法氏囊提取液对新城疫活疫苗免疫效力的影响

本文对鸡法氏囊提取液与ND活疫苗的使用方法进行了分析。通过血凝抑制抗体滴度的测定结果表明,预先接种鸡法氏囊提取液时,经肌肉注射、口服和点眼滴鼻接种方式均能提高ND疫苗免疫鸡的抗体产生能力,且经肌肉注射法最佳。但是,在整个试验期间,法氏囊提取液只能增强鸡群对ND活疫苗的早期免疫应答,而法氏囊提取液与ND活疫苗同时使用时更能刺激免疫鸡群的早期抗体产生水平。     

Ethanol euthanasia and its effect on the binding of antibody generated against an immunogenic peptide construct.

Mice were immunised with an immunogenic peptide construct CKNNNSTNSGI coupled to diphtheria toxoid as a carrier. This peptide sequence contains the epitope STNS which is the target of inhibitory monoclonal antibodies directed against the second merozoite surface antigen of Plasmodium falciparum. Antisera raised against the peptide construct were taken using an injection of 70 per cent ethanol or sodium pentobarbitone as methods of euthanasia and these methods compared by determining their effects on the binding specificity of the antibody to the antigen using the immunological criteria of immunofluorescence, immunoblotting criteria of immunofluorescence, immunoblotting and ELISA assays. There was no significant decrease in antibody binding with either sodium pentobarbitone, or ethanol with a final concentration of less than 30 per cent in mouse antisera. Antisera with an added ethanol concentration of 40 to 60 per cent relaxed antibody conformation and this raises the possibility of using the differential effects of ethanol as a tool in mapping antigenic fine structure of a range of antibodies directed against defined epitopes. The cross-reactive response of non-specific antibodies in polyclonal antisera was lowered at the suggested dosage for ethanol euthanasia. Ethanol has immense potential as an alternative method of euthanasia when barbiturate drugs, such as sodium pentobarbitone, are unavailable in specific experimental protocols. This may especially aid research workers in developing countries involved in vaccine development, antibody production and subsequent serological analysis.     

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specific-pathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.     

Pathogenicity of fowl adenovirus isolated from gizzard erosions to immuno-suppressed chickens

Pathogenicity of a fowl adenovirus (FAV), JM1/1 strain of serotype 1 derived from gizzard erosions of a broiler chicken, was examined to specific pathogen-free (SPF) chickens pre-treated with infectious bursal disease viruses (IBDVs) or cyclophosphamide (CY). Virulent IBDVs, classical type, were inoculated orally at 3 days of age of SPF chickens. CY was treated subcutaneously for 3 days after hatch. FAV was given orally at 30 days of age. At 40 days of age, all chickens were bled and autopsied for serology and gross observation. Gizzard lesions were ranked by the scores depending on their severities. IBDV- or CY-treated chickens showed significantly higher gizzard lesion scores than non treated birds. There were no gross lesions in any other organs except for bursal atrophy. Serologically, antibody production against FAV was highly suppressed by IBDV infection or CY treatment.     

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。     

Effect of bursal cell number on the pathogenesis of infectious bursal disease in chickens

The pathogenesis of infectious bursal disease (IBD) in chickens neonatally chemically bursectomized (CB) by cyclophosphamide and subsequently inoculated with various numbers of bursal cells was examined. CB chickens inoculated with at least 62.5 X 10(6) bursal cells were as susceptible to IBD clinical manifestations (as determined by gross and microscopic evaluation of bursal tissues, virus recovery from spleen, and antibody titer) as intact chickens following inoculation with virus at 5 weeks of age. In contrast, CB chickens inoculated with 2.5 X 10(6) or fewer bursal cells were refractory to the IBD clinical manifestations compared with intact chickens or CB chickens inoculated with 62.5 X 10(6) or more bursal cells. Results from this study suggest that the availability of a large number of bursal cells is an essential factor in the development of IBD.     

A study of the epidemiology of infectious bursal disease in poultry in Queensland, 1976–1979

