The survival of Brachyspira hyodysenteriae and Brachyspira pilosicoli was investigated at 10 degreesC in laboratory microcosms consisting of soil, porcine faeces, and in soil mixed with 10% porcine faeces. respectively. By plate spreading, survival of B. hyodysenteriae was found to be 10, 78 and 112 days in soil, soil mixed with 10% faeces, and in porcine faeces, respectively. The identities of the colonies on the plates were confirmed using PCR targeting 23S rDNA for specific detection of B. hyodysenteriae. A positive PCR signal could be obtained up to 112 days in all microcosms by direct extraction of DNA from microcosms followed by PCR.The survival time for B. pilosicoli was 119 days in pure soil and 210 days in soil mixed with 10% porcine faeces and in pure faeces, respectively, as determined by plate spreading followed by PCR. On the other hand, by direct extraction of DNA followed by specific detection by PCR. B. pilosicoli could be detected up to 330 days in all microcosms.Dot blot hybridisation with digoxigenin-labelled specific oligonucleotide probe targeting rDNA could not be used for direct detection of Brachyspira spp. from microcosms due to low sensitivity. However, it was used for confirmation of the identity of colonies and proved to be a useful technique.These results show that the two Brachyspira species may survive in outdoor environment for the times shown in these investigations using laboratory microcosms. (C) 2001 Elsevier Science B.V. All rights reserved.

The clonal relationship among Salmonella enterica serotype Typhimurium isolates from selected pig production units in Denmark was investigated by the pulsed field gel electrophoresis (PFGE) typing method to determine environmental survival and spread of Salmonella in different herds. Thirty-four Typhimurium isolated during 1996-1998 from porcine faeces and environmental samples from three pig farms designated 1, 3 and 5 were characterised by PFGE using two restriction enzymes. Farm 5 supplied piglets to farm 1 and the herds were located close to each other. Results of PFGE analysis showed both intra- and inter-relationships, i.e. identical PFGE patterns among the faecal and environmental isolates from farm 1 and farm 5. All the isolates from farm 3 irrespective of the source showed identical PFGE patterns, but were different from samples from farms 1 and 5. This study indicates spread between farms and survival of a farm-specific clone. Furthermore, identical PFGE patterns of isolates from piglet supplier and finisher herds indicate that the farrow-to-grower herd of farm 5 was sub-clinically infected prior to delivery to farm 1 and thereby caused the transmission of Salmonella.

A survey was undertaken to examine sea water and sediment for the presence of Vibrio and Aeromonas spp. along approximately 900 km of coast in Southern Italy during early and late summer. A quantitative analysis was also done to evaluate the water fecal contamination at the stations examined. The results indicate that all the investigated areas were submitted to a wide spatial fluctuation of fecal contamination and that Vibrio and Aeromonas spp. were present in both high and low fecal-contaminated stations. Sixty two percent of the investigated samples were positive for Aeromonas spp., while 42% of samples were positive for Vibrio spp. It was interesting to note that 38% of the positive stations for both Aeromonas and Vibrio spp. showed a fecal coliform contamination of water at < 10(2) cells 100 ml(-1). Thus, these findings support the hypothesis that the bacterial indicators (such as fecal coliforms) do not always satisfactorily reflect the hygienic quality of water. The presence of Vibrionaceae on copepods was also investigated. Copepods were sampled at a station located inside the harbour of the city of Naples and were found contaminated by V. cholerae non-O1, V. alginolyticus, V. fluvialis and A. caviae. Furthermore, the antibiotic resistance patterns of isolated bacteria showed the presence of a number of resistant strains among the isolates. In order to discriminate the isolates on the basis of their biochemical profiles and/or antibiotic resistance patterns, cluster analysis was carried out which showed that no unique assay could fully discern these isolates. However, the best discrimination resulted from complete pattern profile based on both biochemical profiles and antibiotic resistance patterns.

The agricultural utilization of sewage sludge imposes a high level of sanitation and stabilization of organic matter in order to maintain soil, water and air qualities, and to effectively use such bioresidues as a soil amendment and as a source of nutrients for plants. Improper sanitation poses a serious threat to human and animal health. Stabilization and sanitation of sewage sludge have the advantage of coupling safe sewage sludge recycling in agriculture with its disposal, as many economic and environmental constraints make discharging strategies more and more difficult to apply. This article reviews the various biological methods to sanitize sewage sludge and the efficiency and efficacy of these methods in eliminating pathogens and parasites. Available monitoring techniques are also discussed. Finally, a conceptual approach is presented on the basis of epidemiological and ecological studies in order to assess the environmental and health impacts of recycling sewage sludge through land application.

