Roy D. Montgomery

A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

BackgroundMany viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products.ResultsWe evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus) from cell culture and lymphocystis disease virus (iridovirus) and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1). The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species.ConclusionThe 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.

Attempts to Reproduce a Runting/Stunting-type Syndrome Using Infectious Agents Isolated from Affected Mississippi Broilers

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.

A Comparison of the Gland of Harder Response and Head-Associated Lymphoid Tissue (HALT) Morphology in Chickens and Turkeys

The immunofunctional response of the gland of Harder (GH) was compared in chickens and turkeys using an in vivo assay previously developed for use in chickens. The GH were surgically removed (GHx) from leghorn chicks at 1 day of age and from poults at 2 days of age. Intact birds of each species served as controls. During the fourth week of age, both GH-intact and GHx chicks were exposed to killed Brucella abortus antigen by the ocular or intraperitoneal route. One week later, serum and tears were collected and assayed for antibodies to B. abortus. In addition, all birds were killed at the end of the trial period, and the heads were fixed and processed for histologic examination. Various components of the head-associated lymphoid tissue (HALT) including the GH, nasal glands, lacrimal glands, lacrimal ducts, eyelid conjunctiva, and nasal cavity mucosa/submucosa, were evaluated microscopically using a scoring system to estimate quantity and degree of development of immune tissue in those sites. Results of all analyses indicate that functional response and morphology of the HALT are comparable in turkeys and chickens.

Consequences to Chicks Hatched from Escherichia coli- Inoculated Embryos

An Escherichia coli causing negligible mortality in embryonated chicken eggs was adapted to grow in media containing nalidixic acid. This isolate (EcNAL) was inoculated into 12-day-old embryonated eggs. Additional embryos inoculated with tryptose phosphate broth (TPB) served as controls. Six days later, all surviving eggs were moved to hatching units. One hatcher contained half of the TPB-inoculated eggs; the chicks hatching from these eggs served as negative controls. The EcNAL-inoculated eggs and the remaining TPB-inoculated eggs were moved to a second hatcher and allowed to hatch together; chicks hatching from these TPB-inoculated eggs served as contact controls. On day of hatch and at intervals thereafter, chicks from each of the treatment groups were sampled. Their body and yolk weights were recorded, and various tissues were cultured for the presence of the EcNAL bacterium. Hatchability of the EcNAL-inoculated embryos was markedly lower than that of either control group. Chicks from EcNAL-inoculated embryos also had low but detectable levels of mortality, lowered body weights, and increased yolk-to-body weight ratios. These same chicks had persistently high levels of EcNAL in the yolk and lower but detectable levels of the organism in the lungs and tracheas, which lasted a few days. The contact controls, on the other hand, were similar to the negative controls as far as having negligible mortality, steadily increasing body weights, and declining yolk-to-body weight ratios. However, in contrast to the negative controls, EcNAL was recovered primarily from the respiratory tract of the contact controls for a brief period of 3-4 days after hatch.

Effects of an Arkansas Strain of Infectious Bronchitis Vaccine on the Head-Associated Lymphoid Tissue (HALT)

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).

The Embryo Lethality of Escherichia coli Isolates and Its Relationship to Various In Vitro Attributes

Abstract 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.

Influence of Antigen Concentration, Inoculation Interval, Number of Exposures, Type of Housing, and Placement Concentration on the Tear Antibody Response to Brucella abortus in Chickens

A series of trials was run in leghorn chicks to examine select conditions affecting the tear antibody response to killed Brucella abortus antigen given by eyedrop administration. Specific conditions examined were concentration of antigen, number of antigen exposures, and interval between antigen exposures. Trials were also run to determine the earliest age at which the assay was functional in both broilers and leghorns. Two types of housing (isolators and battery cages) were examined, as were two levels of placement concentration, or bird density (0.32 and 0.64 ft. [0.03 and 0.06 m2]/chicken). All trials included intraperitoneally inoculated chicks as a comparison; tears as well as serum were assayed for antibodies. Of the various antigen regimens evaluated, two exposures of 20% B. abortus given 3 days apart was found to give a satisfactory antibody response in the tears 1 week later. Furthermore, response to B. abortus was found to be somewhat proportional to age, with the minimum age for a satisfactory response being 3 weeks in leghorns or 4 weeks in broilers. Statistically, there were no differences in antibody responses due to the types of housing or levels of placement used.