The epidemiology of infectious bursal disease (IBD) was studied by serology and sometimes by visual examination of the bursa of Fabricius in poultry flocks in Queensland during 1976–1979.
Ten flocks, each of approximately 30,000 meat breeding chickens, were surveyed. All chickens had maternally-derived antibody against IBD virus (IBDV) at hatching and active antibody was not detected while the chickens were brooded on rearing farms. When distributed to breeding farms, 7 of the flocks developed antibody when 11 to 25 weeks of age. The remaining 3 flocks were vaccinated by infection of 10% of the birds and within 4 weeks more than 80% of the chickens had developed precipitating antibody to IBDV.
Blood samples of 20 to 30 broiler chickens were collected at slaughter (7 to 9 weeks of age) from each of 312 broiler flocks raised on 37 contract farms. While the samples from 21 flocks were without detectable antibody to IBDV, all serum samples for 263 flocks contained antibody. The ratio of bursal weight to bodyweight was significantly lower in birds from 144 flocks having antibody to IBDV than in birds from 10 flocks that were without detectable antibody. In sequential studies, IBDV antibody became demonstrable in 27 of 30 flocks when the chickens were one to 6 weeks of age and was accompanied by bursal atrophy.
Serological investigation of 4 flocks of layer breeding chickens on a multi-age farm at approximately monthly intervals resulted in antibody to IBDV being detected at every examination.
Serological tests and bursal examinations were carried out weekly in 2 flocks each of 4000 layer chickens between one and 20 weeks of age. Serum antibody developed in one flock at 4 weeks of age and in the other at 17 weeks of age. In both flocks, bursal atrophy occurred concurrently with the development of antibody.     

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

禽网状内皮组织增生病病毒感染对SPF雏鸡法氏囊细胞Bcl-2和C-myc基因表达及凋亡的影响

【目的】 探究禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)感染禽类后导致免疫器官发生细胞凋亡的机理。【方法】 以1日龄SPF雏鸡为试验对象,将100只SPF雏鸡随机均分为REV感染组和未感染病毒的对照组,REV感染组雏鸡经腹腔感染500 μL REV稀释液,对照组雏鸡经相同途径注射等量灭菌生理盐水,于病毒感染后第1、7、14、21、28和42天,2组雏鸡随机各抽取5只,心脏采血处死雏鸡后快速摘取法氏囊。分别应用HE染色和病理切片成像系统测定分析法氏囊细胞核浆比,TUNEL细胞凋亡原位检测试剂盒测定凋亡细胞数,免疫组化法测定Bcl-2和C-myc阳性细胞数量,实时荧光定量PCR和ELISA法分别检测法氏囊Bcl-2和C-myc 基因mRNA表达和蛋白含量。【结果】 ①REV感染1日龄SPF雏鸡后21~42 d,其法氏囊淋巴细胞凋亡百分比显著或极显著高于对照组雏鸡(P<0.05;P<0.01);②SPF雏鸡感染REV后21和28 d,其法氏囊细胞核浆比显著低于对照组雏鸡(P<0.05);③REV感染SPF雏鸡法氏囊中Bcl-2和C-myc阳性细胞数在病毒感染后21和28 d显著高于对照组雏鸡(P<0.05);④REV感染SPF雏鸡后21 d,其法氏囊Bcl-2和C-myc 基因mRNA表达极显著高于对照组雏鸡(P<0.01)。⑤SPF雏鸡感染REV后,其法氏囊中Bcl-2蛋白含量较对照组雏鸡有不同程度的增加,其中21和28 d分别差异极显著(P<0.01)和显著(P<0.05),病毒感染组雏鸡的C-myc蛋白含量也始终高于对照组雏鸡,且21和28 d极显著增高(P<0.01)。【结论】 REV感染所致SPF雏鸡法氏囊细胞Bcl-2和C-myc的mRNA表达以及蛋白含量异常均与病毒感染导致的法氏囊细胞凋亡密切相关,而法氏囊细胞凋亡数量增加与REV感染引发的机体免疫机能抑制密切相关。     

Protective immunity against infectious bursal disease virus in chickens in the absence of virus-specific antibodies

The role of cell-mediated immunity (CMI) in pathogenesis of infectious bursal disease virus (IBDV) was investigated. One-day-old specific pathogen-free chickens were treated with 3mg of cyclophosphamide (Cy) per chicken for 4 consecutive days and, 3 weeks later, infected with the IBDV-IM strain. Chickens were examined for: (a) mitogenic response of splenocytes to ConA, as an indicator of T-cell functions in vitro, (b) antibody against IBDV by ELISA, (c) IBDV genome in various tissues by RT-PCR and (d) immunological memory. At the time of IBDV infection, Cy-treated chickens had depleted bursal tissue (an avian primary B-cell lymphoid organ), severely compromised antibody-producing ability, but normal T-cell response to ConA. In primary infection, no detectable antibody against IBDV antigen in Cy-treated, IBDV-infected chickens was observed up to 28 days post-infection (PI), while IBDV genome was detected by RT-PCR in spleen, thymus, liver and blood until 10 days PI. Like intact control chickens infected with IBDV, Cy-treated, IBDV-infected chickens suppressed splenocytes responses to ConA from 5 to 10 days PI, suggesting that intact control as well as Cy-treated chickens responded similarly to IBDV infection in the early phase. Following re-infection with IBDV, no detectable secondary antibody response to IBDV as well as IBDV genome in tissues were observed in Cy-treated chickens, while intact control chickens developed vigorous secondary antibody response. Similar to intact control chickens infected with IBDV, Cy-treated chickens after second infection with IBDV did not suppress splenocyte response to ConA. These results suggested that in the absence of detectable anti-IBDV antibodies, protection of Cy-treated chickens from IBDV infection may occur via immunological memory mediated by CMI. We concluded that under normal conditions, IBDV induces a protective antibody response, however, in the absence of antibody, CMI alone is adequate in protecting birds against virulent IBDV.     

Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.     

Recovery of antibody-producing ability and lymphocyte repopulation of bursal follicles in chickens exposed to infectious bursal disease virus.

We studied the long-term effect of infectious bursal disease virus (IBDV) in chickens. Specifically, the restoration of virus-induced bursal lesions and the duration of humoral immunodeficiency were examined. One-week-old specific-pathogen-free chickens were intraocularly inoculated with an intermediate vaccine strain (IBDV-Vac) or a virulent strain (IM-IBDV). At intervals postinoculation (PI), chickens were examined for histopathologic lesions. At 1, 3, 5, 10, or 15 wk PI, the chickens were injected with a mixture of antigens, and primary antibody responses were examined at 10 days postimmunization. Initially, the virus caused extensive necrosis of bursal B lymphocytes. This lesion was accompanied by an infiltration of T lymphocytes. With time, the necrotic lesion in the bursa was resolved. The follicles became partly repopulated with B lymphocytes. The repopulation occurred faster in the chickens exposed to IBDV-Vac than in the chickens exposed to IM-IBDV. By 7 wk PI, 40% and 80% of bursal follicles in IM-IBDV- and IBDV-Vac-inoculated chickens, respectively, were repopulated with immunoglobulin M+ B lymphocytes. Both IBDV-Vac and IM-caused suppression of the primary antibody response to antigens. However, the antibody responses of the chickens exposed to either of the two IBDV strains used were compromised only during the first 6 wk of virus exposure. Subsequently, the antibody response returned to near normal levels.     

Effects of infectious bursal disease on Marek's disease vaccination: suppression of antiviral immune response

Studies were made to determine whether infectious bursal disease virus (IBDV) infection would affect the response of chickens to turkey herpesvirus (HVT) vaccination in the development and level of HVT viremia and virus-neutralizing (VN) antibodies to HVT. The HVT viremia in the vaccinated chickens was not affected by IBDV, whether IBDV was inoculated simultaneously with HVT vaccination at one day of age or whether it was inoculated 3 weeks postvaccination with HVT. However, VN antibody response to HVT was significantly suppressed (P less than 0.001) when vaccinated chickens were exposed to IBDV either at the time of vaccination or at 3 weeks postvaccination. Such immunosuppression by IBDV of VN antibody response to HVT vaccination may result in a reduced antiviral immunity against Marek's disease virus.     

Quantitation of natural antibodies to infectious bursal disease in Nigerian indigenous chickens

Serum samples collected from 687 indigenous chickens located in small scattered groups in four states of Nigeria were examined for antibodies to infectious bursal disease (IBD) virus by the agar-gel precipitation (AGPT) and counterimmunoelectrophoresis (CIEOP) tests. 51 of the positive samples were further titrated by each of the two techniques. CIEOP detected more positive reactors (74.59%) than AGPT (58.95%). CIEOP also detected higher antibody levels among the reactors [geometric mean titre (GMT) of 51 samples was 23.02] when compared to AGPT. GMT of the same 51 samples was 21.8. The prevalence of antibodies to IBD virus in the indigenous chickens ranged from 64.7 to 77.7% CIEOP reactors between states. Since reports of IBD outbreaks among these chickens are rare unlike the situation among commercial poultry flocks, it was concluded that local chickens probably act as carriers of IBD virus.     

Immunogenicity and antigenicity of infectious bursal disease virus serotypes I and II in chickens

Chickens were inoculated with infectious bursal disease virus serotype I or serotype II to determine if their immune system can distinguish between the two serotypes. Chickens had neutralizing antibodies to only serotype I viruses following exposure to serotype I viruses, and chickens had antibodies to only serotype II viruses following exposure to serotype II viruses. No cross-reactions were observed between antisera prepared to each of these two serotypes using a cross-virus-neutralization assay. Signs of disease were detected only in birds exposed to a virulent serotype I isolate. Chicks exposed to the serotype II viruses were not protected from challenge with a virulent serotype I isolate. In one experiment, antibodies to a serotype II isolate, which were detected before challenge, did not protect chicks from challenge with a virulent serotype I isolate.     

Biotin-Streptavidin Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies to Campylobacter jejuni and C. coli in Chickens

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.     

