Virus inactivation in groundwater in a postglacial lava field in arctic climate.

Outbreaks of viral gastroenteritis are often connected to contaminated drinking water. The assessment of the water quality relies on the cultivation of indicator bacteria, and little is known of the fate of viruses in groundwater, especially in arctic regions. In Iceland, the groundwater temperature is between 3 and 6°C. The aim of this study was to determine virus inactivation at low temperature in a groundwater microcosm and in a borehole in a postglacial lava field. Two phage species that are commonly used as surrogates for norovirus were used, MS2 and PhiX174. Dialysis bags were used for the samples, and a device was constructed to hold many samples at a time and protect the samples in the borehole. No significant decrease of infective PhiX174 phages in the borehole or of the MS2 phages in the microcosm was observed. A slightly significant decrease of PhiX174 in the microcosm was noticed, with one log reduction time of 476 days. On the other hand, a significant reduction in MS2 was found in the field test, where the time needed for 90% reduction was 12·5 days. The results showed that an infective virus can exist in groundwater for months or years in arctic regions and a great difference may exist between results from microcosm and field tests. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that arctic regions are highly sensitive to virus contamination as an infective virus may exist in groundwater for years at low temperature. The results also show that the virus inactivation observed in field tests may differ considerably from the inactivation observed in laboratory microcosms. The results emphasize the importance of large protection zones around drinking water intakes as well as good wastewater treatment so that the likelihood of faecal contamination of groundwater is reduced.

Isolation and characterization of an antigen from the fish pathogen Moritella viscosa.

AIMS: Moritella viscosa is a Gram-negative psychrophilic bacterium that causes winter ulcer disease in farmed fish. The aim of the study was to describe an outer membrane protein of roughly 20 kDa in pathogenic M. viscosa and to compare the coincident protein of strains isolated from different fish species and geographical locations. METHODS AND RESULTS: The protein was isolated from a pathogenic strain of M. viscosa. An oligopeptide sequence obtained with MS/MS analysis showed homology to Escherichia coli OmpA and Neisseria surface protein A. The protein was named Moritella viscosa outer membrane protein 1 (MvOmp1), and sequence analysis confirmed that it is an integral membrane protein consisting of eight antiparallel ß-strands, three short periplasmic turns and four long hydrophilic extracellular loops. The encoding gene, mvomp1, was fully sequenced in nine strains representing different serotypes and phenotypes. The results revealed some differences in the extracellular loops between strains. The mvomp1 gene was cloned and expressed in E. coli, and the recombinant product was recognized by anti-M. viscosa polyclonal antisera. CONCLUSIONS: The results indicate that MvOmp1 is a major protective antigen of M. viscosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The results open up possibilities for use of the protein as a part of a subunit vaccine in the future.

Antigen profiles of the fish pathogen Moritella viscosa and protection in fish.

AIMS: Moritella viscosa is a gram-negative bacterium that causes winter ulcer disease in salmonid fish cultured in sea water below 8 degrees C. The aim of this study was to study the antigen profiles of these bacteria and to reveal the protection which the antigens induce in fish. METHODS AND RESULTS: Lipooligosaccharides (LOS) and an approximately 17-19 kDa outer membrane antigen were shown to be the major specific antigens of M. viscosa. The size of the wall antigen differed between strain groups and even between strains reacting positively in the same sera. Four different serotypes of M. viscosa were determined by producing polyclonal sera. Western blot analysis revealed that sera from vaccinated fish groups that had good or fair protection reacted against the LOS and the 17/19 kDa antigen, while no antibody response was observed with sera from groups that showed no efficacy. CONCLUSIONS: The study provides evidence that LOS and an approximately 17-19 kDa outer membrane antigen are the major specific protective antigens of M. viscosa, and that the M. viscosa species consists of many different serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are important for the production of vaccines against winter ulcers and should also lead to better methods of verifying the bacteria and monitoring winter ulcers.

Penicillin tolerance among oral streptococci.

Penicillin tolerance was elicited in 18 of 46 strains of viridans streptococci isolated from the mouths of 19 of 20 healthy subjects and in 31 of 54 consecutive blood-culture isolates of streptococci. Enterococci and Streptococcus sanguis were the organisms most frequently tolerant but the property was also common among isolates of S. mutans, S. mitior and Lancefield Group G streptococci. Pneumococci and S. salivarius were rarely tolerant. When incubated with penicillin at 5 x MIC in batch or continuous cultures, both tolerant and sensitive strains of oral streptococci declined in number less rapidly than S. pyogenes. However, combinations of penicillin and gentamicin killed tolerant and sensitive oral streptococci.

Non-sporeforming anaerobic bacteria in clean surgical wounds--air and skin contamination.

