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.

Avoidance and treatment of retinopathy of prematurity.

Retinopathy of prematurity is a blinding eye disease of premature infants. It is increasing in incidence as more babies of lower birth weights survive. Although in the 1940s and 1950s the major predisposing factor was high oxygen exposure, the main risk factor now is birth weight less than 1,000 g. Retinopathy of prematurity can be classified into several distinct stages, which progress in a typical manner. Once the stage of threshold is reached, the eye has a 50% chance of becoming blind. At this stage, laser peripheral retinal photo ablation or peripheral cryo ablation may cause the neovascularization to regress and preserve vision. The treatment is not effective in all cases, however, and a large number of children continue to become blind each year because of this devastating disease. New microsurgical techniques such as lens-sparing vitrectomy and modified scleral buckle for tiny eyes have improved the outcome for some children. Prevention is the best hope to eradicate blindness caused by this disorder, and manipulations of the metabolic and neonatal intensive care unit environment are currently being studied.

Early-onset accommodative esotropia.

PURPOSE: To determine the frequency of accommodative esotropia with onset by 6 months of age; to determine if the presence or absence of characteristics usually associated with infantile esotropia can help in the diagnosis; and to determine if antiaccommodative therapy is adequate treatment for the esotropia. METHODS: The charts of 100 patients with infantile esotropia, seen over a 2-year period (September 1995 to September 1997), were reviewed. We identified those with at least 2.25 diopters (D) of hyperopia and determined the presence of large angle esotropia (> 30 to 40 prism diopters [delta]), amblyopia, inferior oblique overaction, dissociated vertical deviation, latent nystagmus, and cross-fixation. The success of antiaccommodative therapy, if attempted, was also evaluated. RESULTS: Of 100 patients with infantile esotropia, 15 (15%) were found to have at least +2.25 D. This represented 8% of all patients with accommodative esotropia seen over the same time period. The average age at initial examination was 21 months, although the reported age of onset in all cases was 6 months or less. Two had surgery before presenting to our institute. Eleven of 13 (84%) had 40 delta or less. Six (40%) of the 15 had amblyopia, 5 (33%) had inferior oblique overaction, 3 (20%) had dissociated vertical deviation, 1 (7%) had latent nystagmus, and 4 (27%) had cross-fixation. Of the 13, 7 were given glasses initially, with 3 being fully corrected. Six were not given glasses, all had surgery, and all were given glasses postoperatively for a residual esotropia. CONCLUSION: Fifteen percent of infantile esotropia patients and 8% of accommodative esotropia patients have infantile accommodative esotropia. Other characteristics of infantile esotropia may be present, but are less frequent, and at least 40% are fully corrected with spectacles indicating that when the hyperopia is 2.25 D or greater, antiaccommodative therapy should be instituted before surgery.

In ovo vaccination technology.

More than 80% of the U.S. broiler industry has converted to the in ovo vaccination process for control of Marek's disease. Providing certain criteria are met, including timing and site of vaccine placement, vaccine mixing, machine sanitization, and hatchery management specifications, this has proven to be an efficacious and convenient method of vaccination. Efforts to extend the technology for other viral vaccines including Newcastle, bronchitis and bursal disease, and bacterial and parasitic vaccines are in progress. Collectively, these studies demonstrate that in ovo vaccination technology using approved vaccine is a safe, efficacious, and convenient method for vaccination of poultry.

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.

Primary carcinoid of the duodenum: detection and characterization by magnetic resonance imaging.

We report a rare case of a primary small bowel carcinoid in which high-resolution MRI was able to detect a 1-cm primary duodenal neoplasm. This case illustrates that breath-hold contrast-enhanced MRI is capable of evaluating disease of the small bowel.

Accuracy of bedside chest hard-copy screen-film versus hard- and soft-copy computed radiographs in a medical intensive care unit: receiver operating characteristic analysis.

PURPOSE: To compare the clinical diagnostic accuracy of hard-copy readings of screen-film bedside chest radiographs and both hard- and soft-copy readings of bedside chest computed radiographs obtained in a medical intensive care unit. MATERIALS AND METHODS: Two samples of 95 cases were assembled from chest images obtained in 541 patients with either screen-film radiography or computed radiography. The cases were stratified according to the clinical problem for which the examination was ordered; the corresponding diagnosis was verified by a panel of two or three radiologists. Four radiologists blindly read the hard-copy images obtained with screen-film or computed radiography. Six months later, the radiologists read the computed radiographs by using an 8-bit, 1,684 x 2,048-pixel display. The data were analyzed by using multireader-multicase receiver operating characteristic (ROC) analysis of variance. RESULTS: No statistically significant differences in the area under the ROC curve were found between any of the methods. CONCLUSION: The results provide some justification for using bedside chest computed radiography and for reading soft-copy images from a high-quality display.