A comparison of gel diffusion, fluorescent antibody and virus isolation methods in experimental and natural cases of infectious bursal disease.

In studies with chicks inoculated with the Sk-1 strain of infectious bursal agent the bursa of Fabricius was found to be the tissue of choice for virus isolation as well as for use in the fluorescent antibody test and the agar gel diffusion test. In separate experiments positive results were obtained until postinoculation days 3 or 4 by the agar gel diffusion test, 5 or 6 by the fluorescent antibody test and 14 by the virus isolation method, respectively. Bursas from chickens involved in seven natural outbreaks of infectious bursal disease were then examined by these three methods. Virus was isolated from six outbreaks and infectious bursal agent antigen was demonstrated in three by the agar gel diffusion test method and seven (three by direct examination and four after one passage in chicks) by the fluorescent antibody test method. Passage in chicks was required when nonspecific fluorescence complicated the interpretation of fluorescent antibody test results.     

Immunoreactivity and morphological changes of bursal follicles in chickens infected with vaccine or wild-type strains of the infectious bursal disease virus

Infectious bursal disease (IBD) is characterized by immunosuppression due to the depletion of lymphocytes in the atrophied bursa of Fabricius (BF). We have sometimes encountered contradictory findings: chickens infected with the vaccine IBD virus (IBDV) strain have sometimes exhibited a highly atrophied BF, but not immunosuppression. In this study, chickens administered vaccine or wild-type strains of IBDV were later vaccinated with the B1 strain of the Newcastle disease virus (NDV). Bursal changes were examined histologically with a focus on the bursal follicle. The immunoreactivity to NDV was also evaluated with the hemagglutination inhibition test. In gross examination, we observed a few chickens with a severely atrophied BF in vaccine strain-administered groups (vaccine groups), and the level of severity was the same as that in the wild-type strain-administered group (wild-type group). However, these chickens retained humoral antibody responses to NDV and were revealed to possess a higher number of bursal follicles than those of the wild-type group. These results indicated that macroscopic evaluation dose not accurately reflect the immunoreactivity and degree of bursal damage in IBDV-administered chickens. We also found non-immunosuppressed chickens in the wild-type group. These non-immunosuppressed chickens retained a significantly higher number of normal follicles and total follicles according to our statistical analysis. Furthermore, a high correlation coefficient between the NDV-HI titer and the number of normal follicles was found in the wild-type group. These results implied that the retained number of normal follicles is important for the immunoreactivity of chickens infected with IBDV.     

Evaluation of the specificity of Pasteurella multocida somatic antigen-typing antisera prepared in chickens, using ribosome-lipopolysaccharide complexes as inocula

Purified lipopolysaccharides (LPS) from 16 serotypes of Pasteurella multocida were complexed with Aspergillus fumigatus ribosomes. The complexes were used as inocula to prepare antisera, in chickens, for somatic antigen typing by the gel diffusion precipitin test (GDPT). Antisera made against 15 of 16 LPS reacted with their respective specific heat-stable antigens in the GDPT and homologous LPS in the passive hemagglutination test. Antisera could not be made against serotype 15 LPS. Correlation was not observed between intensity of the precipitin reaction in the GDPT and titer to homologous LPS in the passive hemagglutination test. Most antisera cross-reacted with other heat-stable antigens of other serotypes in the GDPT. Many of these cross-reactions were eliminated by dilution. Cross-reactions that occurred in the GDPT with antisera made against LPS of serotypes 2, 5, 7, and 8 could not be eliminated by dilution.     

Subclinical infectious bursal disease in an integrated broiler production operation.

A field study was designed to determine the prevalence of subclinical infectious bursal disease (IBD) in broiler chickens from a commercial poultry company. Bursae of Fabricius (BF) from two vaccinated and three nonvaccinated broiler flocks were evaluated histologically, and antibody profiles of these broiler and matched parent breeder flocks were established. Lesions of IBD, including lymphoid necrosis, stromal edema, and infiltrates of heterophils and macrophages, were first detected in BF at 24 days of age in both vaccinated and nonvaccinated chickens. At 41 days, all BF had lesions characteristic of IBD, including severe lymphoid depletion, proliferation of epithelial cells, and mild fibroplasia. Although mean maternal antibody levels (measured by enzyme-linked immunosorbent assay) in broilers were apparently protective through day 12, IBD antibodies decreased to nonprotective levels (below 1,000) by day 16 or 20. Titers began to increase by day 28 or 32 because of field exposure. Sentinel birds, placed with broiler flocks, also developed IBD antibody titers. Broiler breeders had low and nonuniform antibody titers. Prevalence of field IBD exposure was high, and existing vaccination programs were not effective.