The contamination of clean surgical wounds with anaerobic and aerobic bacteria was studied in 52 hip operations. In addition to wound samples, samples from air and the patients' skin were taken. The median of the total number of bacteria isolated from the wounds was 26 colony-forming units (cfu). The median percentage of anaerobic bacteria in the wound counts was 30. Propionibacterium spp. were found in 71 per cent of the wounds and anaerobic or microaerophilic cocci, most often Peptococcus spp., were found in 23 per cent. In six of 43 patients the same bacterial flora was seen in the skin samples and wounds. The geometric mean of the total number of bacteria in the air was 70.3 cfu/m3. Of these the median percentage of anaerobic bacteria was 30.3. When operating clothing of a disposable fabric (Barrier 450, Johnson & Johnson) was used, the counts of airborne bacteria were a little less than half those found when conventional cotton clothing was worn. Probably because of the overall low air counts in the operating theatre and the great variability in the individual bacterial counts from the operation wounds, a significant decrease in wound contamination was not observed. A positive correlation was found between the duration of operation and wound contamination.

Isolation of anaerobic and aerobic bacteria from clean surgical wounds: an experimental and clinical study.

A pad imprint method was evaluated for quantitative sampling of anaerobic and aerobic bacteria from clean surgical wounds. The best recovery of bacteria from the pads was obtained when they were treated with a rinsing solution in a 'Stomacher' apparatus. Pads of three different fabrics were compared, but no difference was noted in their efficiency in sampling anaerobic and aerobic bacteria from clean surgical wounds. Sampling of known quantities of Staphylococcus aureus from experimental wounds in rabbits performed with the pad method gave a threefold better recovery than with a swab. The pad method was superior to the wound wash-out method when sampling wounds in hip surgery.

Growth and lysis of the fish pathogen Moritella viscosa.

AIMS: The sensitivity to lysis is a profound bottleneck to studies of the fish pathogen Moritella viscosa. The aim of this study was to examine the growth and the lysis process of M. viscosa cells under different physical and chemical conditions. METHODS AND RESULTS: Growth and cell lysis were studied under different conditions. The growth rate was highest at 15 degrees C and lowest at 4 degrees C, but the cells reached a higher density at 4 degrees than at 15 degrees C and the cells were more stable. The presence of minerals reduced lysis. CONCLUSIONS: Premature lysis of the cells is dependent on environmental factors. Moritella viscosa should be cultivated and kept in media containing a certain set of minerals and at temperatures as low as 4 degrees C. Formalin favours the stability of cells. The instability of the M. viscosa cells at temperatures above 10 degrees C might be one of the factors responsible for their inability to infect fish at higher temperatures. The presence of DHA in the cell membranes is predicted to be responsible for the susceptibility of the cells to lysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The cultivation of M. viscosa cells is a key factor in studying the pathogenicity of the bacteria and in making an effective vaccine to prevent winter ulcers in farmed fish. The study provided recommendations on how to cultivate M. viscosa and how lysis of the cells can be minimized.

Airborne non-sporeforming anaerobic bacteria.

A large proportion of postoperative infections after clean surgery are thought to be exogenous. For aerobic bacteria different routes of transmission have been thoroughly studied. Airborne infection has been considered very important in infections after total hip replacement (Charnley, 1972). Anaerobic non-sporing bacteria have been found in deep late infections after total hip replacement (Kamme et al. 1974; Schwan et al. 1977; Petrini, Nord & Welin-Berger, 1978). However, infections caused by anaerobic bacteria have been considered endogenous, and little is known about the routes of transmission for these bacteria. The aim of this investigation has been to study the survival of anaerobic non-sporeforming bacteria in the air and environment to make it possible to study their routes of transmission in the operating room later.

Dispersal of non-sporeforming anaerobic bacteria from the skin.

Dispersal of non-sporeforming anaerobic bacteria was studied. Skin samples were taken from the subjects, and dispersed from different parts of the body was examined. The number of anaerobic bacteria dispersed was not correlated to their density on the surface of skin area exposed. The highest density of anaerobic bacteria on the skin was found in the face and upper trunk, but the highest yield of anaerobic bacteria dispersed came from the lower trunk. The dominant anaerobic bacteria dispersed were Propionibacterium acnes, but Propionibacterium avidum, Propionibacterium granulosum and Gram-positive cocci were also isolated from the dispersal samples. Peptococcus magnus was the most common coccus isolated. For the less frequently isolated bacteria, the best correlation was found between the perineal flora and airborne bacteria. A comparison was also made of bacterial dispersal by naked and dressed subjects. The dispersal of both aerobic and anaerobic bacteria was higher when the subjects were dressed in conventional operating theatre cotton clothing than when they were naked. The increased dispersal of anaerobic bacteria when the subjects were dressed was mainly due to increased dispersal of Propionibacterium sp.

