sera and field sera obtained after an outbreak of African swine fever on two farms in the Netherlands in 1986. Although the ELISA was less sensitive than the immunoperoxidase monolayer assay on sera taken early after infection, the tests were equally useful for screening purposes. The isotype-specific assays gave epizootiological information about the stage of infection on the two farms.     

Measurement of isotype-specific antibody responses to Aujeszky's disease virus in sera and mucosal secretions of pigs.

Enzyme-linked immunosorbent assays (ELISAs) for the detection of porcine IgM, IgA, IgG1 and IgG2 antibodies directed against Aujeszky's disease virus (ADV) are described. ADV-specific IgA and IgM were detected in an antibody capture assay, and ADV-specific IgG1 and IgG2 were detected in an indirect double antibody sandwich assay. A selected set of samples was tested in the four ELISAs and in a 24 h virus neutralization assay. Comparison of the results showed that the ELISAs were isotype-specific, sensitive, and reproducible. Samples with ADV antibody of one isotype showed that ADV-specific IgG1, IgG2 and IgM were able to neutralize the virus in vitro. In vitro neutralization of virus can be enhanced by complement. ADV-specific IgA neutralized virus only weakly. ADV-infected cells activated complement in the absence of antibody. Specific IgG2 and IgM enhanced complement activation. Analysis of the time course of antibody responses after infection or vaccination revealed that the isotype-specific ELISAs are suitable to study the humoral antibody response of pigs to the virus in mucosal secretions. Wild-type virus (strain NIA-3) and an attenuated vaccine strain (Bartha) administered intranasally induced mucosal IgM and IgA responses to the virus. In contrast, a killed vaccine (Nobivac) administered intramuscularly induced only weak mucosal IgM responses. The attenuated vaccine strain primed for a mucosal IgA memory response evoked upon challenge infection with wild-type virus.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

H5N1亚型禽流感病毒感染SPF雏鸡的外周血液免疫病理变化

以7日龄SPF雏鸡为试验动物,应用细胞培养和免疫酶技术,通过对外周血液免疫球蛋白含量、T和B淋巴细胞数量及其功能的检测,较全面系统地研究了鹅源H5N1亚型中强毒禽流感病毒(AIV)感染SPF雏鸡后,其外周血液上述指标的动态变化。结果发现,SPF雏鸡感染鹅源H5N1亚型AIV后,血清IgG和IgA含量在1~4d显著或极显著低于对照雏鸡(P<0.05或P<0.01),而IgM在病毒感染早期未见显著性差异,随后3种免疫球蛋白含量逐渐回升;血液T淋巴细胞数量显著低于对照雏鸡(P<0.05或P<0.01),而B淋巴细胞在1~5d明显降低(P<0.05或P<0.01);T、B淋巴细胞对ConA或PMA的增殖反应分别于1~8d、1~4d明显低于对照雏鸡(P<0.05或P<0.01)。上述结果表明,AIV感染SPF雏鸡外周血液无论是细胞免疫还是体液免疫功能均呈现一定抑制。     

Measurement of Pasteurella haemolytica-specific lung and serum antibodies by ELISA

The modified enzyme-linked immunosorbent assay (ELISA) was used to determine the relative quantities of class-specific antibodies to Pasteurella haemolytica. IgG1, IgG2 and IgA were present in significantly higher quantities in bronchoalveolar washings (BAW), but in decreasing quantities, respectively; IgM was present in very low amounts. IgM, IgG1 and IgG2 were present in serum, again in decreasing quantities, respectively. IgA antibody quantities were lowest in serum. The indirect antibody ELISA was found to be superior to the indirect bacterial agglutination (IBA) technique for determining antibody titres against P. haemolytica.     

Lachrymal antibody response of specific pathogen free chickens and chickens with maternal immunity after infectious bronchitis virus vaccination

Lachrymal fluid specific IgG and IgA were detected by ELISA in chickens with specific maternal antibodies and in chickens free of antibodies (SPF), after vaccination at 1 day of age with the H-120 vaccine strain of infectious bronchitis virus. Samples were obtained at 3 day intervals and until Day 37 of age. Optical densities were in all instances low but significant differences could be detected within and between the experimental groups. Both class-specific immunoglobulins showed a similar kinetic pattern. Nevertheless, the SPF group increased its IgA level on Day 13 while chickens with maternal immunity increased their level on Day 16. The antibody levels of both IgG and IgA were also different, being higher in the SPF group. In both chicken groups, higher levels of IgA than IgG were detected.     

Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.     

Immunoglobulin class distribution of systemic and mucosal antibody responses to Newcastle disease in chickens

In serum, tracheal wash fluid, and bile from chickens that were inoculated with live or inactivated Newcastle disease virus (NDV), the kinetics and immunoglobulin (Ig) class distribution of an antibody response were demonstrated. The Ig classes (IgM, IgG, and IgA) were captured using monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (Ig-capture ELISA). The antibody specificity of the captured Ig was confirmed by binding of NDV. After inoculation with live virus, antibodies of the IgG and IgM classes were mainly found in serum. IgM was produced early from day 4 postexposure (PE) onward, IgG was detected later from day 7 PE onward, and in the tracheal wash fluid and bile, all three Ig classes were demonstrated. After inoculation of inactivated virus, a delayed response of all three classes was observed in serum, and only IgM and IgG were recognized in the tracheal fluid and bile. The type of vaccine and the mute of antigen entrance may have determined the immunoglobulin class produced. The Ig-capture ELISA assay developed in this study can be useful for evaluating various strategies to improve the efficacy of Newcastle disease vaccines and to study the evoked immune mechanisms.     

免疫增强剂对IBD疫苗免疫雏鸡局部黏膜免疫功能的影响

分别将免疫增强剂“禽福”和“Inmunair”与鸡传染性法氏囊病（IBD）中等毒力活疫苗配合使用,检测免疫雏鸡哈德尔氏腺和盲肠扁桃体T细胞及IgA、IgM和IgG抗体生成细胞数量。泪液、气管液、胆汁、肠液中免疫球蛋白IgG、IgM、IgA含量的动态变化,结果发现,应用免疫增强剂IBD疫苗免疫雏鸡上述局部免疫组织及四种局部体液的相应各项免疫检测指标均较IBD疫苗单独免疫雏鸡不同程度地增高,表明免疫增强剂与疫苗联合应用,能显著提高疫苗免疫雏鸡眼部、呼吸道和消化道局部黏膜免疫功能;而IBD强毒攻击后,疫苗单独免疫雏鸡上述指标明显低于应用免疫增强剂的疫苗免疫雏鸡,同时。后者对IBD强毒攻击的有效抵抗力明显高于前者。     

一株H5亚型禽流感病毒N P蛋白两个鸡M HC分子限制性T细胞表位的鉴定

为验证一株禽流感病毒(AIV)核蛋白(NP)中的3条T细胞表位候选肽NP89-97(PKKTGGPIY)、NP198-206(KRGINDRNF)和NP64-71(ERMVLSAF)的免疫原性,本研究构建了含有NP基因的重组质粒pCAGGS-NP,将其转染293T细胞,间接免疫荧光和western blot检测表明NP蛋白在293T细胞中获得表达.重组质粒免疫鸡后,用间接酶联免疫实验检测发现重组质粒在鸡体内能诱导产生相应的抗体.将多肽NP89-97、NP198-206、NP64-71和不相关肽N71-78(WRRQARYK)在体外刺激免疫后鸡的脾淋巴细胞,流式细胞术检测发现CD8+T淋巴细胞增殖分别为13.7%、11.9%、0.6%和0.4%;ELISA检测结果显示多肽NP89-97、NP198-206刺激后的淋巴细胞中IFN-γ的分泌量明显增加.本实验表明多肽NP89-97和NP198-206能在体外诱导活化的鸡淋巴细胞产生细胞毒性T淋巴细胞反应,为AIV NP的T 细胞表位.该研究结果对研究禽类抗流感病毒的免疫机制及抗流感疫苗具有重要的意义.     

