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Biomedical subjects

D Todd

Publications and source records attributed to D Todd.

268 records · Page 15Linked to original sources
PubMedDate not supplied

In situ hybridization: an optimised detection protocol for a biotinylated DNA probe renders it more sensitive than a comparable 35S-labelled probe.

The development of a highly sensitive biotinylated in situ hybridization protocol using egg drop syndrome virus infection of domestic fowl as a model is described. This protocol incorporates the use of a monoclonal antibody to biotin as the initial step in a peroxidase-based detection system. The detection of viral nucleic acid in formalin-fixed, paraffin-embedded sections using biotinylated and 35S-labelled probes is compared to the detection of viral antigen by immunocytochemistry. Biotinylated probes detected more positive cells than 35S-labelled probes and were more specific. Biotinylated probes detected more positive cells than immunocytochemistry.

Adenoviridae↗
PubMedDate not supplied

Can defects in transferrin receptor 2 and hereditary hemochromatosis genes account for iron overload in HbH disease?

Iron overload was found to be the major cause of disability in Chinese HbH disease patients although they were not on regular blood transfusion. The transferrin receptor 2 (TFR2) and hereditary hemochromatosis (HFE) genes were examined to see if inheritance of these gene defects may be a possible cause of iron overload in 45 HbH patients. A novel intronic (IVS6 (+6) T-->A) mutation of the TFR2 gene was identified in one patient, and six others were found to carry a known missense mutation (exon 5, I238M) that was also present in one normal control subject. One HbH patient and one normal control carried the H63D mutation of the HFE gene. Since only eight out of 45 iron-overloaded HbH patients carry a defect in the TFR2 or HFE gene in the heterozygote state and their iron loading status was comparable to the matched controls without such defects, it would appear that the accumulation of excess iron in HbH disease is more likely a result of increase dietary absorption secondary to ineffective erythropoiesis.

Alleles↗
PubMedDate not supplied

Immunopathologic investigations with an attenuated chicken anemia virus in day-old chickens.

The immunopathologic effects induced by two attenuated chicken anemia virus (CAV) isolates, known as cloned isolate 34 (CI 34) and cloned revertant isolate 18 (CRI 18), that were derived from highly passaged pools of Cux-1 CAV isolate, were compared with those induced by a pathogenic, molecularly cloned, low-passage Cux-1 isolate (CI Cux). This comparison involved the intramuscular inoculation of 1-day-old specific-pathogen-free chicks with each of the viruses and investigation of birds at selected days postinoculation for gross pathology and depletions in the thymic T-cell populations as determined by flow cytometry. Whereas infection with the pathogenic CI Cux produced severe anemia and pronounced bone marrow and thymus lesions, infections with the attenuated CRI 18 and CI 34 isolates produced no anemia, no or mild lesions, respectively, and moderate T-cell depletion. The results suggest that, with CAV, reduced pathogenicity for 1-day-old chicks correlates with reduced depletion of T-cell populations in the thymus and with reduced severity of lesions in the thymus and bone marrow.

Animals↗
PubMedDate not supplied

Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments.

cDNA fragments were generated from RNA extracted from preparations of avian encephalomyelitis virus (AEV) by a reverse transcription-polymerase chain reaction (RT-PCR) strategy, which exploited the probability that AEV is a picornavirus. Rapid amplification of the 3' cDNA ends, which utilized an oligo d(T)-based primer that hybrizes to the putative Poly (A) tract at the 3' terminus of picornavirus RNA, produced a 3.8-kbp fragment (3.8-kbp 3' RACE fragment), from which a 2.5-kbp cDNA fragment specific to the extreme 3' terminal region of the AEV genome was cloned. Positive hybridization reactions between RNA from gradient-purified virus and radiolabeled probes confirmed that the cloned 2.5-kbp fragment was AEV specific. The success of the RT-PCR amplification strategy adopted and the results of northern blotting hybridization experiments indicated that the AEV genome is a polyadenylated, single-stranded RNA, approximately 7.5 kb in size. Sequence analysis of a 869-base region at the 3' terminal of the genome indicated that this region encoded a protein with close homologies to picornaviral RNA polymerase proteins. On the basis that the highest levels of protein homologies were observed with hepatitis A virus, it is likely that AEV will be reassigned to a genus other than the enterovirus genus within the virus family Picornaviridae. The AEV-specific cloned DNA fragments and nucleotide sequence information resulting from this investigation may facilitate the development of in situ hybridization and RT-PCR methods that will be useful in AEV diagnosis.

Amino Acid Sequence↗
PubMedDate not supplied

Effects of chicken anemia agent on lymphokine production and lymphocyte transformation in experimentally infected chickens.