Arachidonic acid level of non-esterified fatty acids and phospholipids in serum and heart muscle of patients with fatal myocardial infarction.

The relationship between non-esterified fatty acids (NEFA) in serum and heart muscle was examined in 15 patients who died of myocardial infarction (MI) and seven people who died suddenly in accidents. There was no correlation between NEFA levels of serum and non-infarcted cardiac muscle in patients with fatal MI. No significant difference was encountered in cardiac NEFA content between patients with fatal MI and people who died in accidents. The phospholipid (PL) content was significantly lower in patients with fatal MI than observed in people who died in accidents. The arachidonic acid (20:4 (n-6)) concentration of serum NEFA was significantly lower in patients with fatal MI compared to normal subjects. The cardiac NEFA and PL in patients with fatal MI contained significantly lower percentage levels of arachidonic acid compared to people who died in accidents. The results indicate that the death of the MI patients was not accompanied by elevated cardiac NEFA levels.

Characterization of Vibrio viscosus and Vibrio wodanis isolated at different geographical locations: a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb. nov.

Vibrio viscosus and Vibrio wodanis are recently described species of psychrotropic bacteria that have been found associated with a disease called 'winter ulcer', affecting salmonid fish reared in saline water in Norway, Iceland and recently in Scotland. V. viscosus and V. wodanis strains initially isolated from fish in Iceland and Norway were subjected to characterization using biochemical tests, SDS-PAGE of whole-cell proteins and a novel DNA fingerprinting method, amplified fragment length polymorphism (AFLP). The V. viscosus strains isolated from diseased fish grouped into homogeneous subgroups according to geographical origin and challenge experiments revealed that representatives of these groups are virulent. The results revealed that the V. wodanis strains are heterogeneous genotypically and phenotypically. Sequencing of almost complete 16S rRNA genes of V. viscosus and V. wodanis revealed that V. viscosus showed a 99.1% sequence similarity to Moritella marina and V. wodanis showed a 98.8% sequence similarity to Vibrio logei CIP 103204. A reclassification of Vibrio viscosus as Moritella viscosa comb. nov. is proposed.

An ultrastructural study of the cerebrospinal fluid in visna.

An electron microscopic examination was done on 8 samples of cerebrospinal fluid (CSF) from Icelandic sheep infected by the intracerebral route with visna virus. The specimens were collected 1 month, 2 months, and 4 years after infection. A differentail cell count done on low-power electron micrographs showed that the cellular exudate was composed of mononuclear cells mainly macrophages and lymphocytes with a few plasma cells. Macrophages were with one exception more numerous than lymphocytes and an increased proportion of macrophages showed evidence of phagocytosis with time after infection. Reactive lymphocytes were in general more numerous than small lymphocytes. Various stages in the maturation of plasma cells were observed. The cellular composition in the CSF is compatible with the view that visna is an immunopathological process. Myelin figures and fragments of myelinated axons were observed in two specimens indicating an active myelin-breakdown. The possibility that escape of myelin into the CSF may lead to sensitization to myelin antigens and perpetuation of this chronic neurologic affection is discussed. Visna virions could not be demonstrated.

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue.

Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.

Intracerebral serial passage of visna virus KV1514 through three Icelandic sheep was used to select for strains with increased neurovirulence. A strain (KV1772) with increased neuropathogenicity was obtained. We isolated several proviral molecular clones from a plaque-purified biological clone of KV1772 that induced typical visna virus pathology in young sheep. One of the clones (kv72) was infectious, while others contained mutations or were permuted and required gene recombination with other proviral clones to generate infectious virus after transfection. Stable plasmids containing functional, full-length, visna virus KV1772 genomes were constructed from the proviral molecular clones. The in vitro cytopathic effects of virus derived from these clones varied depending upon the tissue origin of the infected cells. A goat cell line became persistently infected with molecularly cloned KV1772 virus; these cells resisted the cell-killing effects and continuously shed high levels of infectious virus. We determined the complete nucleotide sequence of a KV1772 provirus; it contains open reading frames for all structural and accessory genes previously identified in the visna virus genome and is highly homologous to other published visna virus sequences. Progeny of molecularly cloned KV1772 virus rapidly induced both a pronounced neuropathology and an unexpected, strong, neutralizing antibody response in experimentally infected young Icelandic sheep. The availability of stable plasmids of replication-competent and pathogenic proviral molecular clones of visna virus should now enable the study of the genetic determinants of neurovirulence and their interaction with the host immune system in visna virus pathogenesis.

Biological and genetic differences between lung- and brain-derived isolates of maedi-visna virus.

During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.