鸭源鸡杆菌抗体间接ELISA检测方法的建立及应用

利用鸭源鸡杆菌YU-PDS-RZ-1-SLG分离株制备超声波裂解抗原,建立了可以检测鸭源鸡杆菌多个血清型抗体的间接ELISA方法。包被抗原质量浓度为10mg/L,包被条件为37℃2h,再4℃过夜;封闭液为1%明胶,封闭条件为37℃1h;阴、阳性血清最佳稀释度为1∶100,酶标二抗工作滴度为1∶1 000;底物显色时间为15min。经交叉性试验、阻断试验和重复性试验证实建立的ELISA方法重复性好,特异性强。板内变异系数为2.01%～5.75%,板间变异系数为2.43%～6.20%。间接ELISA方法的灵敏度是微量凝集试验的25～100倍。利用所建立的ELISA方法检测了人工感染鸭源鸡杆菌的4日龄SPF鸡在感染后不同阶段感染组、同居组和空白对照组的血清抗体,并根据感染后不同阶段所测D450值绘制抗体消长曲线,其抗体水平在感染后32～47d开始上升,60d时达到高峰,但维持时间较短,2周后迅速下降。建立的间接ELISA方法可以用于临床病例的血清学快速检测,为进行鸭源鸡杆菌的血清流行病学调查提供了手段。     

A longitudinal study of rotavirus antibody titers in swine in a closed specific pathogen-free herd.

In a newly established closed specific pathogen-free (SPF) swine herd, gilt/sow suckling and weaned pig rotavirus specific antibody titers were followed for three lactations by enzyme-linked immunosorbent assay (ELISA) to gain insight into the dynamics of herd antibody titers to group A rotavirus. Among gilts/sows, serum antirotavirus IgG titers increased during each lactation with a subsequent drop in titer between farrowings. Serum antirotavirus IgM titers declined during each lactation and with subsequent parity. Serum antirotavirus IgA titers remained constant during lactations and among parities. In colostrum and milk, antirotavirus IgA antibody was abundant. Differences in titer were not noticed between gilts and second litter sows but third litter sows had significantly higher titers than the first two groups. Antirotavirus IgG was high in colostrum but nearly nonexistent in milk. This titer did not vary significantly within or among parities. There was a linear regression in the titers of baby pig serum antirotavirus IgG from the post colostral sample through to seven weeks old, after which titer began to increase. No difference in baby pig serum antirotavirus IgG was noted among the three litters. Serum antirotavirus IgA and IgM were undetectable in baby pig sera after 2-3 weeks of age. Coproantibody to rotavirus was sporadically present in pig feces for 2-3 weeks after birth with highest titers in the IgA fraction. We conclude that although it is probable that age resistance of pigs to rotavirus diarrhea occurs, humoral immunity as measured by ELISA rotavirus antibody titers may not be intimately involved in virus clearance since in our studies baby pigs passively received large amounts of antibody but still excreted pathogenic virus. The finding of increasing levels of serum antirotavirus IgG in gilt/sow serum suggest that exposure to antigen of dams occur without significant increases in antirotavirus IgG titers in either colostrum, milk, or baby pig serum.     

Development of intestinal antibodies against Escherichia coli antigens in piglets with experimental neonatal E. coli diarrhoea

Intestinal immune responses to Escherichia coli antigens were studied in conventionally reared piglets orally infected on the first day of life with a virulent enterotoxigenic E. coli (O149: K88). During the first week of life intestinal antibodies were produced against the homologous lipopolysaccharide (LPS) as well as against the K88 antigen and the heat-labile enterotoxin (LT). On Day 7, anti-LPS antibodies of the IgA and IgG classes were detected in most piglets, whereas anti-K88 antibodies of the IgG and IgM classes predominated; antibodies against the enterotoxin were usually of the IgG class. In 21-day-old piglets antibodies of all immunoglobulin classes had usually been produced. In most cases, the levels of intestinal antibodies were substantially higher on Day 21 compared to Day 7, but the levels varied considerably both between and within litters. The intestinal immune responses did not correlate with the severity of clinical symptoms. One-, 7- and 21-day-old piglets reared in a specific-pathogen-free (SPF) herd lacked significant intestinal antibodies to the antigens examined. The oral challenge did not stimulate systemic immune responses. After colostral intake, all piglets had high antibody levels in the circulation. These levels decreased continuously during the 3-week study period. The possibility that high amounts of antibodies in colostrum could interfere with this early intestinal antibody formation should be considered when planning vaccination programmes against E. coli diarrhoea in piglets.     