A total of 100 Aeromonas caviae strains isolated from river waters (38 isolates), raw sewage (30 isolates) and effluents of stabilisation ponds (i.e. treated sewage; 32 isolates) in Marrakech, Morocco, were tested for the presence of putative virulence factors to delineate differences, if any, in their enteropathogenic potential in relation to the source of isolation. A number of A. caviae isolates were able to elaborate at least one of the tested virulence factors. Of the 100 strains tested, 27 were capable of producing haemolysins, 19 produced cytotoxin, 24 produced cytotonic toxin, while 18 of the isolates possessed the capacity to adhere to HenLe 407 human intestinal cells with more than five bacteria adhering per cell. In order to assess the virulence potential of A. caviae isolates in relation to the source of isolation, we suggest the potential virulence index (PVI) as a tool for comparison. It is calculated as PVI=x/ny, where x is the number of positive occurrence of virulence factors in a population y, and n is the number of suitable virulence factors used for the calculation of the PVI. Accordingly, in this study A. caviae strains isolated from treated sewage samples showed a higher PVI (=0.406) than those isolated from raw sewage (PVI=0.175) or those from river waters (PVI=0.09). These results suggest that stabilisation pond systems used for sewage purification under arid climate conditions in Marrakech, may have selected potentially enteropathogenic A. caviae strains.

An enzyme-linked immunosorbent assay (ELISA) to determine IgG antibody levels against K88 (F4) fimbrial antigen from porcine enterotoxigenic Escherichia coli (ETEC) has been developed, The ELISA method was checked with serum samples obtained from rabbits and pigs, and the parameters affecting the method were also analysed, ELISA plates were optimally coated with K88 antigen 0.5 mu g/ml for testing rabbit antiserum or with 1.25 mu g/ml for testing pig serum, Optimal concentrations of H2O2 (0.5%) and orthophenylene-diamine (OPD) (0.125%) were chosen when a 10-min incubation period was used, The expression of antibody levels as enzyme-immunosorbent units (EIU) significantly decreased the variability of results between duplicate plates, when compared with the expression of results as direct OD values, ELISA-K88 applied to a field study with serum samples from 141 vaccinated and 52 unvaccinated sows was shown to be useful in differentiating between samples from vaccinated and unvaccinated animals.

The colonisation of planktonic copepod integument by bacteria belonging to the family of Vibrionaceae is a well described phenomenon. In this study, besides reporting on the occurrence of Vibrionaceae and other enteropathogens, we further report on the bacterial attachment to the Estuarine copepod Acartia margalefi in a faecal polluted coastal lagoon near Naples, Southern Italy. In addition, we also performed a laboratory experiment to study the ability of 7 bacterial strains (Vibrio cholerae non-Ol, V. mimicus, V. parahaemolyticus, V. alginolyticus, Aeromonas hydrophila, Escherichia coli and Pseudomonas sp.) to colonise the copepod integument. For this laboratory study, 4 different species of copepods, namely Temora stylifera, A. clausi, Centropages typicus and Paracalanus parvus sampled from the Gulf of Naples (Southern Italy) were used. Scanning electron microscope (SEM) studies on the copepods sampled from the lagoon indicated that the bacterial attachment on the integument of Acartia margalefi was mainly on the ventro-lateral body region of the host and in the joints of the thoracic segments, as well as on the swimming and feeding appendages. This infestation, made by rod-like bacteria, was absent in winter but reached peak values of 70% frequency in June. The laboratory studies showed that while V. cholerae non-Ol and A. hydrophila attached on live and dead copepods, respectively, the V. parahaemolyticus, V. alginolyticus, V. mimicus, E. coli and Pseudomonas sp. failed to colonise either live or dead copepods. Thus, this study provides further valuable information about the ecological relationship between different microorganisms (pathogens) and copepods in the coastal marine environment in Southern Italy. Copyright (C) 1996 Elsevier Science Ltd

A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the beta-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food “landgång” which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty‐two of the 27 persons became ill within 20‐34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left‐over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left‐over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 10 6 to log >10 7 organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus , fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce β‐hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.

A variety of potential virulence markers such as the production of cytotoxin, haemolysin, exoenzymes, bactericidal action of sera, presence of capsule and adhesion to human intestinal cells were investigated on Vibrio vulnificus strains isolated from eels in Sweden. The strains had the capacity of producing all or some of the above-mentioned virulence markers, to varying degrees though none of the strains produced any capsule. The strains also bound specifically to human intestinal cells in vitro with maximum adhesion levels of 30 bacteria/cell. The results on binding of V. vulnificus cytotoxin to HeLa cells, showed that a very short exposure time (30 min) was required for inducing the cytotoxic effects. V. vulnificus is a relatively new addition to the list of bacteria pathogenic for humans, and since there are increasing reports on its isolation from aquatic environments and seafood (e.g. raw oysters, crabs and shellfish), the results on virulence profiles of V. vulnificus strains presented above emphasize the importance of these organisms in public health and epidemiological studies.

