Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes.

Blood samples from closely monitored patients at the Veterans Administration Hospital in Houston, Texas, were collected, coded, and sent to Microbiological Associates over an 8-month period. Lymphocytes were isolated and cryopreserved at -190 degrees. Lymphocyte samples were simultaneously thawed, phytohemagglutinin activated, and analyzed for benz(a)anthracene-induced aryl hydrocarbon hydroxylase (AHH) levels, [3H]thymidine incorporation, and reduced nicotinamide adenine dinucleotide-dependent cytochrome b5 (cytochrome c) reductase activity. Determinations were made at both 96 and 120 hr in culture, and peak activities were compared among a total of 51 individuals who expressed such lesions as squamous cell carcinomas (22%), adenocarcinomas (14%), oat cell carcinomas (6%), chronic obstructive pulmonary disease (22%), and other nonmalignant diseases. Of the 14 highest AHH/cytochrome c activities observed, all were found in patients with primary lung cancer. Mean AHH/cytochrome c activities were 0.89 for lung cancer patients (a total of 21) and 0.47 for noncancer patients (a total of 30) (p less than 0.001). No relationship was observed between AHH/cytochrome c activity and age of patient, numbers of cigarettes smoked, family history of cancer, location or histological type of tumor, or level of phytohemagglutinin blastogenesis ([3H]thymidine cpm/cytochrome c). Whether the higher AHH levels are the cause or the result of the primary lung cancer remains to be determined.

Induction of aryl hydrocarbon hydroxylase in human peripheral blood lymphocytes by chrysene.

Many of the polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene (B[a]P), benzanthracene (BA), 3-methylcholanthrene (3-MC)) are not only carcinogenic, but also induce AHH in human tissues. Recently, chrysene has been implicated as an etiologic determinant of chemical carcinogenesis. Here we describe the ability of chrysene to induce AHH in cultured human lymphocytes. Lymphocytes were obtained from 9 healthy subjects, divided into 2 sets, and cultured in duplicate, triplicate, or quadruplicate for 48 h. Chrysene (25 microM final concentration) in acetone was then added to the induced culture set and the control set received acetone alone. Lymphocytes were then cultured an additional 24 h before harvesting. AHH was quantitated by a fluorometric analysis of the phenolic metabolites produced by incubating the lymphocytes with B[a]P for 35 min. A significant increase in enzyme induction occurred in the chrysene-induced cultures compared with control (non-induced) cells (one-tailed student t-test; P less than 0.001). It was also observed that the interindividual variation in AHH inducibility seen with other PAHs is also observed with chrysene.

Patterns of benzo[alpha]pyrene metabolism in normal human pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (PAMs) obtained by bronchopulmonary lavage from 6 normal non-smoking volunteers were incubated with [3H]-benzo[alpha]pyrene to ascertain the normal metabolism and conjugation of polycyclic aromatic hydrocarbons. Through the use of a crude glucuronidase preparation, both glucuronic acid and sulfate conjugates were examined. Phenols and quinones were identified by high-pressure liquid chromatography as the principal free metabolites formed during 1 h incubation with benzanthracene induced PAMs. In addition, phenols and quinones were major substrates utilized by these cells for conjugation during the incubation period. The ranges of benzo[alpha]pyrene metabolites produced by PAMs from non-smokers were compiled and the variation in production as well as detoxification of proximate carcinogenic benzo[alpha]pyrene metabolites are presented.

Characterisation of the bovine enteric calici-like virus, Newbury agent 1.

The bovine enteric calici-like virus, Newbury agent 1 (NA1) was characterised to determine if it is a member of the Caliciviridae and to establish its antigenic relationship to the established bovine enteric calicivirus Newbury agent 2 (NA2). Solid phase immune electron microscopy (SPIEM) allowed quantification of NA1 virions and identification of faecal samples with optimal virus levels. NA1 particles were 36.6 nm in diameter, had an indefinite surface structure resembling that of human small round structured viruses (SRSVs), and a buoyant density of 1.34 g ml(-1). A single capsid protein of 49.4 kDa was detected by Western blotting in purified NA1 preparations prepared from post-infection but not pre-infection faecal samples and with post- but not pre-infection sera. NA1 was antigenically unrelated to the bovine enteric calicivirus NA2 by SPIEM. These properties were consistent with classification of NA1 within the Caliciviridae but demonstrated heterogeneity in the capsid composition of bovine enteric caliciviruses.

Evaluation of an immunogold electron microscopy technique for detecting bovine coronavirus.