Detection of antibody-forming cells directed against Newcastle disease virus and their immunoglobulin class by double immunoenzyme histochemistry

Antibody-forming cells (AFCs) against Newcastle disease virus (NDV) and their immunoglobulin (Ig) class were demonstrated by a double immuno-enzyme histochemical technique. The AFCs were stained and quantified in spleen sections of chickens euthanatized at day 7 postexposure to the Roakin strain of NDV. The sections were incubated with NDV to determine the specificity of the AFCs. Bound virus was subsequently visualized with a primary monoclonal antibody (MAb), a secondary horseradish peroxidase-conjugated MAb, and 3-amino-9-ethylcarbazole as substrate. IgM and IgA were stained with MAbs and an alkaline phosphatase (AP)-conjugated secondary antibody. IgG class antibodies were demonstrated with an AP-conjugated rabbit serum. The final substrate for the three Igs was naphthol AS-MX-phosphate and fast blue BB. About 64-159/mm2 AFCs against NDV were detected. Of these virus-binding cells, about 55% produced IgM, 37% produced IgG, and the remaining 8% produced IgA.     

感染鸡贫血病毒雏鸡接种新城疫疫苗后局部粘膜免疫功能的变化

雏鸡1日龄感染鸡贫血病毒（CAV），8日龄接种Lasota疫苗，以未感染免疫雏鸡为对照，于免疫后7、14、28d检测其哈德尔腺和盲肠扁桃体T细胞及IgG^ 、IgM^ 、IgA^ 抗体生成细胞数量，泪液、气管液、肠液、胆汁中IgG、IgM、IgA含量以及泪液、胆汁HI抗体滴度的动态变化。揭示了感染CAV雏鸡接种ND疫苗免疫后哈德尔腺、盲肠扁桃体的T细胞和IgG^ 、IgM^ 、IgA^ 抗体生成细胞数量，泪液、气管液、肠液、胆汁中免疫球蛋白IgG、IgM、IgA含量以及泪液、胆汁HI抗体滴度，均较未感染免疫雏鸡明显减少。表明眼部、呼吸道和消化道局部粘膜免疫防御能力减弱。     

Monoclonal precipitating antibodies to porcine immunoglobulin M

Fusion of splenic immunocytes from a porcine IgM-immunized BALB/c mouse with SP2/0 mouse myeloma cells resulted in 231 primary hybrids. Culture fluids of the primary hybrids were screened for antibody production by enzyme-linked immunosorbent assay (ELISA) against porcine IgM and by radial immunodiffusion versus porcine serum. Culture fluids of 10 of the primary hybrids were positive in IgM-ELISA and radial immunodiffusion. Six of these primary hybrids (1A11, 1D10, 2D7, 2E2, 3B11, and 5C9) were cloned, and ascitic fluids were produced using cloned primary hybrids. The monoclonal antibodies (Mabs) in ascitic fluids were characterized as to their reactivity with porcine immunoglobulin isotypes. All six Mabs had mouse IgG1, K isotype and were mu-chain specific as they formed single precipitin lines against porcine serum and porcine IgM and no lines against porcine IgG, IgA, and fetal porcine serum in immunodiffusion and immunoelectrophoresis. In indirect ELISA, all Mabs reacted with porcine serum, porcine IgM, and mu-chains but did not react with porcine IgG, IgA, or light chains. All six Mabs were species-specific and recognized either of two antigenic regions of mu-chain. These Mabs have been successfully used to detect IgM-containing cells in tissue sections, to detect IgM in serum, and to quantitate surface membrane IgM-bearing cells in peripheral blood.     

毒害艾美耳球虫初次感染对雏鸡外周血液免疫功能的影响

应用免疫SPA菌体花环、间接ELISA及细胞培养技术和四甲基偶氮唑盐(MTT)测定法对毒害艾美耳球虫(Eimerianecatrix)初次感染雏鸡外周血液的T、B细胞数量及其对ConA或PMA的增殖功能和IgG、IgM、IgA含量的动态变化进行了较全面系统的研究。结果发现,E.necatrix初次感染雏鸡外周血液的T、B细胞数量及其相应增殖功能、血清中上述三种免疫球蛋白含量均不同程度地高于未感染的对照雏鸡,表明E.necatrix初次感染雏鸡外周血液的细胞免疫和体液免疫功能均明显提高。