Detection of Marek's disease virus antigens and DNA in feathers from infected chickens

Two novel tests, enzyme-linked immunosorbent assay (ELISA) and dot-blot hybridization, were developed to detect and quantify the antigens and DNA of Marek's disease virus (MDV) in feather tips from infected chickens. In both methods, buffered extracts of the feathers served as the same test material. The ELISA technique was compared to the conventional agar-gel precipitation (AGP) test, using the same convalescent serum from a MDV-infected bird. Of 86 feather samples tested, 34 were negative by both methods, while 6 out of 52 were ELISA positive but AGP negative. Viral antigen detection by the AGP and ELISA methods was compared with the detection of MDV DNA by the dot-blot DNA hybridization technique. At an ELISA reading (OD 405) of 0.3 and above, only 5 out of 48 DNA extracts failed to hybridize with the MDV-DNA probe. The use of the radioactively labelled MDV-DNA probe for hybridization with DNA extracts from feather tips of MDV-infected chickens was both sensitive and specific, and there was good correlation among the different tests.     

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).     

Review: putative mutagens and carcinogens in foods. III. Butylated hydroxytoluene (BHT)

Although the average American's daily consumption of BHT can be measured in milligrams, there are numerous reports that BHT causes organ damage in laboratory animals. Only a few genotoxic effects of BHT have been reported, however, including mutagenicity in the abnormal sperm assay and ambiguous results regarding its teratogenicity. More dramatic are the modulatory effects of BHT on the actions of established mutagens and carcinogens. BHT can either enhance or inhibit mutagenic potency, depending on the substance tested. For example, in the Ames test, BHT is antimutagenic towards benzo(a)pyrene, but increases the number of Salmonella revertants induced by aflatoxin B1. BHT is one of the few compounds to have both tumor prophylactic and tumor promoting capacities. It is the temporal sequence in which BHT and carcinogens are administered to test animals which determines how BHT affects the response to these carcinogens. In common with other antioxidants, BHT inhibits the ability of carcinogens to induce tumors in various rodent organs when the animal is given BHT prior to carcinogen treatment. Unlike other antioxidants, however, the number of tumors increase when BHT is administered after carcinogen exposure. The comutagenic and cocarcinogenic properties of BHT have been demonstrated in tests ranging from the Ames test to cell transformation procedures to in vivo assays. These effects are probably mediated by metabolites of BHT, rather than by BHT itself.     

Butylated hydroxytoluene (BHT) induction of pulmonary inflammation: a role in tumor promotion

Chronic pulmonary inflammatory diseases predispose towards lung cancer by unknown mechanisms. Butylated hydroxytoluene (BHT) administration to mice causes lung injury and a subsequent inflammatory response, and when administered chronically to certain inbred strains following carcinogen treatment, increases lung tumor multiplicity. We hypothesize that inflammation promotes lung tumor growth in this model system and have begun to examine this hypothesis by assessing inflammatory parameters in inbred strains that vary in their susceptibility to promotion. Positive correlations were found between susceptibilities to tumor promotion and BHT induction of alveolar macrophage and lymphocyte infiltration into alveolar airspaces, and increased vascular permeability (P < .03, P < .04, and P < .005, respectively). The amounts of pulmonary cyclooxygenase (COX)-1 and COX-2 did not strongly correlate with promotion. Because persistent elevation of macrophage content is the hallmark of a chronic inflammatory response, the alveolar macrophage population was depleted by adding chlorine to the drinking water prior to carcinogenesis. This treatment reduced lung tumor multiplicity following 2-stage carcinogenesis (P < .05). These correlations between inflammatory and tumorigenic responses to BHT, along with decreased tumorigenesis after macrophage depletion, are consistent with a role of inflammation in promotion. Inflammatory mediators may provide targets for early diagnosis and chemoprevention.     

Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model

Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.     

An avirulent live Pasteurella multocida vaccine for drinking water and aerosol administration against turkey cholera

A naturally avirulent isolant of Pasteurella multocida (Heddleston sero-type 3) was investigated as a seed strain for the development of a live vaccine against turkey pasteurellosis. Laboratory trials showed that when given in aerosol form at a dosage of 5 x 10(8) c.f.u. per m(2), of barn floor, the vaccine produced satisfactory protection against both homologous and heterologous serotypes challenge administered by aerosol. Likewise, turkeys vaccinated once, twice or three times, and challenged at various time intervals were protected for over 20 weeks. The vaccine strain could be recovered from the lungs for a period of 6 days following aerosol vaccination.     

Antibacterial acylphloroglucinols from Hypericum olympicum

New antibacterial acylphloroglucinols (1-5) were isolated and characterized from the aerial parts of the plant Hypericum olympicum L. cf. uniflorum. The structures of these compounds were confirmed by extensive 1D- and 2D-NMR experiments to be 4,6-dihydroxy-2-O-(3″,7″-dimethyl-2″,6″-octadienyl)-1-(2'-methylbutanoyl)benzene (1), 4,6-dihydroxy-2-O-(7″-hydroxy-3″,7″-dimethyl-2″,5″-octadienyl)-1-(2'-methylbutanoyl)benzene (2), 4,6-dihydroxy-2-O-(6″-hydroxy-3″,7″-dimethyl-2″,7″-octadienyl)-1-(2'-methylbutanoyl)benzene (3), 4,6-dihydroxy-2-O-(6″-hydroperoxy-3″,7″-dimethyl-2″,7″-octadienyl)-1-(2'-methylbutanoyl)benzene (4), and 4,6-dihydroxy-2-O-(6″,7″-epoxy-3″,7″-dimethyloct-2″-enyl)-1-(2'-methylbutanoyl)benzene (5). These new natural products have been given the trivial names olympicins A-E (1-5). All compounds were evaluated against a panel of methicillin-resistant Staph. aureus and multidrug-resistant strains of Staph. aureus. Compound 1 exhibited minimum inhibitory concentrations (MICs) of 0.5-1 mg/L against the tested Staph. aureus strains. Compounds 2 to 5 were also shown to be active, with MICs ranging from 64 to 128 mg/L. Compound 1 was synthesized using a simple four-step method that can be readily utilized to give a number of structural analogues of 1.