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Biomedical subjects

M Brugh

Publications and source records attributed to M Brugh.

At least 37 records · Page 2Linked to original sources
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Simulation of maternal immunity by inoculation of immune yolk preparations into the yolk sac of 1-day-old chickens.

Yolk harvested from eggs laid by hens hyperimmunized with killed Newcastle disease virus (NDV) was inoculated into the yolk sac of 1-day-old specific-pathogen-free (SPF) chickens. Serum hemagglutination-inhibition antibody titers reached maximum levels 1 to 4 days after yolk inoculation and declined at a rate similar to that reported for naturally acquired maternal antibody. Expected levels of immune interference were observed when yolk-inoculated chickens were vaccinated with a conventional oil-emulsion NDV vaccine. These results show that yolk-sac inoculation with yolk antibody is a suitable approach for producing maternally immune chickens for laboratory studies.

Animals↗
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Re-evaluation of the pathogenicity of A/chicken/Alabama/75 (H4N8) influenza virus.

Avian influenza (AI) virus A/chicken/Alabama/7395/75 (H4N8), a putatively non-pathogenic virus associated with a self-limiting outbreak of severe disease in commercial layers, was selectively passed in chickens or in cell cultures and then in chickens to determine whether virus with increased pathogenicity would emerge. When 20 derivatives of the parental virus were each inoculated intranasally and intratracheally in leghorn hens, mortality rates ranged from zero (0/24) to 25% (6/24); mortality was 4% (1/24) for hens inoculated with the parental virus. Many virus reisolates (51/144) from hens that died exhibited high pathogenicity, killing at least six of eight intravenously inoculated 4-week-old chickens. Most derivatives examined produced plaques in trypsin-free cell cultures more efficiently than the parental virus, but the highest plaquing efficiencies observed (10%) were lower than would be expected (100%) for highly pathogenic subtype H5 or H7 AI viruses. These results confirm that the Alabama H4N8 virus can acquire increased pathogenicity upon passage in chickens and suggest that it may have acted alone in producing the severe disease observed in laying chickens in Alabama.

Animals↗
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Amantadine resistance among hemagglutinin subtype 5 strains of avian influenza virus.

Several avian influenza virus strains of hemagglutinin subtype 5 were assayed for sensitivity to the antiviral drug amantadine. Most strains exhibited little sensitivity to the drug as measured by plaque reduction. The A/Chicken/Scotland/59 (CS59), however, was highly sensitive, making it easily distinguishable from the other H5 strains. Drug sensitivity of the viruses was also assayed in chicken embryos. The in ovo patterns of amantadine sensitivity differed from those detected in cell culture. The CS59 isolate could not be distinguished from all the other strains on the basis of its response to amantadine in ovo. Although amantadine protected chickens inoculated with CS59 from morbidity and mortality, drug-resistant viruses were readily isolated from the infected birds. As found with other amantadine-resistant variants, the structure of the matrix gene was altered in the resistant isolates. These results demonstrate that amantadine resistance is widespread among avian influenza viruses of the H5 subtype, that drug sensitivity in cell culture does not necessarily reflect responses to amantadine in ovo and in vivo, and, as previously found, amantadine-resistant derivatives of H5 strains may be isolated from birds protected by the drug.

Amantadine↗
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Emergence of highly pathogenic virus during selective chicken passage of the prototype mildly pathogenic chicken/Pennsylvania/83 (H5N2) influenza virus.

The prototype mildly pathogenic A/chicken/Pennsylvania/21525/83 (H5N2) avian influenza virus, which was isolated more than 5 months before the emergence of highly pathogenic virus in the major 1983 Pennsylvania outbreak, was examined for the presence of minority subpopulations of highly pathogenic virus. Selective serial passage of the parental mildly pathogenic virus in leghorn hens did not lead to recovery of highly pathogenic virus. However, several highly pathogenic reisolates were recovered from hens inoculated with either of two mildly pathogenic virus clones selected for their ability to efficiently produce plaques in trypsin-free chicken embryo fibroblasts. Unlike the parental virus, these reisolates caused high mortality in chickens and produced postmortem lesions typical of highly pathogenic avian influenza. Electrophoretic mobilities of the hemagglutinin glycoproteins of the highly pathogenic derivatives resembled those of the prototype highly pathogenic A/chicken/Pennsylvania/1370/83 (H5N2) virus isolated in October 1983. These results suggest that unrecognized subpopulations of highly pathogenic virus may have infected Pennsylvania chickens for several months before emerging as the clinically manifest component of the virus population.

