Suitability of FTIR to distinguish pure cultures of problematic mould species from closely related species in the meat industry.

AIMS: The aim of the study was to apply Fourier Transform Infrared spectroscopy (FTIR) as a rapid screening method for moulds in a specific food production environment (cured meat) and to evaluate whether the method was sufficiently accurate to distinguish Penicillium species that constitute a hazard for the food quality and safety (Penicillium solitum and Penicillium nordicum) from closely related species. METHODS AND RESULTS: FTIR was applied to classify the indigenous mycobiota of two production sites for dried and cured meat products in Norway. Results showed that FTIR was suitable to analyse large amounts of data. While correct classification rates varied depending on the species, overall results indicated that FTIR was able to distinguish the undesired mould species P. solitum and P. nordicum from other species and may hence present an option for rapid screening of large numbers of samples to identify changes in mould composition on site. CONCLUSIONS: FTIR presents a potential method for detecting changes in levels of undesired fungi in meat-processing environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that applies FTIR to a specific food production environment and it increases the knowledge on both possibilities and limitations of the method in classification of fungi.

Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

A fluorescence-based assay for in vitro screening of Saprolegnia inhibitors.

The incidence of fish pathogenic oomycetes, Saprolegnia, has increased significantly in aquaculture since the ban of malachite green. For the efficient characterization of anti-Saprolegnia therapeutics, simple accurate methods are required. However, the current screening methods are limited by time, and none of them are confirming the viability of treated spores or hyphae. In this study, a modified fluorescence-based assay for the in vitro screening of Saprolegnia inhibitors has been developed. This method involves the use of FUN-1 viability dye combined with calcofluor white M2R, and is based on the formation of orange-red cylindrical intravacuolar structures (CIVS) in metabolically active spores, hyphae and biofilms. Heat-killed and bronopol-treated Saprolegnia spores, hyphae and biofilms exhibited diffuse bright green fluorescence which confirms complete loss of viability. For boric acid-treated spores, no germination was observed. However, tiny CIVS were observed in 50% of treated spores which indicated reduction in their viability. Our results proved that FUN-1 dye is an efficient tool to distinguish between live and dead Saprolegnia spores, hyphae and biofilms and to monitor the change in Saprolegnia viability during qualitative evaluation of potential anti-Saprolegnia compounds.

Factors influencing Saprolegnia spp. spore numbers in Norwegian salmon hatcheries.

A quantitative survey of Saprolegnia spp. in the water systems of Norwegian salmon hatcheries was performed. Water samples from 14 salmon hatcheries distributed along the Norwegian coastline were collected during final incubation in the hatcheries. Samples of inlet and effluent water were analyzed to estimate Saprolegnia propagule numbers. Saprolegnia spores were found in all samples at variable abundance. Number of spores retrieved varied from 50 to 3200 L(-1) in inlet water and from 30 to >5000 L(-1) in effluent water. A significant elevation of spore levels in effluent water compared to inlet water was detected. The estimated spore levels were related to recorded managerial and environmental parameters, and the number of spores in inlet water and temperature was the factor having most influence on the spore concentration in the incubation units (effluent water). Further, the relative impact of spore concentration on hatching rates was investigated by correlation analysis. From this was found that even high spore counts did not impact significantly on hatching success.

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon, Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re-emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).

A thicker chorion gives ova of Atlantic salmon (Salmo salar L.) the upper hand against Saprolegnia infections.

Since the ban of malachite green in the fish farming industry, finding alternative ways of controlling Saprolegnia infections has become of utmost importance. Much effort has been made to elucidate the mechanisms by which Saprolegnia invades fish eggs. Little is known about the defence mechanisms of the hosts, making some eggs more prone to infection than others. One clue might lie in the composition of the eggs. As the immune system in the embryos is not developed yet, the difference in infection levels could be explained by factors influenced by the mother herself, by either transferring passive immunity, influencing the physical aspects of the eggs or both. One of the physical aspects that could be influenced by the female is the chorion, the extracellular coat surrounding the fish egg, which is in fact the first major barrier to be overcome by Saprolegnia spp. Our results suggest that a thicker chorion in eggs from Atlantic salmon gives a better protection against Saprolegnia spp. In addition to the identification of differences in sensitivity of eggs in a fish farm set-up, we were able to confirm these results in a laboratory-controlled challenge experiment.

Invading slugs (Arion vulgaris) can be vectors for Listeria monocytogenes.

