Detection of avian leukosis virus antigens by the ELISA and its use for detecting infectious virus after cultivation of samples and partial characterization of specific pathogen-free chicken lines maintained in this laboratory.

An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4 ng/well and about 10(3) infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF chicken lines were found to produce endogenous virus at a high frequency.     

抗鸡传染性法氏囊病病毒冻干卵黄抗体与同类产品的比较试验

本研究鸡传染性法氏囊病冻干卵黄抗体和市售精制液体卵黄抗体分别以10羽份和3 mL超剂量肌肉注射各10只18日龄SPF雏鸡,观察14 d均安全。两种抗体以1羽份剂量分别肌肉注射各20只18日龄SPF雏鸡24 h后,用IBDV强毒TL株攻击,连续观察144 h,2个试验组分别有20/20、17/20的雏鸡健活,剖检没有发现法氏囊病变;二者分别以1羽份剂量连续2 d肌肉注射各20只18日龄已经被IBDV强毒TL株攻击12 h的SPF雏鸡,连续观察96 h,2个试验组有19/20、17/20的雏鸡健活,而预防和治疗试验中的阳性对照组中20只鸡均有法氏囊特征性病变发生（20/20）,且死亡率在85%（17/20）以上,阴性对照组雏鸡均正常。     

Relationship of common avian pathogen antibody titers in so-called chicken anemia agent (CAA)-antibody-positive chicks to titers in CAA-antibody-negative chicks.

Antibody titers for infectious bursal disease virus (IBDV), infectious bronchitis virus, Newcastle disease virus, and reovirus from chicks with chicken anemia agent (CAA) antibodies were compared with antibody titers from their CAA-antibody-negative counterparts. These comparisons were made in 396 chickens that were 1 day, 2 weeks, 8-9 weeks, 10 weeks, 17 weeks, or 29-32 weeks old. Only one serum sample was collected from any given chick or chicken. There were no significant differences between the antibody titers at any age for any antigen, with one exception: at 29-32 weeks, the IBDV titers were higher (t = 2.62, df = 142, P less than 0.01) in chickens with CAA antibody. Although not at all likely, we believe that the observation of high IBDV antibody titers in CAA-antibody-positive chicks could have been a spurious one.     

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.     

Influence of antibody treatment of Campylobacter jejuni on the dose required to colonize chicks

This study was designed to clarify the role of antibodies in controlling chicken colonization by Campylobacter jejuni. Cecal colonization by C. jejuni was compared after the organism was exposed either to phosphate-buffered saline, normal rabbit serum, rabbit hyperimmune anti-C. jejuni serum, or anti-C. jejuni antibodies extracted from chicken bile. Antibodies from chicken bile were extracted by affinity absorption against outer-membrane proteins from the challenge organism. Sera were heated 1 hour at 56 C to destroy complement activity. Bacterial inoculum levels were enumerated after 1 hour exposure at 4 C to the various treatments. The heated sera and the bile antibodies were not bactericidal, and bacterial agglutination was not evident. Serial dilutions of the antibody-treated C. jejuni were given by gavage into 1-day-old chicks. Six days later, the ceca were removed from the chicks, and samples were cultured on Campylobacter-charcoal differential agar. The colonization dose-50% was increased by twofold to 160-fold when the organism was preincubated with hyperimmune antiserum or the bile antibodies as compared with preincubation with phosphate-buffered saline. We conclude that antibodies inhibit chicken cecal colonization by C. jejuni.     

Comparison of a kinetic-based enzyme-linked immunosorbent assay (KELISA) and virus-neutralization test for infectious bursal disease virus. II. Decay of maternal antibody in progeny from white Leghorns receiving various vaccination regimens

A computer-assisted single-serum-dilution indirect kinetic-based enzyme-linked immunosorbent assay (KELISA) was used for quantitating the natural decay rate of infectious bursal disease virus (IBDV) maternal antibody in progeny obtained from white leghorn breeders. The KELISA results were compared with those of a standard virus-neutralization (VN) test. Pullets were subjected to two IBDV immunization regimens. Group 1 was vaccinated at weeks 0, 2, and 10 with two live vaccines in drinking water and at week 20 with an oil-emulsified (SC) IBDV vaccine, group 2 received only the first and last immunizations, and group 3 served as the unvaccinated control. Pullets were artificially inseminated at 28 weeks of age. Progeny chicks from each group were bled every other day for 47 days. Both KELISA and VN test detected linear relationship in the decay of maternal antibodies. The VN test detected no significant differences (P greater than 0.05) in the IBDV maternal antibody titers at day 1 or in the rate of decay between the progeny from groups 1 and 2. The VN maternal antibody titers decreased at a rate of 0.16 log2 titer per day. In contrast, KELISA revealed higher IBDV maternal antibody titers in day-old progeny from pullets vaccinated 4 times (log2 = 14.3). However, KELISA titers of progeny from this group decreased at a faster rate than titers of progeny from pullets vaccinated twice (0.20 vs. 0.13 log2 titer per day).     