A solid phase colloidal gold immunoelectron microscopy (IGEM) technique for detecting bovine coronavirus (BCV) was developed and shown to be specific. This test was compared with three other diagnostic tests using fifteen faecal samples. Bovine coronavirus was detected in 2 samples by direct electron microscopy (DEM), in 3 samples by immunosorbent electron microscopy, in 5 samples by haemadsorption-elution-haemagglutination and in 6 samples by IGEM. Ninety four faecal samples were tested by DEM and IGEM. Of 26 samples found to contain BCV by IGEM only 14 were positive by DEM. The IGEM technique is simple, efficient and less susceptible than others to non-specific reactions.

A liquid-hybridization method for typing the Vp4 and Vp7 genes of bovine rotaviruses.

A simple liquid-hybridization assay was developed which allows assessment of the degree of hybridization between the two serotype-determining genes of the bovine rotavirus strain UK and the homologous genes of the isolate under test. 32P-labelled transcription probes were produced from cloned complementary DNA (cDNA) copies of UK gene segments 4 and 8 and hybridized to double stranded RNA (dsRNA) extracted from rotavirus-positive field samples. Subsequent treatment with ribonuclease A (RNase A), separation of the RNase A-resistant hybrid fragments by polyacrylamide gel electrophoresis (PAGE) and autoradiography yielded a specific, reproducible banding pattern for each isolate. A total of 74 field samples was tested by both the hybridization assay and by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies (Mabs). The results obtained were in excellent agreement and confirmed that serotype G6 rotaviruses predominated. Hybridization of these G6 viruses with the gene 4 probe suggested that viruses with Vp4s related to that of UK rotavirus are also common. The hybridization assay was more sensitive than the ELISA.

Interaction of rotavirus and enterotoxigenic Escherichia coli in conventionally-reared dairy calves.

A study was made of the effects of rotavirus and/or enterotoxigenic Escherichia coli (ETEC) on dairy calves born and suckled on the farm and subsequently reared in isolation. Calves were orally inoculated at 6 days old with either rotavirus (5), ETEC (7), rotavirus and ETEC (5) or remained uninoculated controls (4), and their reactions were recorded by clinical, microbiological, and pathological observations. Rotavirus infection consistently produced diarrhoea, while ETEC inoculated alone did not colonise the intestine. In dual infections, both rotavirus and ETEC multiplied, although the severity of diarrhoea was not greater than that caused by rotavirus alone. Some ETEC-inoculated calves developed subsequent naturally-acquired rotavirus infections, but in these no ETEC multiplication occurred. The results suggest that prior or simultaneous rotavirus infection is necessary to enable ETEC colonisation of the intestine in conventional calves of this age.

Homotypic and heterotypic serum and milk antibody to rotavirus in normal, infected and vaccinated horses.

The homotypic and heterotypic antibody response to rotavirus was determined in three pony mares and their foals. The normal concentrations of anti-rotavirus antibodies in mares' milk and mares' and foals' serum over the first 10 weeks post-partum were measured using IgA, IgG and rotavirus serotype-specific enzyme linked immunosorbent assays. Experimental infection of the foals with serotype 3 equine rotavirus produced a rapid, serotype-specific response which peaked 10 days after infection and a slower heterotypic response which peaked 32 days later. In contrast, vaccination of the mares with an inactivated, adjuvanted serotype 6 bovine rotavirus produced a heterotypic response similar to that of the homotypic response in both serum and milk, although the predominant response in serum was IgG, while in milk it was IgA. These results suggest that non serotype-restricted passive protection of foals against rotavirus may be achieved by parenteral vaccination of mares.

IgG1 antibody in milk protects lambs against rotavirus diarrhoea.

Newborn gnotobiotic lambs were fed a diet of diluted evaporated milk supplemented either with normal ewes' milk or with milk obtained from ewes injected parenterally during gestation with rotavirus. Lambs fed 150 ml per day of milk collected 5 days after lambing from normal ewes were susceptible to rotavirus infection and diarrhoea, while lambs fed milk from vaccinated ewes collected either 5 or 12 days after lambing were protected. Analysis of the milk by column chromatography showed the anti-rotavirus activity to be in the fractions containing IgG1.

Characterisation of the primary local and systemic immune response in gnotobiotic lambs against rotavirus infection.

This study characterised the primary immune response in gnotobiotic lambs after infection with a lamb rotavirus (RV). Lambs were infected and killed over a 7 week period together with controls. RV-ELISA and neutralising antibodies were determined in serum, nasal secretions, and intestinal scrapings. RV-antibody secreting cells (ASC) were enumerated in blood. Lymphocyte proliferations were determined in blood and gut-associated lymphoid tissues and cytokine expression was analysed in jejunal Peyer's patches (JPPs) and mesenteric lymph nodes (MLNs). Infected lambs cleared the virus by 8-9 days after infection without showing any clinical signs. The first indication of a specific immune response to RV was an increased expression of IL-4 mRNA in the JPPs in the infected group compared to the control group 3 days after infection. Rotavirus-specific IgA ASC in blood and IgA antibodies in serum and nasal secretions were detected from 7 days after infection followed at 10 days after infection by RV-specific IgG ASC and antibodies. Rotavirus-specific IgA antibodies were not detected in intestinal scrapings in the first 10 days after infection, but were detected by 52 days after infection. No RV-specific neutralising antibodies were seen in the intestine during the course of the experiment.

