Use of indicators for monitoring the reduction of pathogens in animal waste treated in biogas plants.

The possibility of including bacterial indicators in programmes for monitoring the disinfection of animal waste treated in biogas plants was examined. Five full-scale plants receiving waste from several farms were monitored. Of these, two used thermophilic and one mesophilic digestion exclusively whereas two employed a two stage process involving thermophilic pretreatment followed by mesophilic digestion. For each plant the reducing effect on bacterial pathogens present in the incoming waste and on E. coli and enterococci indigenous to the waste was followed over long periods of time (six to 16 months). It was found that both E. coli and enterococci could be used as indicators to monitor the reduction of vegetative bacterial pathogens, but the use of enterococci would give the broadest "safety margin" in this respect. It is concluded that thermophilic as well as mesophilic digestion with thermophilic pretreatment will result in a sufficient reduction of both vegetative pathogenic bacteria and intestinal parasites occurring in concentrations usually found in animal waste to allow for unrestricted use of the de-gassed material in agriculture. This cannot be said for mesophilic digestion used alone. To achieve a sufficient reduction in the number of pathogens during the digestion process, a decrease in the enterococcal concentration of at least 3-4 log10 units should be accomplished, which for normal waste would correspond to a maximum concentration of these bacteria in treated material in the order of 10(2) cfu/ml. It is suggested that enterococcal counts in newly digested material should be one of the parameters in a monitoring programme aimed at controlling the hygienic state of the end-product from large biogas plants.

The cytochrome P450 system of Atlantic salmon (Salmo salar): II. Variations in hepatic catalytic activities and isozyme patterns during an annual reproductive cycle.

A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5-7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17ß was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b5, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17ß levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17ß as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17ß independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.

Environmental contaminants and biochemical responses in flatfish from the Hvaler Archipelago in Norway.

The levels of several environmental contaminants, including selected polyaromatic hydrocarbons (PAH), organochlorines (DDT/DDE, hexachlorobenzene), 15 polychlorinated biphenyl (PCB) congeners, and polychlorinated dibenzofurans and dibenzo-p-dioxins, PCDF/PCDD), and heavy metals (Cd, Hg, Pb, and As) were analyzed in muscle and liver of three different flatfish species (dab, Limanda limanda; flounder, Platichthys flesus; plaice, Pleuronectes platessa) caught by gill netting at different sites in the Hvaler Archipelago. Indices of biochemical effects in liver S9-fractions were studied by measuring cytochrome P450-dependent monooxygenase and UDP-glucuronyl transferase activities, and by immunoquantitating cytochrome P450 1A1 using an indirect enzyme-linked immunosorbent assay (ELISA). Only low levels of PCDD/PCDF, Cd, and Pb were observed, whereas PCB levels were significantly elevated in fish from the inner sites of the Archipelago compared to a reference site. The contaminant gradient toward the Glomma estuary was correlated with increased cytochrome P450 1A1 activity, measured as 7-ethoxyresorufin O-deethylase (EROD), and with immunoquantitated P450 1A1. In contrast, fish from the site at Idefjorden, although containing elevated contaminant levels, did not show elevated EROD activity, but apparently elevated P450 1A1 protein. These findings may reflect different pollution histories of the sites, and indicate the applicability of biochemical effect indices (i.e., EROD and P450 1A1 immunoquantitation) to monitoring studies. The integrated chemical-biochemical approach employed in this study can obviously be expanded to give fruitful information about cause-effect relationships in other contaminant situations.

Application of a cytochrome P-450 IA1-ELISA in environmental monitoring and toxicological testing of fish.

1. An enzyme-linked immunosorbent assay (ELISA) based on polyclonal rabbit anti-cod cytochrome P-450 IA1 IgG has been developed in our laboratory. 2. The antibodies employed in the ELISA demonstrate good cross-reactivity with the homologous protein in a number of other fish species, giving cross-reacting protein bands o 54-59 kDa in liver microsomes, as determined by Western blotting. 3. The ELISA technique has been used in numerous experiments with both field collected and laboratory exposed fish of different species, showing good correlation with contaminant exposure. 4. In some instances of PCB exposure where the classical P-450 IA1 monooxygenase assay 7-ethoxyresorufin O-deethylase (EROD) failed to reveal any induction, the ELISA technique demonstrated increased levels of P-450 IA1 protein, indicating inhibiting effects of the PCBs on EROD measurement. 5. In tissues like gill filaments, and in whole cod larvae, where EROD activity is barely detectable, if at all, the ELISA technique showed induction after exposure to a water soluble fraction (WSF) of North Sea crude oil. 6. The results reviewed indicate the usefulness of the ELISA technique to allow rapid screening of a large number of samples, and especially when catalytic measurement is difficult or impossible due to (a) small sample or tissue size, (b) loss of activities in bad storage conditions, or (c) presence of compounds inhibiting activity.

The cytochrome P450 system of atlantic salmon (Salmo salar): I. Basal properties and induction of P450 1A1 in liver of immature and mature fish.