Applications in in ovo technology.

By mid-August 1995, 55% of broiler embryos in North America were vaccinated for Marek's disease using the INOVOJECT system, with 201 INOVOJECT machines placed with 16 of the top 25 poultry producers, providing the industry with the capacity to inject in excess of 400 million eggs per month or about 5 billion eggs per annum. In ovo administration of a bursal disease antibody-infectious bursal disease virus (BDA-IBDV) complexed vaccine to specific-pathogen-free (SPF) embryos was safer and more potent than conventional IBDV vaccine alone because it delayed the appearance of bursal lesions, produced no early mortality, produced higher geometric mean antibody titers against IBDV, and generated protective immunity against challenge. In ovo administration of a BDA-IBDV complexed vaccine to broiler embryos generated antibody titers against IBDV sooner than conventional virus vaccinates, and generated protective immunity against challenge Direct DNA injection of plasmid DNA encoding beta-galactosidase into breast muscle in ovo and posthatch was an effective means to achieve both gene transfer and expression, with potential for the development of gene vaccines using plasmids encoding protective antigens from poultry pathogens. In ovo administration of 800 U chicken myelomonocytic growth factor (cMGF), a chicken hematopoietic cytokine for cells of the monocytic-granulocytic lineages, significantly reduced mortality associated with Escherichia coli exposure within the hatcher when compared to PBS controls (6.1 vs 12.4, P < or = 0.05), but not when compared to a yeast expression control. A procedure was developed enabling injection prior to the onset of incubation without compromising embryo viability. This in ovo injection process has opened up the window of embryo development during incubation for intervention, as illustrated by the 100% male phenotype produced in chicks hatching from eggs injected with aromatase inhibitor prior to incubation. These data illustrate some of the in ovo applications presently in use by the poultry industry, and under development or in research at EMBREX.

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.

Evaluation of the humoral immune response to different antigens in Arkansas Regressor and Progressor chickens.

Arkansas Regressor and Progressor chickens were re-evaluated for their immune response to different antigens. Chickens received i.v. injection of either SRBC (10 birds per line) or Salmonella pullorum (SP; 10 birds per line) at 7 wk of age, and sera were collected at 6, 13, and 20 d postimmunization. A third group of birds (10 birds per line) received and i.m. injection of GAT emulsion at 7 and 12 wk of age, and sera were collected at 10 and 14 wk of age. There were significant differences between the two lines in their humoral immunity to SRBC, SP, and GAT. Such results suggest genetic control of humoral immunity to these antigens in these lines. It is unknown whether humoral immunity to these antigens is correlated to regression of tumors induced by Rous sarcoma virus.

Action spectrum of antiviral factor from chicken sera.

Rous sarcoma virus infections of regressor line chickens stimulate the transient production of antiviral factors in the serum. Earlier the present authors reported that a viral neutralization factor (VNF) inactivated Rous sarcoma virus during a 3-h incubation. The VNF is likely to have a broad antiviral and antimicrobial spectrum because it is active against several unrelated pathogenic poultry viruses. The present study measured the activity of VNF against Newcastle disease virus, infectious bursal disease virus, and infectious bronchitis virus. The VNF is active in immunologically incompetent systems and must be preincubated with the virus in order to inhibit it. Based upon the current experiments, it is proposed that VNF is not an immunomodulator but directly inactivates the virus. The VNF agent appears to be one of a newly identified class of nonspecific antiviral agents produced in vivo in chickens in response to a viral infection.

Amino acid sequence of a globin from the sea cucumber Caudina (Molpadia) arenicola.

Coelomic cells from the sea cucumber Caudina (Molpadia) arenicola contain four major globins, A, B, C and D. The hemoglobins from this organism show unusual ligand-linked dissociation properties. The complete amino acid sequence of the D globin has been established. It is N-acetylated, consists of 158 residues and has a 10 amino acid N-terminal extension similar to that found in some other invertebrate globins. The C. arenicola D globin has an equal sequence identity (28%) with both alpha and beta human globins and as anticipated, is more closely related to these vertebrate proteins than are molluscan globins. The C. arenicola D globin shows a 59% identity with the globin I from the sea cucumber Paracaudina chilensis. The availability of the C. arenicola D globin sequence will aid the X-ray analysis of this protein and facilitate an understanding of the changes in subunit interactions that occur with cooperative ligand binding.

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.