Aeromonas hydrophila strains isolated from the same geographical region (southern Italy) but from different sources (sea sediments and human diarrhea cases) were characterized for the production of potential virulence determinants, such as production of cytotoxins, cytotonic toxins, hemolysin, and dermonecrotic factors and their capacity to adhere to human intestinal 407 cells in vitro. The results showed that isolates from both the sources produced all or some of the virulence factors which may be involved in the pathogenesis of Aeromonas-associated infections. Our study indicates that further epidemiological studies are necessary to elucidate the public health significance of infections caused by Aeromonas spp.

An outbreak of Aeromonas hydrophila infection with a high rate of mortality (95%) in turtles ( Pseudemis scripta ) in Italy is reported. Pure cultures of the pathogen were isolated from liver, lung, kidney, and heart specimens of the turtles. The A. hydrophila isolate was resistant to amoxicillin, ampicillin, cephalothin, and trimethoprim-sulfamethoxazole but was sensitive to a number of other antibiotics tested. The study indicates that pet turtles can act as reservoirs of this pathogen and may play an important role in the etiology of Aeromonas -associated human infections.

Different food samples collected from randomly selected retail consumer shops in Uppsala (Central Sweden) were investigated for the presence of Aeromonas species. Forty-two percent (10/24) of the food samples contained organisms belonging to two Aeromonas spp. namely, Aeromonas hydrophila and Aeromonas sobria. The type of foods from which Aeromonas were isolated, were poultry, fish, beef and pork. None of the Aeromonas spp. were isolated from vegetables or raw milk samples. The Aeromonas isolates possessed the capacity to produce different potential virulence determinants such as haemolysin, cytotoxin, cytotonic toxin, enterotoxin and protease. Since A. hydrophila and A. sobria are considered to be potential human pathogens, it is concluded that in case foodborne infections are suspected or encountered, the role of Aeromonas should not be ruled out.

Drinking water from three different municipal distribution systems in Sweden was investigated for the occurrence of Aeromonas spp. and identification of virulence factors of these organisms. Twenty-three of the 27 samples (85%) from the three distribution systems were positive for presumptive Aeromonas with a maximum of 860 cfu/100 ml drinking water. Of the 61 randomly selected presumptive Aeromonas isolates, 41 were identified as A. hydrophila and 20 as A. sobria. These Aeromonas isolates produced a variety of potential virulence factors such as haemolysin, cytotonic- and cytotoxic-toxin, enterotoxin and protease. These findings on the presence of Aeromonas in municipal drinking water supplies in Sweden show that the consumer can be exposed to high number of aeromonads. However, more epidemiological information needs to be gathered in this regard to elucidate their public health significance.

Thirty-five Salmonella strains isolated from human cases of salmonellosis were tested and compared for their fibronectin (fn) binding capacities by using two fn-particle agglutination assays (fn-PAAs) prepared by coating with human fn either (i) latex beads (Difco; 0.81-micron diameter) (L-fn-PAA) or (ii) heat-killed formalin-treated Staphylococcus aureus Cowan 1 cells (C-fn-PAA). Six S. aureus strains were also included in this study as controls. The strains were cultured on colonization factor antigen agar and blood agar and in tryptic soy broth and brain heart infusion broth. The Salmonella and S. aureus strains were cultured at 33 and 37 degrees C, respectively, for optimal expression of fn-binding proteins. Bacterial cells (approximately 10(10) cells per ml) harvested from growth in various culture media and suspended in 0.02 M potassium phosphate buffer (pH 6.8) agglutinated the fn-PAA reagents. These reactions were scored semiquantitatively from + to + depending on the speed or intensity of the reactions within 2 min. Maximum agglutination in fn-PAA systems was observed when the cells were grown in brain heart infusion broth, while tryptic soy broth proved to be least suitable media for culturing cells for fn-PAAS. Although a statistically highly significant correlation was obtained between results of assays of radiolabeled fn and 29-kDa fragment binding, no significant correlation was observed (i) between the results of strains cultured in different media or (ii) when semiquantitative score results of the two fn-PAA systems were compared with those of the conventional radiolabeled fn assay. To enhance the efficiency of the test system, the C-fn-PAA reagent was stained with methylene blue (2% in 0.17 M glycine-NaOH buffer [pH 6.8]). This facilitated easy interpretation of results, which could be performed on hydrophobic paper instead of glass slides. The results obtained with both unstained C-fn-PAA and stained C-fn-PAA were comparable to each other and reproducible. Although the fn-PAAs are simple and easy to perform, the results did not differentiate between negative, low, moderate, and high binding abilities when Salmonella strains were evaluated for fn binding, and the results were not comparable to those obtained by the conventional radiolabeling method.

