Prevalence of avain nephritis virus in England.
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Animals↗
Biomedical subjects
R A Nicholas
Publications and source records attributed to R A Nicholas.
Site-directed mutants of a soluble form of penicillin-binding protein 5 from Escherichia coli and their catalytic properties.
Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.
Amino Acid Sequence↗
Serology-based potency test for inactivated Newcastle disease vaccines.
Twenty-seven inactivated oil-emulsion Newcastle disease vaccines were tested for potency in chickens. Serum samples from groups given 1/50 dose of vaccine were examined by the haemagglutination inhibition (HI) test and the indirect enzyme-linked immunosorbent assay (ELISA). Good correlations were observed between potency and HI titres and between potency and ELISA absorbance. It is recommended that a serology-based potency test replaces the challenge test, reserving challenge only for those batches of vaccines where a clear pass is not indicated.
Animals↗
EMS response to a ski lift disaster in the Colorado mountains.
The EMS system in a Colorado mountain community was tested by the fall of a ski lift injuring 49 people. The response was complicated by the remote location of the accident and the number of injuries. Use of a preconceived disaster plan reduced morbidity and mortality. Patients were stabilized and triaged at the disaster site, and transported to the Snake River Health Services, Inc., where a second level of triage occurred. ATLS was provided at the Snake River Health Services and patients were transported to various surgical facilities throughout Colorado. Because a disaster in a remote or rural area can more easily overwhelm available resources, physicians should ensure that a good disaster plan is in place, should be able to provide initial evaluation and stabilization of patients, and arrange transport to the nearest appropriate facility.
Aircraft↗
Replication of avian encephalomyelitis virus in chick embryo neuroglial cell cultures. Brief report.
Avian encephalomyelitis virus replicated to relatively high titres in chick embryo neuroglial cell cultures, as measured by the indirect fluorescent antibody test, but induced few cytopathic effects. This cell system should provide a good source of virus for serological tests and vaccines.
Animals↗
An enzyme-linked immunosorbent assay for the detection of antibodies to avian reticuloendotheliosis virus using whole cell antigen.
An enzyme-linked immunosorbent assay (ELISA) using reticuloendotheliosis virus-infected chick embryo fibroblasts as coating antigen is described for the detection of antibodies to reticuloendotheliosis virus in chicken sera. The ELISA was specific and during the early stages of infection more sensitive than an indirect fluorescent antibody test.
Animals↗
Detection of avian encephalomyelitis virus.
Methods for the detection of two strains of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures and chickens were compared. It was found that the agar gel precipitin test (AGPT) and the enzyme-linked immunosorbent assay (ELISA) carried out on the serum of inoculated chickens were more sensitive than either the indirect fluorescent antibody test in cell cultures or the detection of clinical signs in chicks. On the basis of results obtained in this experiment the effects were then determined of routes and time of inoculation of chickens on the detection of AEV. It was found that birds infected at two weeks old produced higher antibody titres than one-day-old birds and the AGPT and ELISA detected comparable levels of antibody in them. It was recommended that the tests to detect the presence of AEV as a contaminant of vaccines be replaced by a serological test carried out on chicks inoculated intramuscularly at two weeks old.
Animals↗
Development of in vitro tests for detection of extraneous agents.
The use of animals in tests to detect extraneous agents is not only undesirable from the ethical viewpoint but also because of the expense and length of time involved in such tests. We have carried out tests on a variety of potential contaminating avian pathogens to determine whether tests in chicks offer any advantage over tests in embryos or cell cultures. In many cases, but not all, in vitro tests were shown to be more sensitive. The use fluorescent antibody or enzyme-linked assays serves to enhance the sensitivity of the tests. In the future it may be possible to adapt techniques such as nucleic acid hybridisation to the detection of extraneous agents.
Animal Testing Alternatives↗
A comparison of titration methods for live avian encephalomyelitis virus vaccines.
Factors associated with the indirect fluorescent antibody test used for the titration of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures were examined for their influence on virus replication. It was found that virus should be inoculated onto semi-confluent cell cultures and adsorbed for two hours at room temperature. The cells should then be examined for fluorescence after five days' incubation. Using these conditions, the cell culture assay was compared with the embryo and chick assays for its ability to estimate the virus content of live commercial AEV vaccines. In most cases titres obtained by the chick assay were slightly, but not significantly, higher than those obtained in the cell culture assay, although the reliability of the chick assay was, at times, questionable. In all cases titres obtained in the embryo assay were low. It is recommended that the cell culture assay be adopted as the method of choice for titrating AEV vaccines because it is rapid, reproducible, specific and greatly reduces the requirement for experimental animals.
Animals↗
Improved potency test for live avian encephalomyelitis vaccines.
Potency tests were carried out on live avian encephalomyelitis virus (AEV) vaccines. Vaccines were titrated in chick embryo brain cell cultures using an indirect fluorescent antibody test just before vaccination. Groups of chickens were inoculated orally with graded doses of each vaccine. After three weeks serum was taken from the chickens and examined for antibodies to AEV by indirect enzyme-linked immunoassay (ELISA). The chickens were then challenged by intracerebral inoculation of virulent AEV and monitored for signs attributable to clinical AE. The results showed a relationship between virus content, protection and antibody development. It is recommended that serological evaluation using ELISA replace the challenge step in potency tests for live AEV vaccines.
Animals↗
Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli.
This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.
Amino Acids↗
Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222).
The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.
Amino Acid Sequence↗
Identification of the active site in penicillin-binding protein 3 of Escherichia coli.