Maternal antibody and its effect on infectious bursal disease immunization

Chickens vaccinated with infectious bursal disease virus (IBDV) early in life and revaccinated with an inactivated, oil-adjuvant IBDV vaccine at 18 weeks of age produced and maintained high levels of virus-neutralizing (VN) antibody through 10 months of lay. VN-antibody titers of chicks hatched from eggs laid during the same period closely matched the average VN-antibody titers of the dams. A sequential study of the decline rates of IBDV maternal antibody (MAB) in unvaccinated and IBDV-vaccinated chicks showed that the vaccine virus did not accelerate the antibody depletion rate in vaccinated chicks. Chicks carrying high IBDV MAB showed no active immune response to vaccination with commercial IBDV vaccines. They were also refractory to a pathogenic field isolate of IBDV (FV). However, chicks with low levels of MAB responded to both vaccine virus and the FV, although their response to vaccine virus was milder and delayed.     

Effect of infectious bursal disease on the response of chickens to Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus.

At 35 days of age, chickens which as 1-day-old chicks were inoculated with the infectious bursal disease virus (IBDV) had significantly lower antibody titers against Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus than did those never inoculated with IBDV. The IBDV also had a marked effect on the development of air-sac lesions. Birds infected with IBDV that were later inoculated with M synoviae (day 14), Newcastle disease virus (days 14 and 28) experienced an increased incidence and greater seversity of airsacculitis than did chicks which were not exposed to IBDV.     

犬瘟热病毒免疫蛋鸡血清抗体与卵黄抗体消长规律的研究

试验旨在了解产蛋鸡对犬瘟热病毒(canine distemper virus,CDV)抗原的免疫反应及其血清抗体和卵黄抗体的消长规律,为制备卵黄抗体提供依据。将CDV接种Vero细胞和DF1细胞进行传代并测定其病毒滴度,选取细胞病变出现早、病毒滴度高的Vero细胞毒作为接种病毒抗原免疫蛋鸡。每10 d免疫蛋鸡1次,第4次免疫后每30 d加强免疫1次。免疫前及免疫后每10 d采血分离血清,每5 d收集鸡蛋提取卵黄抗体,用琼脂扩散法和间接ELISA法测定其抗体水平。结果显示,血清抗体比卵黄抗体的滴度高,两者呈正相关性,卵黄抗体出现较血清抗体迟3~5 d,卵黄抗体在第3次免疫后3~5 d就已达到较高水平,此时即可开始收集鸡蛋制备卵黄抗体。     

A comparison of serum neutralisation, immunofluorescence and immunoperoxidase tests for the detection of antibodies to chicken anaemia agent

A comparison was made of the serum neutralisation, immunofluorescent antibody and immunoperoxidase tests for the detection of antibodies to chicken anaemia agent. Serum samples from groups of chicks with and without maternally derived antibody to the agent were tested and the response of chicks after inoculation with the agent was also measured. The serum neutralisation test was reliable and sensitive, but expensive and could take up to three weeks to obtain a result. The immunofluorescent antibody test was cheaper and required only one day to obtain a result, but it was not as sensitive in detecting low levels of antibody to the chicken anaemia agent. The immunoperoxidase test was also cheaper but took two days to obtain a result and required one more manipulation than the immunofluorescence test. It was comparable to the serum neutralisation test in its ability to detect low levels of antibody.     

Immune responses in chickens against Salmonella typhimurium monitored with egg antibodies

Three mature hens were immunized with an Aro- mutant of Salmonella typhimurium beginning with a subcutaneous dose in adjuvant followed by two oral boosters. Isotype-specific antibodies were measured in the white and yolk eggs collected weekly over a period of 230 days. Two hens showed a memory response to the first oral booster, with large increases in egg yolk IgG and smaller increases in IgA and IgM antibodies in egg whites. Smaller amounts of IgA and IgM antibodies were found in egg yolks, and a slight increase in IgG occurred in the whites. One hen showed an increase in serum titers of all isotypes against S. typhimurium. The second hen had high serum titers before immunization was started which did not change. The third hen had a high level of IgM in the white of eggs before immunization was started. This hen showed erratic responses in egg white antibodies following immunization, no increase in IgA or IgM in yolks and only a slight increase in IgG, no increase in serum IgG, and was the only hen with a high level of IgM antibody against S. typhimurium in the bile, conditions reflecting a state of oral tolerance. With the exception of this hen, the results showed that IgA and IgM antibodies were aroused in hens by immunization with an avirulent mutant of S. typhimurium, and that these antibodies were present in the white of eggs from immunized hens.     

