Protective immunity to infectious bronchitis in broilers vaccinated against Marek's disease either in ovo or at hatch and against infectious bronchitis at hatch.

Two experiments were conducted using commercial broiler chickens to determine if Marek's disease (MD) vaccines HVT/SB-1 and HVT plus CVI-988 given either in ovo or at hatch adversely affected the efficacy of infectious bronchitis (IB) vaccines (Ark and Mass serotypes) given by eyedrop on the day of hatch. Nonvaccinated negative controls and controls that received only IB vaccines were included in each study. Birds were challenged with either infectious bronchitis virus (IBV) Mass-41 or IBV Ark-99 on either day 26 or 27 of age. Protection was assessed 5 days post-IBV challenged by virus isolation from the trachea. The day of hatch mean antibody titer to IBV was 12,668 +/- 4704 and 2503 +/- 3243 by enzyme-linked immunosorbent assay in experiments 1 and 2, respectively. In each study, nonvaccinated controls had a significantly higher (P < or = 0.05) incidence (88%-100%) of IBV challenge virus isolation than did controls vaccinated for IB but not for MD. Analysis of data from both studies showed that protection to IB in groups that received only IB vaccines at hatch ranged from 55.0% to 77.3%, whereas protection to IB in groups receiving both MD and IB vaccines ranged from 50.0% to 95.5%. In both experiments and within IBV challenge serotype, broilers given MD vaccines (in ovo or at hatch) and IB vaccines at hatch had protection rates to IBV challenges that were not significantly less (P < or = 0.05) than IB protection rates of groups that received only IB vaccines at hatch. Analysis of these data shows that administration of high-titered MD vaccines either in ovo or at hatch did not affect the efficacy of an IB vaccination (serotypes Ark and Mass) given by eyedrop at hatch.

The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease.

The role of immune complexes (Icx) in B-cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV-Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV-Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV-Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV-Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV-Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.

Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens.

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.

Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV.

A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.

Adaptation of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the determination of virus-neutralizing antibodies using the virus-neutralization assay.

The tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] has been widely used for bioassays. Herein is described the use of the MTT dye with a virus-neutralization (VN) assay to titer infectious bursal disease virus (IBDV)-neutralizing antibodies. A standard VN assay using chicken embryo fibroblasts (CEFs) and IBDV was used for the assessment of IBDV-neutralizing antibodies. The percent of CEF killing due to IBDV was quantitated using MTT, and the absorbance (A) data were used to calculate the VN antibody titer. This method of calculation offers the expression of VN titer in terms of units of activity per unit of volume.

In vivo studies with dimethyldithiocarbamate, a possible new antimicrobial for use against Aspergillus fumigatus in poultry.

Dimethyldithiocarbamate (DmDTC), the carbamate analogue, was tested for therapeutic efficacy in a series of in vivo challenge trials using 5- and 10-week-old white leghorn chickens. Challenge organisms were Pasteurella multocida X-73, Escherichia coli O1:K1, and Aspergillus fumigatus. Birds were evaluated for survival rates, lesion scores, and the rate at which the bacteria or mold could be reisolated following challenge. Results showed DmDTC to be ineffective against P. multocida and E. coli at the treatment levels and in the form used in these trials, but DmDTC significantly reduced lesion scores and inhibited the rate of isolation of A. fumigatus compared with untreated infected birds.

In vitro studies with dimethyldithiocarbamate, possible new antimicrobial for use in poultry.

Tests were conducted to determine the in vitro efficacy of the dithiocarbamate analogue, dimethyldithiocarbamate (DmDTC), against selected poultry pathogens. Organisms studied were two bacteria, Pasteurella multocida and Escherichia coli, and a mold, Aspergillus fumigatus. Zone of inhibition and the minimum inhibitory concentration were determined for each organism. DmDTC was effective in vitro against all organisms tested, with A. fumigatus showing greatest overall sensitivity.

Transfer of blood lymphocytes and macrophages between histocompatible progressor and regressor chickens infected with Rous sarcoma virus.

The development of histocompatible White Leghorn (progressor) and Arkansas Regression (regressor) chicken lines was described. When challenged with Rous sarcoma virus, progressor chickens developed fatal tumors while the regressor chickens eliminated the sarcoma. When sensitized histocompatible peritoneal macrophages and blood lymphocytes were transferred from regressor donors to progressor recipients, they both eradicated growing tumors. Histoincompatible cells were ineffective in inducing tumor remission. Within the two age groups tested, the sensitized blood lymphocytes and macrophages were only effective when transferred between age-matched donor and recipient chickens.

Genetic aspects of antibody responses in chickens to different classes of antigens.

Six breeding groups of chickens, each characterized by a different haplotype of the B blood group system, were challenged with different classes of antigens, namely Newcastle disease vaccine (ND), infectious bronchitis vaccine (IB), infectious bursal disease viral agent (IBD), Salmonella pullorum antigen (P), and sheep red blood cells (SRBC). Parents were challenged at 20 weeks of age, and their offspring were challenged at 3 weeks of age. Blood samples were taken from the parents at 1 week after challenge, and from the offspring at 1, 2, 3, and 4 weeks after challenge for determination of antibody titers to each antigen. The offspring were also challenged at 8 weeks of age in the wing-web with Rous sarcoma virus (RSV). Tumor scores were taken weekly on individual chickens for the next 10 weeks. There were significant differences (P less than .01) between breeding groups of parents for antibody titer responses to ND, IB, P, and SRBC. There were significant differences (P less than .05) between the breeding groups of offspring for antibody titer responses to ND, IB, IBD, P, and SRBC. There were significant (P less than .01) differences between the breeding groups in the accumulative tumor scores over the 10-week period. The lines that cause regression of Rous sarcomas (R-lines) were significantly (P less than .01) superior in resisting tumor growth to those lines that allow progressive growth of tumors (Pr-lines). The only antigen to which the R-lines gave significantly (P less than .01) higher titers of antibody responses than the Pr-lines was SRBC.