We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.
Acyltransferases↗
Detection of antibodies against infectious bursal disease virus: a comparison of three serological methods.
Four different oil emulsion infectious bursal disease virus (IBDV) vaccines were inoculated into four-week-old specific pathogen-free chickens. At weekly intervals for five weeks, sera were obtained from the vaccinated birds and from uninoculated control birds and examined for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA), the quantitative agar gel precipitin (QAGP) test and the virus neutralisation (VN) test. There was a highly significant correlation between the mean responses to all tests; the highest correlation (0.818) was between VN and QAGP and the lowest (0.573) between QAGP and ELISA. Generally the ELISA detected positive sera earlier than the VN test which in turn was more sensitive than the QAGP test. The ELISA and QAGP test were less variable, more reproducible and easier to perform than the VN test.
Animals↗
Purification of the membrane-spanning tryptic peptides of the alpha polypeptide from sodium and potassium ion activated adenosinetriphosphatase labeled with 1-tritiospiro[adamantane-4,3'-diazirine].
Five long, membrane-spanning tryptic peptides from the alpha polypeptide of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] have been purified. (Na+ + K+)-ATPase, isolated from canine kidney, was exposed to ultraviolet light in the presence of a high concentration of 1-tritiospiro[adamantane-4,3'-diazirine], a carbene precursor that partitions into the bilayer of the membrane. The alpha polypeptide, modified with 1.2 mol of [3H]adamantylidene (mol of polypeptide)-1, was isolated and digested with trypsin. Digestion with trypsin ensures that membrane-spanning sequences remain intact during the digestion, since lysine and arginine, being extremely hydrophilic, rarely appear in the membrane-embedded regions of membrane proteins. This digestion produced radioactive tryptic peptides greater than 25 residues in length. The tryptic digest of the labeled alpha polypeptide was chromatographed on Sephadex LH-60 in ethanol-formic acid, 4:1. The majority of the radioactivity (87%) eluted with distribution coefficients corresponding to peptides longer than melittin (26 residues), whereas 73% of the protein traveled with distribution coefficients corresponding to peptides less than 30 residues in length. Five radioactive peptides were further purified by high-pressure liquid chromatography, and each peptide displayed a unique, hydrophobic amino-terminal sequence. No other candidates could be found when a search for additional membrane-spanning peptides was conducted. Gel filtration of the tryptic peptides from the alpha polypeptide of (Na+ + K+)-ATPase labeled with 5-[125I]iodo-1-naphthyl azide, a lipophilic nitrene precursor, produced no additional radioactive components. Amino-terminal sequences and amino acid compositions of the five purified peptides are presented.
Adamantane↗
Detection of avian leukosis virus: comparison of five techniques.
Five techniques were compared for their ability to detect decreasing dilutions of RAV-I, an avian leukosis sarcoma virus, in serially passaged chick embryo fibroblast cell cultures. The indirect fluorescent antibody test, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase assay were equally sensitive in detecting the virus. The indirect immunoperoxidase and complement fixation avian leukosis tests were less sensitive. It is recommended that the sandwich ELISA be used for routine detection of the avian leukosis sarcoma virus group as possible vaccine contaminants because it is rapid and simple to perform and may be carried out on a large number of samples conveniently.
Animals↗
The hazards of retroviruses associated with monoclonal antibody production.
The majority of mammalian cells contain in their chromosomes genetic information related to retroviruses. Mice, and in particular, the inbred strains used in laboratory studies, have a number of different types of these viruses and murine cell lines frequently express virus. Retrovirus production has been noted in a number of myeloma cell lines and in hybridomas prepared from them. Therefore crude monoclonal antibody preparations may contain large numbers of these viruses. Retroviruses have been shown to be capable of activating or of acquiring oncogenes from cells. They may also recombine with endogenous leucosis virus to produce a recombinant virus with pathogenic properties. Comparison of endogenous cell DNA and retrovirus sequences has shown that retroviruses are capable of crossing genus and even class barriers. Retroviruses that are harmless in one species may be pathogenic in another. Hence care is required in the manufacturing and quality control procedures applied to monoclonal antibodies, particularly with products intended for medical and veterinary use. Widespread inoculation of retroviruses into a range of animal species may result only in trauma for the occasional individual but it is not possible to preclude the formation of a new type of pathogenic agent that can spread throughout the population.
Animals↗
Comparison of techniques for the detection of avian infectious bronchitis virus as a contaminant of vaccines.
Techniques for detecting various levels of both field and vaccine strains of infectious bronchitis virus in a deliberately contaminated Newcastle disease vaccine were compared using chick embryos, chick kidney cells, chick tracheal organ cultures and chickens with a view to determining the most appropriate method for screening vaccines for freedom from IBV contamination. Techniques examined included detection of abnormalities and deaths in embryos, cytopathic effect in chick kidney cells, ciliostasis in chick tracheal organ cultures and clinical signs and virus isolation in chickens as well as the fluorescent antibody test, negative contrast electron microscopy and serology where appropriate. Results showed that the techniques capable of detecting both strains of infectious bronchitis virus were, in order of sensitivity, the fluorescent antibody test on allantoic cells from infected embryos, ciliostasis and direct electron microscopy of allantoic fluid. One surprising feature was the poor results obtained using chickens. Some detection was achieved with tracheal virus isolation and tracheal organ cultures prepared from inoculated birds and to a lesser extent with histology and clinical signs, but no technique detected the field strain.
Animals↗