Efficacy of maternal Salmonella antibodies and experimental oral infection of chicks with Salmonella enteritidis

Distribution of maternally transmitted Salmonella antibodies and their protective effects were studied in the progeny of broiler breeder birds which had been vaccinated with live S. Typhimurium and inactivated S. Enteritidis vaccines. Vaccination resulted in a significant increase of the antibody concentration in yolk of hatching eggs and in serum and jejunum of the progeny of immunized breeder birds. Higher antibody titres for isotypes IgG and IgA were still seen on day 21 of age. Antibody production of isotypes IgA and IgM by the chickens themselves was found between 14 and 21 days of age. Two challenge models (10(2) cfu/bird on day 1 of age and a seeder bird model, respectively) were used to evaluate the efficacy of maternal antibodies against challenge with S. Enteritidis. Using both models numbers of challenge organisms were lower in the caeca of the progeny of immunized parent birds between day 7 and day 21 of age (maximum about 1.5 log10 units) compared with control chicks. The results indicate the efficacy of maternally transferred antibodies but it remains the question of their practical relevance. The effects of acquired maternal antibodies on an active immunization of the progeny of immunized breeder birds with live Salmonella vaccines are discussed.     

Prediction of optimal vaccination timing for infectious bursal disease based on chick weight

Growth rate in broiler birds has increased substantially in the last decade due to improvement in genetics, feed formulation, cleaner environment, and vaccine formulations. As a result, it has become necessary to review and revise prediction method for vaccination in chicks. This study was undertaken to determine the possible use of the rate of weight gain rather than age in predicting vaccination time. Two groups of 1-day-old broilers originating from old and young breeders, respectively, and with different levels of maternal antibodies against infectious bursal disease virus (IBDV) were used in this study. The chicks were divided into four groups and subjected to two feed regiments: groups A1 and B1 were fed broiler feed for normal growth rate, and groups A2 and B2 were fed breeder feed for slower growth rate. At 1, 4, 8, 12, 16, 22, 29, and 36 days of age, 22 chicks in each group were weighed, and blood samples were collected. Serum samples were tested for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA) and virus neutralization test. Maternal antibody decline curves for each group were plotted according to chick age and chick weight. Fast-growing birds in groups A1 and B1 showed a faster rate of antibody decline, whereas slow-growing birds in groups A2 and B2 had a slower rate of antibody decline. Based on the effect of weight gain on maternal antibody decline, a new way of predicting vaccination time for IBDV based on measuring maternal antibody titers at 4 days of age was proposed and tested. The predicted antibody decline was shown to correspond to the real ELISA titers measured in our experiments (R = 0.9889), whereas a lower correlation (R = 0.8355) was detected between real ELISA titers and the titers predicted by the current method using age-based Deventer formula.     

Single and combined infections of specific-pathogen-free chickens with infectious bursal disease virus and an intestinal isolate of reovirus

The susceptibility of 1-day-old and 7-day-old specific-pathogen-free chickens to infection with a virulent strain of infectious bursal disease virus (IBDV) or an intestinal isolate of avian reovirus, or a combination of the two, was investigated. Chickens infected with IBDV and reovirus had more severe pathological lesions than chickens infected with either virus alone, and prior infection with IBDV enhanced the pathogenicity of enteric reovirus. Virus recovery was attempted from bursa, spleen, thymus, liver, intestine, pancreas, cecal tonsils, heart, and tarso-metatarsal tendons. Viruses were recovered from all tissues sampled for either IBDV or reovirus isolation, and indications were that infection with IBDV before infection with reovirus led to longer persistence of reovirus in some tissues. Antibodies to IBDV or reovirus were measured by the virus neutralization test and enzyme-linked immunosorbent assay. Chickens infected with IBDV had lower (P less than 0.05) antibody responses to reovirus than chickens infected with reovirus alone.     

Indirect method for prediction of hemagglutination inhibition antibody titers to Newcastle disease virus in chickens by titration of antibodies in egg yolk.