The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The Km for 7-ethoxyresorufin was 0.4 µM, and Vmax 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20-30 pmol/min/mg protein). Treatment with the P450 1A1 inducer ß-naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30-40-fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (Mr=58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at -80°C in 20% glycerol without losing more than 20-40% of its catalytic activity.

The prevalence of plasmids in Danish bovine and human isolates of Salmonella dublin.

Nine different plasmid profiles were demonstrated among 135 Danish isolates of Salmonella dublin, with 58% of the strains enclosed in the major profile group. Two strains did not carry any plasmids. The plasmid profiles of 129 strains (96%) were made up of one or more of only four plasmids of approximate sizes 98 kb, 80 kb, 4.0 kb, and 3.8 kb. There was no significant difference in the prevalence of different plasmid profile groups between isolates from cattle and man. The serovar-specific and virulence-associated 80 kb plasmid could be demonstrated in all plasmid carrying strains, but one strain carried this plasmid at a very low copy number. Five of 47 strains tested showed resistance to antibiotics (2 to sulphathiazole, 2 to streptomycin, and 1 to streptomycin and tetracycline). Only the combined streptomycin/tetracycline resistance was transferable by a 48 kb conjugative plasmid.

An outbreak of meningo-encephalitis in fallow deer caused by Listeria monocytogenes.

An outbreak of listeric meningo-encephalitis occurred in a population of 1800 fallow deer (Dama dama) in a park during the winter and early spring of 1985 to 1986. Listeriosis was diagnosed in 41 of 42 fallow deer that showed the typical central nervous system signs of circling disease or were found dead. The diagnosis was verified by bacteriological examination of the brains of 35 animals. In five of the seven remaining cases listeriosis was diagnosed by histological examination, and in one animal by clinical signs alone. Listeria monocytogenes was isolated in three of 23 soil samples taken from the park. In addition, L monocytogenes was isolated from the intestinal contents of apparently normal fallow deer. Fifty isolates from animals and soil were serotyped and all of them belonged to serovar 4b except one from brain (serovar 1/2b) and three from intestinal contents (serovar 1/2a). In phage typing of 54 isolates, the 35 isolates from the brain and spleen of diseased animals belonged to the same lysovar, as did most isolates from other sources, but strains from intestinal contents belonged to three other phage types. No external source of L monocytogenes was demonstrated in the outbreak and stress due to the poor beech-mast crop, an increased stocking rate and a sudden change in the weather are suspected as predisposing factors.

[Occurrence and distribution of drug resistant enterobacteria in extra enteral habitats (author's transl)]. / Forekomst og udbredelse af resistente enterobakterier i extra-enterale habitater

In the period 1971-73 various extra enteral habitats have been examined for the occurrence of resistant E. coli. Of 172 isolates of E. coli from retail butcher's meat products 9.9% were resistant and 3.5% R+ E. coli. Of 203 isolates of E. coli from bakery products 7.9% were resistant and 3.5% R+ E. coli. Of 52 isolates of E. coli from freshly killed carcases of chickens 40.4% contained weak non-transferable monoresistance against sulphonamides (Table I). Of 92 isolates of E. coli from seawater receiving human sewage 15.2% were resistant and 4.4% R+ E. coli (Table III). All but one samples of sewage and crude sludge contained resistant E. coli of which 11% harboured R factors (Table II). Of 117 Salmonella isolates from human sewage 11.1% were resistant and in 4.2% resistance were transferable (Table VI). Quantitative examinations of untreated and treated sewage did not indicate any selective reduction of resistant E. coli compared to sensitive (Table IV). However, more di- and multiresistant E. coli were found in sewage than in seawater (Fig. 1). The food hygienic implications of resistant E. coli are discussed.

[Persistance of resistant Escherichia coli in a calf herd]. / Persistens af resistente Escherichia coli i kalvebesaetining

In an experimental station for calf feeding the occurrence and distribution of drug resistant E. coli in faeces have been investigated among selected groups of calves. Samples were obatined just after the calves arrived 1--2 weeks old in January--February and at intervals until an age of 7--11 months. Daily from arrival to the age of 8 weeks all calves at the station received antibiotics in their feed. In 1971 it was chlortetracycline and in 1972 zincbacitracin. In addition, from 3.5 percent to 60 percent of all calves were every month given therapeutic doses of antibiotics, mainly penicillin + streptomycin and neomycin. Both years the highest number of treatments were administered in the months of February and March (cf. fig. 1). In each of the years studied, a high prevalence of calves harbouring resistant E. coli (total resistance: greater than 88 percent) and R-+E. coli (1971: 83 percent and 1972: 50 percent) was demonstrated at the first sampling. As the calves grew older a general tendency to exchange the resistant strains with sensitive ones was recognized. The prevalence of calves with R-+ E. coli generally devreased at a faster rate than that of total resistance. A discussion is given of the epidemiological significance of cross infection and of selection pressure of antibiotics for the establishment of a resistant flora in individuals as well as for replacement of this flora with sensitive strains.