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Preparation of inactivated oil-emulsion vaccines with avian viral or Mycoplasma antigens.

The influence of the composition of water-in-oil emulsions on their physical characteristics was determined by preparing experimental emulsions with various water-to-oil ratios and various emulsifiers. Emulsions containing Tween 80 in the aqueous phase and Arlacel A or Arlacel 80 in the oil phase were lower in viscosity than emulsions containing only an oil-phase emulsifier. Viscosity decreased as the concentration of oil increased. Oil-emulsion vaccines prepared with aqueous- and oil-phase emulsifiers had low viscosity, were stable for more than 12 weeks at 37 C, and induced a marked primary antibody response in chickens.

Animals↗
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Characterization of infectious bronchitis virus using monoclonal antibodies.

Three monoclonal antibodies (MABs) reactive against two structural proteins--the nucleoprotein (NP) or the surface (S) protein--of avian infectious bronchitis virus (IBV) were produced and characterized. The MABs did not neutralize virus infectivity or inhibit hemagglutination. Their reactivity patterns with the homologous strain and eight heterologous strains of IBV were determined using the indirect immunoperoxidase test, the indirect immunofluorescent test, transfer-immunoblotting of separated proteins, and a dot-immunoblotting assay (DIA). Two MABs, NP- or S-protein-specific, reacted with all nine strains; one (NP-specific) reacted with only two strains. The two MABs reacting with all nine strains of IBV also detected 18 IBV field isolates of unknown serotype in the DIA. The MAB detecting only two strains did not react in the DIA. The diagnostic application of these MABs appears promising.

Animals↗
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In ovo competition between distinct virus populations in an avian influenza isolate.

Embryo lethality patterns induced by an avian influenza virus isolate (A/Tk/Ws/68/H5N9) suggested that it contained more than one genetic form. Two different virus populations were recovered from the isolate by plaque isolation and limit-dilution cloning and were characterized with respect to their biological and molecular properties. They were very closely related but exhibited strikingly different mean death times (MDT) in 10-day-old chick embryos. One was rapidly embryo lethal (REL), while the other was slowly embryo lethal (SEL). The REL isolate demonstrated a small but measurable mortality rate in 4-week-old chicks, as did the parental isolate. The SEL isolate, however, was nonlethal to 4-week-old chicks. The embryo MDT induced by the parental isolate revealed a biphasic death pattern reflecting expression of both REL and SEL populations. Mixing experiments, using different amounts of the two cloned populations, demonstrated that expression of their unique phenotypic property (either REL or SEL) was competitive. The number of early or late embryo deaths was directly related to the input levels of each respective virus. The only molecular difference thus far detected between the two populations is in the nonstructural (NS) gene, with the REL clone possessing a faster migrating electrophoretic form of that RNA than the SEL clone. Both forms of the NS gene were present in the original parental isolate. This study thus demonstrates the competitive coexistence of two closely related virus populations within a single natural isolate.

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Effect of dietary histamine on broiler chickens infected with avian reovirus S1133.

One-day-old broiler chickens were infected with either of two vaccine strains of avian reovirus S1133 and fed diets containing 0.2% histamine dihydrochloride for 21 or 35 days. Control groups received either or neither of these treatments. The most notable virus-histamine interaction observed was increased (P less than 0.01) early mortality of chickens infected with the more virulent (pullet) vaccine virus. Histamine in the diet did not affect seroconversion rates or the incidence of stunting in virus-infected chickens. Other evidence of virus-histamine interaction was proventricular enlargement and decreased (P less than 0.05) weight gains in chickens infected with the less virulent (chick) vaccine virus, but these signs were observed inconsistently. The possible clinical significance of these observations is discussed.

Animals↗
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Influence of dietary calcium stress on lethality of avian influenza viruses for laying chickens.