Effects of Newcastle disease vaccines and Newcastle disease/infectious bronchitis combination vaccines on the head-associated lymphoid tissues of the chicken.

Ten Newcastle disease virus (NDV) and 10 NDV and infectious bronchitis virus (IBV) combination vaccines (NDV/IBV) were evaluated for their effect on the head-associated lymphoid tissue (HALT) of 2-wk-old chicks. After vaccination, the chicks were subjected to an in vivo assay that measures the ability of the gland of Harder (GH) to respond to killed Brucella abortus antigen given in the eye by titering B. abortus antibodies in the tears. Following this, several sites in the HALT and trachea were examined histologically and scored for microscopic changes. The results indicated that three of the NDV/IBV combination vaccines (one BI/Mass&Conn and two LaSota/Mass&Conn) interfered with the GH response to killed B. abortus, whereas none of the NDV vaccines did Histologically, several changes were noted in the vaccinated chicks; however, no changes in the GH were observed that could explain microscopically the GH depression. With the IBV-only vaccines reported earlier (16), and the NDV-only and NDV/IBV combination vaccines reported here, a total of 36 vaccines have been evaluated using the same testing protocol. The conclusions of these combined studies suggest that several of the modified live virus vaccines containing IBV, either alone or in combination with NDV, interfere with the ability of the GH/HALT to respond to antigenic stimulation.

Effects of modified-live infectious bronchitis virus vaccines on the head-associated lymphoid tissue (HALT).

Specific-pathogen-free (SPF) leghorn chicks were inoculated with different modified-live infectious bronchitis virus (IBV) vaccines to determine if the vaccines interfered with immune competence of the head region. A total of 16 vaccines were evaluated comprising nine Massachusetts, three Arkansas, two Holland, one Florida, and one combination vaccine (containing both Connecticut and Massachusetts). Chicks were vaccinated when they were 4 weeks, 2 weeks, or 1 day of age. When all chickens were 4 weeks 3 days of age, their glands of Harder (GH) were assayed for the ability to respond to antigenic stimulation. Tissues from chicks given GH-depressing and non-GH-depressing vaccines were also collected and scored for histological changes in the head-associated lymphoid tissue (HALT) sites and the trachea. All 16 vaccines depressed the GH response to antigenic stimulation when given to 4-week-old chicks. Six of these vaccines (two Massachusetts, two Arkansas, and two Holland) also depressed the GH response when given to 2-week-old chicks, and one, an Arkansas vaccine, depressed the GH response when given to 1-day-old chicks. The main histological changes associated with the vaccines were increases in lymphocyte populations in the nasal mucosa, eyelid, and, for some, the lacrimal gland and the GH. In addition, lymphoid follicles were increased in the eyelid, to a lesser degree in the GH, and occasionally in the trachea. No relationship was found between histologic changes and vaccine-induced suppression of the GH response.

Effect of Infectious Bursal Disease Virus Vaccines on Persistence and Pathogenicity of Modified Live Reovirus Vaccines in Chickens

Two commercially available live reovirus vaccines, alone or in combination with two infectious bursal disease virus (IBDV) vaccines, were evaluated for safety and efficacy in specific-pathogen-free leghorn chicks. Four trials were conducted to evaluate the vaccine combinations. At periodic intervals during the trials, tissues were collected and assayed for residual reovirus and examined for histological changes. Six weeks following reovirus vaccination, all treatment groups were challenged with a virulent field isolate of reovirus and sampled 1 week later for the final time. The two reovirus vaccines were safe and effective if given at 1 week of age, regardless of whether the vaccinates had been exposed to IBDV at 1 day. However, both reovirus vaccines persisted in the tendons of 1-day-old vaccinates. The effects of IBDV vaccines were generally minor and reflected by increases in the number of pre-challenge or post-challenge virus recoveries from some of the treatment groups receiving both type vaccines.