Effect of oral rotavirus/iscom vaccines on immune responses in gnotobiotic lambs.

A comparison of the effect on the immune responses in gnotobiotic lambs was made between an iscom vaccine prepared from recombinant rotavirus VP6 protein, an inactivated rotavirus/iscom-matrix vaccine and a vaccine comprising inactivated rotavirus alone. All three vaccines induced immunological priming and some degree of protection was observed after a single oral dose. However, different immune responses were induced in response to a virulent infection. The group vaccinated with the rotavirus/iscom-matrix vaccine showed a Th2-like response characterised by rotavirus-specific antibodies and a down-regulation of IFNgamma in jejunal Peyer's patches. Both Th1-like and Th2-like immune responses were induced in the group receiving the VP6 vaccine as seen by significantly increased expressions of IFNgamma and IL-6 in the jejunal Peyer's patch together with an increased percentage of CD8+ T cells in the intestine and rotavirus-specific antibodies at mucosal surfaces. Iscom vaccines given orally have the ability to induce both Th1-like and Th2-like immune responses in a ruminant model.

A comparison of bovine coronavirus strains using monoclonal antibodies.

Eight monoclonal antibodies (MAbs) were raised against a bovine coronavirus (B.C.V.) which had been isolated in Scotland and was designated S2. The MAbs were divided into two groups on the basis of their reactions with S2 virus in indirect immunofluorescence (I.F.), neutralisation and haemagglutination inhibition (H.A.I.) tests. Five of the MAbs were positive by all three tests but failed to bind to proteins in Western immunoblotting experiments. The remaining three MAbs were positive in I.F. tests only, two of which were shown to bind to the 52K nucleocapsid protein by Western immunoblotting. Different patterns of antigen distribution within infected cells were demonstrated when the MAbs were used in the I.F. test. However only minor strain variations were detected by I.F. and H.A.I. tests when the MAbs were tested against each of five cell culture adapted strains of B.C.V. Twenty-nine isolates of B.C.V. have been grown in neonatal calf tracheal organ cultures: attempts are being made to further characterize these isolates.

Experimental cryptosporidiosis in germfree lambs.

Contaminating bacteria were removed from an isolate of calf Cryptosporidium by 3 sequential passages of the parasite in gnotobiotic lambs, together with antibiotic treatment of the lambs. This preparation, which contained no detectable bacteria or viruses, was given by mouth to 8 2-day-old gnotobiotic lambs, 3 of which were dosed at the same time with bacterial flora from a healthy calf. Lambs were killed at intervals from 12 to 288 h post-inoculation and the sequential development of the parasite, of enteric lesions, and of clinical illness was observed. Lesions were characterized by severe villus stunting and fusion. Clinically the most consistent sign was anorexia, with some lambs developing also a severe watery diarrhoea. Lesions and clinical signs were similar in lambs with and without intestinal bacteria. This demonstration of the enteropathogenicity of Cryptosporidium in germfree lambs suggests that it is a pathogen of significance.

Intestinal cellular immunity after primary rotavirus infection.

Infection of neonatal gnotobiotic lambs with a bovine strain of rotavirus was used to characterize the kinetics of the primary cellular intestinal immune response to this agent. At 2-3 days after infection virus was first detected in the faeces and increased numbers of CD45R+ cells were observed in peripheral blood. These cells persisted in significantly increased numbers in the circulation until 7-8 days after infection. At this time, virus was no longer detectable in the faeces. The increase in CD45R+ cells preceded the appearance of virus-neutralizing antibodies in the serum at 1 week after infection. Maximal antibody titres were reached 2 weeks after infection. Virus-primed cells were first observed 1 week after infection in the jejunal and ileal Peyer's patches, mesenteric lymph nodes and peripheral blood, and persisted in the mesenteric lymph nodes and jejunal Peyer's patches for a further 4 weeks. Analysis of lymphocyte surface antigens indicated that different sub-populations of lymphocytes were responding in the various lymphoid tissues; a majority of CD4+ cells was observed in the mesenteric lymph nodes, whereas B cells predominated in the ileal Peyer's patches.

Chemotherapy of experimental bovine petechial fever.