AIMS: Listeriosis is a frequent silage-associated disease in ruminants. The slugs Arion vulgaris are invaders in gardens, vegetable crops and meadows for silage production. Field and laboratory studies were conducted to clarify whether slugs could host Listeria monocytogenes and thereby constitute a threat to animal feed safety. METHODS AND RESULTS: Selective culture of L. monocytogenes from 79 pooled slug samples (710 slugs) resulted in 43% positive, 16% with mean L. monocytogenes values of 405 CFU g(-1) slug tissues. Of 62 individual slugs cultured, 11% also tested positive from surface/mucus. Multilocus sequence typing analysis of 36 isolates from different slug pools identified 20 sequence types belonging to L. monocytogenes lineages I and II. Slugs fed ≅4·0 × 10(5)  CFUL. monocytogenes, excreted viable L. monocytogenes in faeces for up to 22 days. Excretion of L. monocytogenes decreased with time, although there were indications of a short enrichment period during the first 24 h. CONCLUSIONS: Arion vulgaris may act as a vector for L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Highly slug-contaminated grass silage may pose a potential threat to animal feed safety.

Saprolegnia species in Norwegian salmon hatcheries: field survey identifies S. diclina sub-clade IIIB as the dominating taxon.

Saprolegnia isolates within the recognized clades encompassing the taxa S. parasitica and S. diclina act as opportunist and aggressive pathogens to both fish and their eggs. They are responsible for significant economic losses in aquaculture, particularly in salmonid hatcheries. However, the identity, distribution and pathogenic significance of involved species often remain unexplored. In this study, 89 Saprolegnia isolates were recovered from water, eggs and salmon tissue samples that originated from salmon (Salmo salar) hatcheries along the coast of Norway. The cultures were characterized morphologically and molecularly in order to provide an overview of the species composition of Saprolegnia spp. present in Norwegian salmon hatcheries. We demonstrate that S. diclina clearly dominated and contributed to 79% of the recovered isolates. Parsimony analyses of the nuclear ribosomal internal transcribed spacer (ITS) region split these isolates into 2 strongly supported sub-clades, S. diclina sub-clade IIIA and IIIB, where sub-clade IIIB accounted for 66% of all isolates. A minor portion of the isolates constituted other taxa that were either conspecific or showed strong affinity to S. parasitica, S. ferax, S. hypogyna and Scoliolegnia asterophora. The unique sub-clade IIIB of S. diclina was most prevalent in water and salmon eggs, while S. parasitica isolates were more frequently isolated from post hatching stages. The study demonstrated that morphological criteria in many cases were insufficient for species delimitation due to lack of sexual structures or incoherent morphological expression of such features within the tested replicates.

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon, Salmo salar L. parr.

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re-isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated 'AP' ('activated through passage'). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose-yeast extract.

Characterization of food spoilage fungi by FTIR spectroscopy.

AIMS: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. METHODS AND RESULTS: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93.9 and 94.0% of the spectra were correctly identified at the species and genus level, respectively. CONCLUSIONS: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. SIGNIFICANCE AND IMPACT OF THE STUDY: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.

Microbial background flora in small-scale cheese production facilities does not inhibit growth and surface attachment of Listeria monocytogenes.

The background microbiota of 5 Norwegian small-scale cheese production sites was examined and the effect of the isolated strains on the growth and survival of Listeria monocytogenes was investigated. Samples were taken from the air, food contact surfaces (storage surfaces, cheese molds, and brine) and noncontact surfaces (floor, drains, and doors) and all isolates were identified by sequencing and morphology (mold). A total of 1,314 isolates were identified and found to belong to 55 bacterial genera, 1 species of yeast, and 6 species of mold. Lactococcus spp. (all of which were Lactococcus lactis), Staphylococcus spp., Microbacterium spp., and Psychrobacter sp. were isolated from all 5 sites and Rhodococcus spp. and Chryseobacterium spp. from 4 sites. Thirty-two genera were only found in 1 out of 5 facilities each. Great variations were observed in the microbial background flora both between the 5 producers, and also within the various production sites. The greatest diversity of bacteria was found in drains and on rubber seals of doors. The flora on cheese storage shelves and in salt brines was less varied. A total of 62 bacterial isolates and 1 yeast isolate were tested for antilisterial activity in an overlay assay and a spot-on-lawn assay, but none showed significant inhibitory effects. Listeria monocytogenes was also co-cultured on ceramic tiles with bacteria dominating in the cheese production plants: Lactococcus lactis, Pseudomonas putida, Staphylococcus equorum, Rhodococcus spp., or Psychrobacter spp. None of the tested isolates altered the survival of L. monocytogenes on ceramic tiles. The conclusion of the study was that no common background flora exists in cheese production environments. None of the tested isolates inhibited the growth of L. monocytogenes. Hence, this study does not support the hypothesis that the natural background flora in cheese production environments inhibits the growth or survival of L. monocytogenes.