The effect of calcium stress was studied in an attempt to reproduce lethal infections in laying chickens with A/Chicken/Alabama/75 (H4N8) influenza virus and with two nonpathogenic H5N2 influenza viruses from the 1983-84 outbreak in the eastern United States. Hens were fed calcium-deficient or standard diets for 7 to 14 days; then the calcium-deficient feed was replaced with standard feed supplemented with ad libitum oyster shell, and both groups of hens were inoculated with virus. When hens were infected with the H4N8 virus, respective mortalities of those on the calcium-deficient and standard diets were 19% (27/141) and 5% (7/143). The H5N2 viruses did not kill hens fed either diet. In standard pathogenicity tests, Alabama H4N8 viruses reisolated from the hens that died generally were more lethal for 4-week-old chickens than the stock virus. These results argue for characterization of the Alabama H4N8 virus as pathogenic rather than nonpathogenic as originally determined.

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Highly pathogenic virus recovered from chickens infected with mildly pathogenic 1986 isolates of H5N2 avian influenza virus.

A combination of in vitro and in vivo selection procedures was used to examine the possibility that certain mildly pathogenic field isolates of avian influenza (AI) virus may contain minority subpopulations of highly pathogenic virus. Two mildly pathogenic H5N2 isolates, A/chicken/New Jersey/12508/86 (NJ12508) and A/chicken/Florida/27716/86 (FL27716), recovered from chickens epidemiologically associated with urban live-bird markets, were cloned in trypsin-free chicken embryo fibroblast cultures. Selected clones were inoculated intranasally and intratracheally (IN/IT) into specific-pathogen-free laying hens, and virus reisolated from the hens that died was serially passed in hens by IN/IT inoculation. Several highly pathogenic reisolates were recovered from hens infected with the cloned NJ12508 or FL27716 virus. A highly pathogenic NJ12508 reisolate killed 19 of 24 IN/IT-inoculated hens, and a FL27716 reisolate killed all 24 inoculated hens; signs and lesions were typical of fowl plague. In contrast, uncloned NJ12508 stock virus killed 1 of 24 hens and FL27716 stock virus killed 4 of 24 hens, and neither produced the complete spectrum of lesions associated with fowl plague. Recovery of highly pathogenic viruses from these isolates demonstrates the coexistence of pathogenically distinct subpopulations of virus. Competition for dominance among such subpopulations could explain the variable pathogenicity of some AI viruses.

Animals↗
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Emergence of amantadine-resistant H5N2 avian influenza virus during a simulated layer flock treatment program.

An experiment was designed to simulate field conditions in which preventive treatment is not initiated until after some chickens in a flock are infected with avian influenza (AI). Twelve hens began to receive amantadine hydrochloride on the day they were inoculated (day 0) with highly pathogenic AI virus, A/chicken/Pa/1370/83. These hens remained clinically normal through 8 days postinoculation (PI), but five died after day 9; mean death time (MDT) was 18 days. Three of 12 hens given amantadine beginning 1 day PI died (MDT 5.4 days), seven of 12 hens given amantadine beginning 3 days PI died (MDT 3.7 days), and all 12 inoculated hens not given amantadine died (MDT 4.9 days). The delayed mortality in the day 0 treatment group was likely due not to the original inoculum but to the emergence of a drug-resistant virus population. Virus isolated from a dead hen from that group was resistant to the actions of amantadine in both in ovo and in vivo tests. The lack of late mortality due to the drug-resistant virus in the day 1 and day 3 treatment groups, which were in close contact with the day 0 treatment group, was attributed to their becoming infected before treatment with the drug and to the development of protective immunity.

Amantadine↗
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Isolation of avian influenza virus (subtype H5N2) from chicken eggs during a natural outbreak.

Avian influenza virus (A/Chicken/Pennsylvania/83; H5N2) was recovered from the yolk, albumen, and shell surface of eggs obtained from naturally infected chicken flocks in Pennsylvania and Virginia. These findings represent the first reported isolation of avian influenza virus from the internal contents of eggs from naturally infected flocks. The need for adequate safeguards to prevent spread of the virus during commercial movement of table and hatching eggs, cracked and "checked" eggs, and egg flats and other materials is emphasized.