One-day-old chicks with no maternal antibodies to chicken anemia agent (CAA) were inoculated intramuscularly with CAA grown in MDCC-MSB1 cells. A control group of birds from the same source was inoculated intramuscularly with a lysate from uninfected MSB1 cells. Birds were killed at 8, 15, 22, 29, and 43 days postinoculation (PI), and the spleens were removed. Spleen cells were dispersed and stimulated with various concentrations of Concanavalin A (Con A), and lymphocyte transformation responses were determined. Supernatants from Con A-stimulated cultures were assayed for T-cell growth factor (TCGF) and interferon. Decreased lymphocyte transformation and TCGF production were demonstrated at 8 and 15 days PI. This was followed by a stimulation in activities before a return to control levels at 43 days PI. Interferon levels were elevated 8 days after infection. This was followed by a significant decrease in activity compared with controls at 15, 22, and 29 days PI, and a return to control levels by 43 days PI. The results suggest that CAA infection in young chickens can produce a dramatic decrease in immune competence, which, although transitory, is likely to seriously compromise the ability of birds to mount a successful immune response to invading pathogens.

Anemia↗
PubMedDate not supplied

Economic effects of subclinical chicken anemia agent infection in broiler chickens.

To evaluate the economic effects of subclinical chicken anemia agent (CAA) infection on broiler performance, clinically normal broiler flocks were grouped into two categories: A) flocks in which none of 10 birds sampled at slaughter had antibody to CAA, and B) flocks in which six or more of 10 similarly sampled birds had CAA antibody. Production and performance parameters of 25 flocks in each category were compared. No statistically significant differences were found between category A and category B flocks in major production parameters such as sex, feed manufacturer, type of litter, stocking density, and age at slaughter. However, category A flocks achieved 13% (P less than 0.05) greater net income per 1000 birds, 2.0% better feed-conversion ratio (P less than 0.05), and 2.5% (P less than 0.05) greater average weight per bird than category B flocks. No significant differences in hockburn bonus per 1000 birds (a bonus payable to growers whose broilers have the lowest prevalence of contact dermatitis lesions on the hocks) and mortality were found between category A and category B flocks. These results show that subclinical CAA infection has a substantial, statistically significant effect on commercial broiler performance and profitability.

Age Factors↗
PubMedDate not supplied

Production and preliminary characterization of monoclonal antibodies to chicken anemia agent.

Mice were immunized with partially purified preparations of the Cux-1 isolate of chicken anemia agent (CAA), and their splenocytes were fused with NSO myeloma cells. Three patterns of staining of CAA-infected cells were recognized when the resulting hybridomas were screened by indirect immunofluorescence (IIF). Hybridomas representative of each staining pattern were cloned, and the monoclonal antibodies (MAbs) were characterized. Type 1 staining was indistinguishable from that produced by polyclonal chicken antisera to CAA. Type 2 staining was confined to large nuclear inclusions. Type 3 staining was predominantly nuclear and granular, and differed from type 1 in being more intense and occurring in a higher proportion of nuclei. Three MAbs producing type 1 staining were predominantly Cux-1-specific by IIF; they also reacted to lower titers with the Gifu-1 isolate but not at all with three other CAA isolates. These MAbs had very slight neutralizing activity against Cux-1. Another MAb giving type 1 staining reacted with all CAA isolates tested to high titers in IIF and neutralization tests. MAbs with type 2 and type 3 staining reacted by IIF with all CAA isolates tested but possessed no neutralizing activity. The availability of MABs to CAA should facilitate development of diagnostic tests for the virus.

Animals↗
PubMedDate not supplied

Development of an enzyme-linked immunosorbent assay to detect serum antibody to chicken anemia agent.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to chicken anemia agent (CAA) has been developed. This test utilizes a CAA-specific mouse monoclonal antibody to selectively capture virus antigen. Chicken antibodies to CAA bind to the captured antigen and are detected with horseradish peroxidase-labeled anti-chicken immunoglobulin using a conventional indirect ELISA protocol. When 388 chicken sera from specific-pathogen-free and commercial flocks from the United Kingdom, West Germany, the United States and Australia were examined, 98.5% agreement was obtained between the results of the ELISA and the indirect immunofluorescence assay. This ELISA should have worldwide application in testing SPF and commercial chicken flocks for CAA antibodies.

Animals↗
PubMedDate not supplied

Chicken anemia agent: an electron microscopic study.