Attempts were made to establish methods for indirect prediction of hemagglutination inhibition (HI) antibody titers to Newcastle disease virus (NDV) in sera of laying hens and day-old chicks by determining if these are correlated to HI titers in egg yolks. For this purpose, geometric means of HI antibody titers in sera from 60 hens, yolks from 60 matched eggs, and sera from 180 day-old chicks of an identical vaccination program were measured and plotted. There was a significant correlation between HI antibody titers in yolks (X) and hens (Y), with a linear regression of Y = 23.24 + 0.47X and a correlation coefficient of r = 0.65. The linear regression between HI antibody titers in yolks (X) and chicks (Y) was Y = 6.33 + 0.36X (r = 0.58). Immunity to NDV in hens and their offspring can be maintained effectively, and the proper time for the vaccination or booster can be determined by reference to HI titers predicted from the linear regression in the present study. The approach of testing egg yolk for HI titers provides a feasible alternative to determining HI titers from blood samples and eliminates stress in birds during blood sampling.     

Induction of non-specific suppression in chicks by specific combination of maternal antibody and related antigen

Specific immune suppression in newly hatched chicks induced by specific maternal antibodies has been reported. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP and non-specific immunoglobulin (Ig) Y antibodies were transferred by yolk sac inoculation to newly hatched chicks, and then, they were immunized with an optimum immunogenic dose of DNP-KLH at 1 and 4 weeks of age. Concentrations of anti-DNP antibodies in serum samples of these chicks were measured by using Enzyme-linked immunosorbent assay (ELISA). Proportions of T-cell subsets in peripheral blood of these chicks were also measured by flow cytometric analysis at 5 weeks of age (one week after the second immunization). Suppression of anti-DNP antibody response and down-regulation of CD3⁺CD4⁺ cells were observed in the chicks received high dose of maternal anti-DNP antibodies and immunized with DNP-KLH. On the other hand, normal anti-DNP antibody response and normal proportion of CD3⁺CD4⁺ cells were observed in the chicks received high dose of non-specific IgY antibodies and immunized with DNP-KLH. Furthermore, when chicks received high dose of maternal anti-DNP antibodies and immunized with DNP-KLH at 1 and 4 weeks of age and then with rabbit serum albumin (RSA) at 5 and 8 weeks of age, their primary anti-RSA response was also significantly suppressed. We indicate here that specific maternal antibodies can affect both B and T cell responses and induce non-specific suppression against different antigens. However, this non-specific suppression does not continue for a long time.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

The effect of palm oil and safflower oil in the feed of parent fattening hens on fertility, hatchability and growth of progeny]

The aim of two experiments with broiler breeder hens was to evaluate the effect of diets containing palm butter or safflower oil (25 g and 50 g/kg feed, resp.) on fertility, hatchability and growth of progeny. Especially the incorporation of oleic and linoleic acid in egg yolk reflected the dietary fatty acid source. Eggs were collected and stored in the incubator at a hen age of 31, 40, 50, and 60 weeks. Hatched chicks were reared over 5 weeks. The number of fertile eggs (Experiment 1 and 2, 75 and 88%, resp.) differed between the experiments (P < or = 0.05). Neither embryonic mortality nor hatchability (Experiment 1 and 2, 76 and 78%, resp.) were significantly affected by fatty acid composition of yolk. No clear maternal dietary effect was recorded on chicken weight at hatching (Experiment 1 and 2, 43.3 g and 43.7 g, resp.) and at 35 days of age (Experimental 1 and 2, 1676 g and 1764 g, resp.) The fatty acid composition in the analysed egg yolk sac of chicks showed a different fatty level but corresponded to fatty acid composition of breeding eggs before incubation. According to a decreased level of docosahexaenoic acid in egg yolk due to increased incorporation of linoleic acid, the content of this fatty acid was also diminished in phospholipids of the brain of chicken on days 1 and 5 after hatching.     

Specific suppression of the bursa-dependent immune system of chicks with infectious bursal disease virus.

Chicks which had been inoculated with infectious bursal disease virus (IBDV) at 1 day of age had a severe depression of bursa-dependent humoral immune functions by day 42. Antibody responses against rabbit red blood cells or to immunization with bovine serum albumin were significantly suppressed. In contrast, chicks inoculated with IBDV at 21 days of age produced near normal antibody responses as compared with the responses in noninfected control chicks. The IBDV had no significant effect on the thymus-dependent cellular responses as measured by skin graft rejection or delayed type hypersensitivity reactions to tuberculin.