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs.

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.

Poisoning of dogs with tremorgenic Penicillium toxins.

Fungi in the genus Penicillium, particularly P. crustosum, produce tremorgenic mycotoxins, as well as suspected tremorgenic compounds. The accidental intoxication of six dogs with such toxins are reported. The clinical signs included vomiting, convulsions, tremors, ataxia, and tachycardia, all of which are indicators of intoxications affecting the nervous system. This symptomatology caused us to think that the dog poisoning was the result of tremorgenic mycotoxins. One dog was euthanized in the acute phase, while three others recovered completely within a few days. However, neurological symptoms were still observed four months after the poisoning of two of the dogs. One of these recovered completely within the next 2-3 months, while the other still suffers from ataxia three years later. Available samples of feed, stomach content and/or tissues from the intoxications were subjected to mycological and chemical analysis. Penitrem A was found in all reported poisonings and roquefortine C in all cases when this toxin was included in the analysis. The producer of these toxins, Penicillium crustosum, was detected in all cases where material suitable for mycological examinations (feed or vomit) was available. To our knowledge, this is the first report documenting the presence of penitrems and roquefortine C in organs from poisoned dogs. Furthermore, the report indicates that the recovery period after severe poisonings with P. crustosum may be protracted.

Polyneuropathy associated with forage sources in Norwegian horses.

BACKGROUND: Cases of hindlimb digital extensor weakness of unknown etiology have been observed in Norway since 1995. HYPOTHESIS: We hypothesized that the observed bilateral extensor weakness was attributable to neuropathy of the distal nerves and that this was related to environmental factors, possibly dietary. ANIMALS: Seventy-five horses with digital extensor weakness occurring from 1995 to 2004 are described. METHODS: Eleven horses were examined at The Norwegian School of Veterinary Science, and the medical records of 64 horses seen in ambulatory practice were reviewed. RESULTS: There was no apparent sex, age, or breed predilection, but the majority were horses kept for pleasure or breeding purposes. Clinical signs varied from intermittent knuckling of the hindlimbs to paraplegia. Some horses showed no or only slow progression of signs, whereas others developed severe signs within hours. No other neurologic deficits were detected in any of the horses. Epidemiologic data and laboratory results were not supportive of an infectious etiology. The only common factor for all affected horses seemed to be feeding big bale silage or, occasionally, hay of poor microbiologic quality. Forty of the 75 horses were euthanized. Histopathologic examination of peripheral nervous tissue was performed in 22 horses, all of which had neuronal fiber degeneration. The majority of horses with mild signs recovered after 5-6 months of rest. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical signs correlated with polyneuropathy involving sciatic nerves.

Mould growth on the Norwegian semi-hard cheeses Norvegia and Jarlsberg.

Visible moulds were isolated and identified from 102 samples of each of the Norwegian types of semi-hard cheeses called Norvegia and Jarlsberg. Penicillium species made up 98.1 and 89.2% of the isolates from the Jarlsberg and Norvegia cheeses, respectively. The most frequently occurring species on both was P. roqueforti subspecies roqueforti. The four species Penicillium roqueforti subspecies roqueforti, P. commune, P. palitans and P. solitum made up 69.8% of the total number of isolates from the Norvegia cheese and 81.0% of the total number of isolates from the Jarlsberg cheese.

Mould contaminants on Jarlsberg and Norvegia cheese blocks from four factories.

Visible mould from 225 blocks of the Norwegian semi-hard cheeses Jarlsberg and Norvegia from four factories were subcultured and identified. Altogether 23 different fungal species were detected. The two most important contaminating species were Penicillium commune and P. palitans, constituting 21.4% and 17.9% of the total isolates, respectively. The other dominating contaminants were P. roqueforit spp. roqueforti, Geotrichum candidum, P. solitum and P. crustosum. These species, together with P. commune and P. palitans, represented 80.9% of the total isolates. P. commune, P. palitans, P. roqueforti spp. roqueforti and P. solitum were most common contaminants on cheese produced in all four factories, while G. candidum was found to be important on Jarlsberg cheese from only one factory. P. crustosum was one of the dominating species on Norvegia cheese.