Toxin production, cell-surface hydrophobicity and fibronectin-binding properties of 21 Salmonella strains of different species, isolated from Swedish travelers to different parts of the world, were studied. Cell sonicate supernatants from blood agar grown cultures of 80% of the strains induced rabbit skin permeability reaction in the form of induration and/or blueing while 33% of the strains also produced cell necrotizing factor on rabbit skin. Four strains were negative in the rabbit skin permeability test, while only two were negative when tested on CHO cells. When cultured on blood agar, a majority of the strains (17/21) showed low cell-surface hydrophobicity, showing no aggregation even at 1.5 M ammonium sulfate concentration in salt aggregation test (SAT), while only four strains showed high cell-surface hydrophobicity. Furthermore, these strains could be classified as low fibronectin binders due to their poor interaction with fibronectin or its 29 kDa N-terminal fragment.

125I-fibronectin and 125I-collagen (type II) binding was detected in Escherichia coli strains isolated from chickens and poults. High fibronectin binding-strains also bind the 29 kD aminoterminal fragment of fibronectin. Binding properties in strain CK28 were partially characterized. The highest binding of 125I-fibronectin and 125I-collagen for strain CK28 was obtained with bacteria grown at 33 degrees C. Binding of 125I-fibronectin, its 125I-29 kD fragment, and 125I-collagen, was very rapid, reaching a maximum in 5 min. Binding of 125I-fibronectin and 125I-collagen was considerably inhibited by preincubation of bacteria with unlabelled fibronectin and unlabelled type I collagen respectively, but not inhibited with human immunoglobulin G or bovine serum albumin. Inhibition experiments showed that the reversibility of 125I-fibronectin binding was estimated at approximately 50%, while reversibility for 125I-collagen binding was higher than 90%. Receptors for fibronectin, its 29 kD fragment, and collagen were released from the bacterial surface by treatment at different temperatures, and surface material released at 100 degrees C inhibited binding. There was cross-inhibition for both fibronectin and collagen binding when unlabelled fibronectin and unlabelled collagen were used as inhibitors, suggesting that binding receptors for both proteins may be closely located.

Bacterial surface hydrophobicity as well as mannose-sensitive haemagglutinating (MSHA) and mannose-resistant haemagglutinating (MRHA) activities were studied in Escherichia coli strains with and without recognized fimbrial antigens grown under different conditions. Relative bacterial surface hydrophobicity was measured by the salt aggregation test. Four kinds of bacterial aggregations depending on fimbrial antigens were observed: bacteria with CFA/I, CFA/II, CFA/III and K88 aggregated in a particulated form, bacteria with type 1 pilus in a granular form, and bacteria with K99 in a tiny granular form. Some strains, mainly when grown under non-optimal conditions at 18 degrees C or when heated at 80 degrees C, aggregated in a non-typical filamentous form. Among MRHA- MSHA+ (type 1 pilus positive), two classes of bacteria were detected: hydrophobic bacteria aggregating in 0.2-0.4 M ammonium sulphate, and non-hydrophobic bacteria aggregating in 2.0 to 1.6 M ammonium sulphate. The hydrophobicity levels in strains possessing different recognized fimbrial antigens, when grown under optimal conditions to express fimbriae and their typical haemagglutination pattern, were of a decreasing order, viz. CFA/III = CFA/I greater than CFA/II greater than MRHA-MSHA+ hydrophobic = MRHA+ greater than P987 greater than K99-F41 = K88 greater than MRHA- MSHA+ non-hydrophobic greater than MRHA- MSHA- without recognized fimbriae.

The optimal conditions for the binding of fibronectin to Salmonella enteritidis strain 27655R, and the cell-surface components involved in the binding, were identified. Cultivation on colonisation factor antigen (CFA) agar or in CFA broth at 33°C for 24 h were found to be optimal for the expression of fibronectin binding. Such cultures exhibited 88% and 70% binding of 125I-labelled fibronectin and its 29-kDa N-terminal domain, respectively. The fibronectin binding was reversed by the addition of unlabelled fibronectin or its 29-kDa fragment. Scatchard plot analysis of the binding showed that the strain possessed one high-affinity (Kd = 5.8 × 10−10 M) and one low-affinity (Kd = 2 × 10−8 M) binding site. The fibronectin-binding could be inhibited by cell surface components of S. enteritidis 27655R released by 30 min treatment at 65°C or 95°C. Inhibition could also be achieved using purified fimbriae. A non-fimbriated mutant of strain 27655R showed a much reduced binding of fibronectin (15%). Electron microscopic analysis showed association of the gold-labelled 29-kDa N-terminal fragment with S. enteritidis 27655R fimbriae. In conclusion, the findings suggest that S. enteritidis (strain 27655R) possesses fibronectin-binding fimbriae.