Application of the positive/negative ratio method of analysis to quantitate antibody responses to infectious bursal disease virus using a commercially available ELISA.

An enzyme-linked immunosorbent assay (ELISA) test kit has been developed for infectious bursal disease virus (IBDV), and it is widely used for the detection and quantification of IBDV antibody. With results obtained using the commercial kit, a method of analysis was performed utilizing a positive/negative (P/N) ratio standard curve. A prediction equation was derived from this analysis by which the ELISA titer of a serum sample could be determined using one serum dilution. Values obtained using this method were compared with values obtained by the virus-neutralization equivalent method recommended by the kit manufacturer. The results were comparable in that the trends in titer were similar. The P/N ratio standard curve method is another way of determining titer using the IBDV ELISA test kit.

Probability of survival based on ELISA titer following challenge of vaccinated chickens with the X-73 strain of Pasteurella multocida.

The probability of survival of chickens following a challenge dose of the X-73 strain of Pasteurella multocida was calculated based on enzyme-linked immunosorbent assay (ELISA) titer. Chickens were vaccinated subcutaneously with the Clemson University strain of P. multocida. On days 3, 5, 7, 10, 12, 14, 21, and 28 post-vaccination, 10 vaccinated chickens and 5 unvaccinated controls were selected at random, bled, and then challenged with 2000 colony-forming units of X-73. The ELISA titers to P. multocida vaccination and responses to challenge were recorded. A logistic procedure predicted probability of survival related to ELISA titer. The ELISA titer and survival were highly correlated. A flock profile for each day of challenge was developed based on a probability of survival (PS) at the following levels: PS less than 25%, 25% less than or equal to PS less than 50%, 50% less than or equal to PS less than 75%, and PS greater than or equal to 75%. The antibody response of the chickens through 28 days post-vaccination demonstrated a classic response to vaccination.

Evaluation of four disinfectants under poultry grow-out conditions using contact agar sampling technique.

Four commercial disinfectants used in poultry house sanitation procedures were evaluated for efficacy using a specialized petri dish (Rodac plate) for contact sampling. The samples were taken from a variety of surface materials commonly found in poultry rearing facilities. Two media were used, one for bacteria and one for mold. Predisinfection samples were taken following removal of chickens and litter. Postdisinfection samples were taken 4 hr after room treatment with a high pressure spray apparatus. The most effective disinfectant for reducing bacteria colony counts was a product that contained glutaraldehyde. The other products, ranking in order of efficacy for bacteria, contained cresylic acid, iodophors, and a combination of quaternary ammonium compound and formaldehyde. The most effective disinfectant for reducing mold colony counts was the product containing cresylic acid. The other products, ranking in order of efficacy for mold, contained iodophors, the combination of quaternary ammonium compound and formaldehyde, and glutaraldehyde. The most effective overall disinfectant was the cresylic acid product.

Isolation of high and low molecular weight components from chicken sera that have Rous sarcoma virus neutralizing activity.

Blood sera components from Arkansas regressor line (R-line) and progressor line (Pr-line) chickens are compared for the first time for Rous sarcoma virus neutralizing activity. Sera was fractionated by Sephadex G-100 filtration into a high molecular weight fraction I (HMW-I) and a low molecular weight fraction II (LMW-II) component (HMW-I greater than 14,000 daltons, LMW-II less than 5,000 daltons). Both fractions from each line of chickens exhibit activity against Rous sarcoma virus (RSV) judged by a wing web assay. Both HMW-I (principally antibodies) and LMW-II neutralized RSV when obtained from hyperimmune R-chickens and Pr-chickens with large progressing tumors. However, HMW-I and LMW-II obtained from R- or Pr-chickens before challenge contain so RSV neutralizing activity. The novel low molecular weight fraction II disappeared from the sera of R-line chickens 2 weeks after tumor regression, whereas the HMW-I persisted after tumor regression.

Heritabilities for regression and progression of rous sarcomas in the chicken.

Variance components for hatch, line, and sire expressed as percentages of the total variance for each parameter of regression and progression of Rous sarcomas in chickens were obtained. Some parameters measured specific stages of regression or progression, and other parameters measured the entire process of regression or progression of Rous sarcomas. Heritabilities of each parameter were estimated. Additive genetic variation is strongly implicated in the defense of the chicken against early tumor growth and during the entire process of regression. Environmental variation is important in influencing the point in time of tumor growth when the first reduction in size of tumor occurs. There is evidence of genetic variation exclusive of the R-Rs-1 gene locus that assists the chicken to resist tumor growth. The coefficient of variation for each of the parameters is very large as compared with the coefficient of variation for economic traits such as body weight at broiler age.