The use of AFLP to relate cheese-contaminating Penicillium strains to specific points in the production plants.

Amplified fragment length polymorphism (AFLP) analysis was performed on isolates of Penicillium commune and Penicillium palitans originating from cheese and indoor environment in four cheese factories. The AFLP method was found to be a useful tool for identification of P. commune and P. palitans on, as well as below, species level. However, AFLP in combination with M13 fingerprinting described in a previous paper provided better resolution at the intraspecific level than either of the methods alone. Specific P. commune and P. palitans strains were found in the same factories over a period of more than a year and showed that the cheese factories have contaminating strains that are well established. The majority of the P. commune and P. palitans strains were found only within a single factory, but several were found in different cheese factories. The combined fingerprinting data could relate strains isolated from cheese to specific points in the production plants. Several of cheese-contaminating Penicillium strains could be related to air in the wrapping room, which must be considered to be a critical point for contamination of cheese.

Occurrence of moulds in drinking water.

AIMS: In order to determine the occurrence of filamentous fungi in public drinking water systems in Norway, water from 14 water supply networks from all over the country was sampled and analysed. Networks with both ground and surface water sources were included in this study. METHODS AND RESULTS: During a one-year period, 273 water samples were collected. Frequencies of fungi in samples from raw water, treated water and from home and hospital installations were determined on the basis of incubation of 100 ml membrane-filtered samples on dichloran 18% glycerol agar media. Filamentous fungi were recovered from 62% of all samples. In ground water 42.3% of the samples were positive for mould growth, while surface waters yielded 69.7% positive samples. CONCLUSIONS: The risk to recover moulds from surface water is three times higher compared with ground water. It is more likely to detect moulds in cold waters and showers than in hot waters. SIGNIFICANCE AND IMPACT OF THE STUDY: By analysing the water reaching the consumers, the results reported in present study indicate that filamentous fungi in drinking water is not negligible, and that moulds should be considered as part of the microbiological analysis parameters in drinking water.

Morphological and physiological characteristics of Saprolegnia spp. strains pathogenic to Atlantic salmon, Salmo salar L.

Seventeen strains of Saprolegnia spp. were examined for morphological and physiological characteristics, and seven were examined for their pathogenicity to Atlantic salmon, Salmo salar L. Two of the Saprolegnia strains tested caused 89 and 31% cumulative mortality in challenged salmonids and were significantly more pathogenic than the other strains tested. The positive control (Saprolegnia parasitica ATCC 90213) caused 18% mortality, but this was not significantly higher than non-pathogenic strains (0-3% cumulative mortality). All the pathogenic Saprolegnia strains and two non-pathogenic strains had secondary cysts with long, hooked hairs, a characteristic which is claimed to be typical of S. parasitica. This characteristic is apparently necessary, but does not in itself determine the ability to cause mortality in Atlantic salmon. However, all the pathogenic Saprolegnia strains in the present study showed a significantly higher initial growth rate of cysts in sterilized tap water than did non-pathogenic strains. The results of the present study suggest that initial growth rate of germinating cysts in pure water, together with the presence of long hooked hairs on the secondary cysts, may be indicators of pathogenicity of Saprolegnia strains to Atlantic salmon.

Malt-yeast extract-sucrose agar, a suitable medium for enumeration and isolation of fungi from silage.

A general medium named malt-yeast extract-sucrose agar (MYSA) containing oxgall was designed. The medium was intended for the enumeration and isolation of molds and yeasts in routine examinations of animal feed stuffs. In this study MYSA was tested as a general medium for mycological examination of silage. The medium was compared with dichloran-rose bengal medium (DRBC) in an examination of more than 500 specimens of big bale grass silage. Selected characteristics of known fungal species commonly isolated from feeds were examined after growth on MYSA and DRBC and on malt extract agar, used as a noninhibitory control medium. MYSA suppressed bacterial growth, without affecting the growth of fungi common in feeds. The fungi growing on MYSA were easily recognized, and the medium seemed to slow radial growth of fungal colonies, which permitted, easy counting. The number of species found was higher on MYSA than on DRBC. When we compared MYSA with DRBC for mycological examination of grass silage samples, MYSA was found to be the medium of choice.