Particles of chicken anemia agent (CAA) negatively stained with uranyl acetate were found to be 26.5 nm in diameter. The surface detail evident on the particles indicated that the virus capsid was composed of 32 structural subunits arranged as in a class P = 3 icosahedron with a triangulation number of 3. Using mouse monoclonal antibodies to CAA and a gold-labeled goat anti-mouse IgG, CAA-specific structures were observed by thin-section electron microscopy in infected MDCC-MSB1 cells and in thymic lymphocytes from experimentally infected chicks. These consisted of electron-dense, granular, non-membrane-bound nuclear inclusions, which were often ring-shaped, and cytoplasmic accumulations of microtubules. Aggregates of virus-like particles were sometimes observed in the nuclei of infected MDCC-MSB1 cells. The nucleolar involvement that is characteristic of the morphogenesis of parvoviruses was not observed with CAA.

Animals↗
PubMedDate not supplied

Development of an enzyme-linked immunosorbent assay for the serological diagnosis of big liver and spleen disease.

An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of big liver and spleen (BLS) disease. The test utilizes a soluble, BLS-specific antigen that can be recovered from the livers of infected hens and that is known to react in the agar gel immunodiffusion (AGID) test. For use in ELISA, the BLS-specific antigen is fractionated by gel filtration chromatography and immobilized on microtiter plates using glutaraldehyde. The ELISA was evaluated using sera from infected and uninfected flocks originating in the United Kingdom and the United States. An ELISA format that incorporated control antigen recovered from the livers of uninfected birds for each serum tested was found to be more sensitive than the AGID test. A less sensitive but more cost-effective format that did not incorporate this control is considered suitable for large-scale flock screening programs.

Animals↗
PubMedDate not supplied

A sequential histopathologic and immunocytochemical study of chicken anemia virus infection at one day of age.

Detection systems for chicken anemia virus (CAV) antigen in paraffin sections were evaluated. Mouse monoclonal antibodies to CAV used in conjunction with an avidin-biotin-peroxidase-complex detection system gave best results. Immunoreactivity of CAV was markedly affected by fixation. Fixation in neutral buffered formalin for 6 hours gave best results. Use of decalcifying fluid containing formic acid eliminated immunoreactivity of CAV, whereas use of an EDTA solution did not. In a sequential study, CAV antigen and lesions were first detected in bone marrow, thymus, and spleen at days 3-4 postinoculation (PI). Subsequently, antigen and/or cells containing nuclear inclusions were found in many tissues, but usually within lymphoid tissue therein. Thymus, spleen, bone marrow, proventriculus, and ascending duodenum contained most antigen. No antigen was detected after 26 days PI. The results indicated that CAV replicates in thymic lymphoblasts, intra- and extra-sinusoidal hemocytoblasts, and reticular cells, with consequent lymphocytic depletion of the thymic cortex and hypoplasia of the bone marrow, and that CAV antigen is widely distributed in the body.

Anemia↗
PubMedDate not supplied

In situ hybridization for the detection of chicken anemia virus in formalin-fixed, paraffin-embedded sections.

The development of a biotinylated in situ hybridization procedure for the detection of chicken anemia virus (CAV) is described. A double-stranded DNA probe was prepared using polymerase chain reaction and was biotinylated by nick translation. Hybridization conditions included the use of a microwave oven to denature target and probe nucleic acids and a five-step protocol for detection of biotinylated hybrids. In situ hybridization detected CAV nucleic acid in thymus tissue from experimentally infected birds after 6 hours, 3 days, and 7 days fixation time in formalin. However, immunocytochemical detection of CAV antigen was severely impaired if tissue was fixed in formalin for more than 6 hours.

Anemia↗
PubMedDate not supplied

Ileoanal reservoirs: construction and management.

The operative technique of ileal pouch-anal anastomosis is described, with specific attention to preoperative and postoperative counseling and ET nursing management of patients. This review is based on our experience with nearly 1400 ileal pouch-anal anastomosis operations at the Mayo Clinic since 1981.

Bandages↗
PubMedDate not supplied

The identification of an 18,000-molecular-weight antigen specific to big liver and spleen disease.

Big liver and spleen (BLS) disease is an infectious syndrome of broiler breeders that has been serologically diagnosed worldwide with the agar gel immunodiffusion test. Liver homogenate from an affected broiler breeder was used as the antigen source in this study. This paper reports the identification, from liver, of a soluble basic protein antigen (molecular weight 18,000) that is specific to BLS disease. The antigen was partially purified from soluble extract of liver using a two-step fractionation procedure comprising Sephacryl S200 gel filtration and carboxymethyl (CM) cellulose cation exchange. After cation exchange, the partially purified (CM) antigen contained approximately 12 proteins. Immunoblotting was used to identify the single BLS disease-specific antigen. In addition, a polyclonal rabbit antiserum raised to the CM antigen was found to be monospecific to the 18,000-molecular-weight antigen by immunoblotting on the CM antigen. This serum was also of use in specifically detecting intracellular BLS disease antigen in frozen cryostat sections by indirect immunofluorescence.

Animals↗