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Use of the Nobuto blood-sampling paper strip for Newcastle disease serology.

Dried whole blood collected by the paper-strip method of Dr. Kenzo Nobuto was evaluated for use in the Newcastle disease hemagglutination-inhibition (HI) test. When dried blood on the Nobuto strip was eluted in 1.0 ml of diluent, the resulting HI titers were similar to titers obtained with sera diluted 1:10. Both storage time and temperature influenced the stability of HI activity in dried blood, though the influence was minimal and is considered to have little significance in diagnostic serology, The advantages of this method of blood collection for Newcastle disease HI tests are discussed.

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Experimental infection of laying chickens with adenovirus 127 and with a related virus isolated from ducks.

Infection of commercially reared white leghorn and white rock hens with adenovirus 127 was associated with decreases in total egg production, external egg quality, egg weight (P less than 0.01), and eggshell thickness (P less than 0.01). The egg-production and egg-quality disturbances were transient, and production returned to normal approximately 4 weeks postinfection. Infection of white leghorns with a hemagglutinating adenovirus isolated from Missouri ducks did not adversely affect egg production, external egg quality, or eggshell thickness, but it was associated with decreased egg weight (P less than 0.01). Prior infection with the duck adenovirus prevented the adverse egg-production effects of adenovirus 127 infection. Mean hemagglutination-inhibition antibody titers of chickens infected with adenovirus 127 or with duck adenovirus ranged from 1:588 to 1:10809, and mean titers of uninfected chickens did not exceed 1:2.

Adenoviridae Infections↗
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Influence of formulation on the efficacy of experimental oil-emulsion Newcastle disease vaccines.

Twenty-one experimental oil-emulsion vaccines with different emulsifier contents, aqueous-to-oil ratios, and antigen concentrations were compared by immunization of 4-week-old chickens. Vaccines that contained oil-phase (Arlacel 80) and aqueous-phase (Tween 80) emulsifiers induced 2-to-4-fold higher hemagglutination-inhibition titers than vaccines with only the oil-phase emulsifier. The emulsion vaccines containing both emulsifiers were also more stable at 37 C and less viscous than those containing only the oil-phase emulsifier. Vaccines that had different aqueous-to-oil ratios and contained different quantities of allantoic-fluid antigen (1.2% to 50% of the vaccine volume) induced similar protection against challenge, but hemagglutination-inhibition titers were proportional to the amount of antigen added. Vaccines that had different aqueous-to-oil ratios but contained equal amounts of antigen induced similar hemagglutination-inhibition titers and similar protection against challenge.

Animals↗
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Atypical disease produced in chickens by Newcastle disease virus isolated from exotic birds.

Chickens were infected with a Newcastle disease virus (NDV) recovered from exotic birds with severe clinical disease and with lesions characteristic of viscerotropic velogenic Newcastle disease (VVND). The infection in chickens was inconsistently lethal, some infected chickens were not clinically affected, and gastrointestinal involvement was only marginally evident. Pathogenicity of the virus for chickens was not detectably altered by laboratory passage in chickens or by limit dilution passage in chicken embryos. The results suggest that the difference between velogenic NDV pathotypes may not always be distinct and that clinical manifestations of VVND in chickens may not always be predictable based on signs and lesions observed in exotic birds.

Age Factors↗
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The influence of test conditions on Newcastle disease hemagglutination-inhibition titers.

Replicate samples of serum from chickens immune to Newcastle disease were titrated to determine the influence of certain test conditions on hemagglutination-inhibition (hi) titers. The test conditions studied were those most likely to vary in normal laboratory operations. The most marked effect on magnitude of HI titers was incubation time of twofold serum dilutions in antigen-saline; the average titer increase after incubation of the serum-antigen mixture for 1 hr at 37 C was log2 2.3 (fivefold). Twofold increases in virus concentration of the antigen-saline diluent caused an average titer reduction of log2 0.8. Shifts in HI titers were only minor with twofold changes in erythrocyte concentration (log2 0.3), with variations of test reading times from 0.5 to 2.0 hr (log2 0.1), and with variations in the period between preparation of the initial 1:10 serum dilution in antigen-saline and the subsequent serum dilutions (log2 0.3).

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