The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified viruses according to relatedness to sequences of reference viruses and the submission of the sequence data to GenBank will build the database to make the BLAST analysis a valuable resource readily accessible by most diagnostic laboratories. We demonstrated the utility of this assay on viruses that infect fish and birds. These hosts are phylogenetically distant from mammals yet, sequence data suggests that the assay would work equally as well on mammalian counterparts of these groups of viruses. Furthermore, we demonstrated that obtaining genetic information on routine diagnostic samples has great potential for revealing new virus strains and suggesting the presence of new species.

Various organisms, including 12 aerobic and 2 anaerobic bacteria, an infectious bronchitis virus (IBV), a reovirus, and 2 bacteriophages, were isolated from intestinal tracts of commercial broiler chicks undergoing a runting/stunting-type condition. In a series of trials, these agents were given alone and in combination to 1-day-old chicks in an attempt to reproduce the field condition. Because the agents were isolated and evaluated over time, an augmented designs variation of the analysis of variance proved particularly useful in analyzing the data collected and minimizing bird usage. Chicks inoculated with tryptose phosphate broth served as negative controls, and those inoculated with the original intestinal tract material were positive controls. Relative to the negative control chicks, body weights of the positive control chicks and of chicks inoculated with several of the agent combinations were depressed at 7, 14, and 21 days postinoculation. Common to combinations that most consistently caused weight depression were reovirus + IBV + others of the agents isolated. However, because none of the agent combinations reproduced the lethargy or dry feces seen in the positive controls, none was considered to be the ultimate cause of this particular runting/stunting-type condition. Further characterization of the disease syndrome was based on the positive control chicks. These chicks consistently had lowered body weights and transient lethargy and dry fecal pellets. Microscopic lesions consisted of lymphocytic renal and pancreatic interstitial infiltrates, dilated or cystic duodenal and jejunal crypts of Lieberkühn, increased crypt depth, and increased cellularity in the intestinal lamina propria. Electron microscopy revealed regular arrays of 26-nm viral particles, usually in association with membrane debris, in intestinal epithelial cells and crypt lumens and in intestinal and renal mesenchymal cells. These viral particles were theorized to be essential to reproduction of the complete malady seen.

Based on the hypothesis that bacteria with minimal embryo lethality might be good candidates for vertical transmission, 103 lactose-positive Escherichia coli isolates were collected from different broiler-related conditions (sources) and analyzed using a variety of in vitro assays: biochemical profiles, sensitivity to antimicrobials, and the presence of plasmids in the 2000- to 16,000-base pair range. The results of these assays were analyzed to determine if they were associated with, or could be used as predictors of, the degree of lethality these isolates produced in 12-day-old embryos. In addition, the in vitro assay results were analyzed to determine if there was any correlation between any particular pair of factors. On the basis of biochemical profiles, the isolates were classified into 17 different groups; however, only a limited number of biochemical reactions separated a majority of the isolates. The isolates varied considerably in the number and size of plasmids they contained and in their sensitivity to the antimicrobials evaluated. The isolates also varied in their ability to kill chicken embryos--killing from 0% to 100% of those inoculated--yet significant differences were detected in lethality based on source and biochemical profile of the isolate. In addition, a predictive model for embryo lethality was constructed and evaluated based on three characteristics of these 103 isolates, namely, their ability to ferment raffinose and sorbose and their sensitivity to gentamicin.

Chicks were vaccinated with an Arkansas strain of infectious bronchitis virus (IBV) vaccine when they were 1 day (Trial 1) or 4 weeks old (Trial 2). Starting at 4 weeks 3 days of age, chicks in both trials were subjected to an assay that measures the immunofunctional response of the gland of Harder (GH), one of the components of the head-associated lymphoid tissue (HALT). The assay involved multiple ocular exposures to killed Brucella abortus antigen, after which tears were collected and titered for antibodies to B. abortus. Following this, select tissues from vaccinated and unvaccinated chicks were collected and examined microscopically for specific lesions. Both functional and structural alterations were detected in the HALT of IBV-vaccinated chicks. Antibody titers to B. abortus in vaccinated chicks were significantly lower (P less than 0.05) than in unvaccinated controls. Structurally, there were elevations (P less than 0.01) in the number of lymphoid cells and follicles found in the mucosal lining of the nasal cavity. This occurred in the vaccinated chicks of both trials. Otherwise, histologic changes were confined to the chicks vaccinated at 4 weeks of age (Trial 2). In that trial, there were elevations in lymphoid-cell and follicle numbers in the eyelid (P less than 0.01) and lacrimal gland (P less than 0.05).

Reovirus 81-176 was inoculated subcutaneously into day-old specific-pathogen-free leghorns and evaluated for its effects on the immune system over a 3-week period. Structural criteria included organ weights of the bursa of Fabricius (BF) and spleen (SP), scoring of histological lesions in the BF, SP, and thymus, and hematological analyses of the circulating leukocytes. Alterations in the functional capacity of the immune system were measured using the graft-versus-host reaction, the response of peripheral blood lymphocytes (PBLs) to mitogens, the ability of circulating monocytes to phagocytize latex beads, and the serological responses to Newcastle disease virus, sheep red blood cells, and Brucella abortus antigens. For comparison, infectious bursal disease virus (IBDV) was similarly evaluated by most of the same tests. Structurally, reovirus 81-176 altered BF and SP organ weights, the total numbers of white blood cells in circulation, and the degree of follicular atrophy in the BF. Functionally, reovirus inoculation reduced both the response of PBLs to the phytohemagglutinin-P stimulation and monocyte uptake of latex beads. According to the protocols used here, no significant alteration in B-cell function could be detected in reovirus-infected chicks. With the exception of leukocyte hematology, IBDV-infected chicks had significantly altered responses in all tests used. By way of comparison, the effects of IBDV were more persistent and pronounced than were those seen with reovirus. The graft-versus-host reaction indicated an elevated and/or uninhibited response of T-cells in the blood of IBDV-infected chicks.