A dithiosemicarbazone was compared with two tetracycline formulations in the treatment of bovine petechial fever (BPF) in experimentally infected sheep, and was then used to treat the disease in experimental cattle. The dithiosemicarbazone was found to be more efficacious than either of the other two drugs in treating ovine BPF, and also to be effective against BPF in cattle.

A rotavirus in lambs with diarrhoea.

A reovirus-like agent was identified from an outbreak of enteritis in young lambs. From its morphology and immunological relationship with calf rotavirus, it was concluded that it was a rotavirus which infects lambs.

Enteric campylobacter infection in gnotobiotic calves and lambs.

Gnotobiotic calves and lambs were infected orally with Campylobacter jejuni, C coli or C hyointestinalis to assess pathogenicity. All animals were successfully colonised and excreted mucoid faeces but showed no other clinical signs. Campylobacters colonised the large intestine better than the small intestine, in which bacterial numbers decreased with time after infection. Campylobacters were found occasionally in the lumen of crypts in close proximity to epithelial cells and included in a mucus-like material. Lesions were mostly in the large intestine in calves whereas in lambs they were present in the ileum. In animals inoculated with C jejuni or C coli scattered crypt abscesses, focal inflammatory infiltrates in the lamina propria and goblet cell discharge were found. In lambs inoculated with C hyointestinalis only minor changes were found in the small intestine. Serum antibody response was either absent or present at a low level only from the 19th day after infection.

Some characteristics of a natural infection by parainfluenza-3 virus in a group of calves.

Parainfluenza-3 (Pi3) virus infection in a group of 25 calves is described. The virus was isolated from the lungs of four calve at days 6, 7, 13 and 55 after they were housed together at birth. Intracytoplasmic inclusion bodies were seen by light microscopy in bronchial and bronchiolar epithelial cells of two of these calves. Virus infected cells were detected by electron microscopy in three of the four calves. Haemagglutination inhibition antibodies to Pi3 virus were found in the sera of the calves. Despite the virus being present in the group from one week, a significant increase in antibody titre was found in only two animals although all the calves were in contact with each other during the study period. The pulmonary lesions in the four infected calves consisted of a bronchitis and bronchiolitis with infiltration of the walls and lumena of these structures by neutrophils and an adjacent neutrophil infiltration of alveoli some of which were collapsed.

Diarrhoea in dairy calves reduced by feeding colostrum from cows vaccinated with rotavirus.

Forty-two dairy calves remained with their dams for two days after birth, and then were removed to a calf rearing shed. Calves were allocated to three groups for the next 14 days, and received twice daily either whole milk, whole milk with a 10 per cent supplement of pooled normal bovine colostrum or whole milk with 10 per cent supplement of colostrum from cows vaccinated with rotavirus. A natural outbreak of diarrhoea occurred, affecting 28 of the 42 calves. Feeding immune colostrum delayed the onset of diarrhoea, and reduced its incidence, duration and severity. Live weight gains were consequently improved. The group fed normal colostrum had diarrhoea intermediate in severity between that of control calves and those fed immune colostrum. The aetiology of the diarrhoea was complex, with calves excreting rotavirus, enteropathogenic Escherichia coli and cryptosporidia.

The prevalence of enteric pathogens in diarrhoeic thoroughbred foals in Britain and Ireland.

A survey of 77 normal and 326 diarrhoeic foals in Britain and Ireland from 1987 to 1989 revealed a significantly higher prevalence of Group A rotaviruses and Aeromonas hydrophila in diarrhoeic foals. The prevalence of cryptosporidia, potentially pathogenic Escherichia coli, Yersinia enterocolitica and Clostridium perfringens was similar in normal or diarrhoeic foals. Rotaviruses had a similar prevalence in all age groups of scouring foals up to three months of age, with an overall prevalence of 37 per cent among diarrhoeic foals. The number of cases of diarrhoea varied considerably from year to year, but in all three years of the survey rotavirus was a significant pathogen. A comparison of diagnostic tests for rotavirus in the faeces showed electron microscopy (EM) and polyacrylamide gel electrophoresis (PAGE) to have similar sensitivity. The Rotazyme ELISA test kit was found to have the same sensitivity as a combination of EM and PAGE. A. hydrophila had an overall prevalence of 9 per cent among diarrhoeic foals, although its prevalence was higher in some age groups. A. hydrophila has not been established previously as a significant enteric pathogen in foals. Other putative pathogens found at very low prevalence were coronavirus, the putative picobirnavirus, Campylobacter spp. and Salmonella spp. No evidence was found of synergistic effects between rotavirus, cryptosporidia and potentially pathogenic E. coli. Neither coccidia nor non-Group A rotaviruses were found in any of the samples examined.