Bjørn Gjerde

Microbiological analysis of seed sprouts in Norway

As part of larger survey of microbial contamination of fruits and vegetables in Norway, four different sprouted seed products were analysed for bacterial and parasitic contaminants (n = 300 for bacterial analyses and n = from 17 to 171 for parasite analyses, depending on parasite). Escherichia coli O157, Salmonella, Listeria monocytogenes, Cyclospora oocysts, Ascaris eggs and other helminth parasites were not detected in any of the sprout samples. Thermotolerant coliform bacteria (TCB) were isolated from approximately 25% of the sprout samples, with the highest percentage of TCB positive samples in alfalfa sprouts. Most TCB were Enterobacter spp. and Klebsiella. E. coli was isolated from 8 of 62 TCB positive mung bean sprout samples. Cryptosporidium oocysts were detected in 8% of the sprout samples and Giardia cysts were detected in 2% of the samples. All the Cryptosporidium positive samples, and most of the Giardia positive samples, were mung bean sprouts. Parasite concentrations in positive samples were low (between 1 and 3 oocysts/cysts per 50 g sprouts). Sprout irrigation water was also analysed for microbial contaminants. E. coli O157 and L. monocytogenes were not detected. TCB were isolated from approximately 40% of the water samples. Salmonella reading was isolated from three samples of spent irrigation water on 3 consecutive days. Cryptosporidium and Giardia were also isolated from spent irrigation water. Additionally, eight samples of unsprouted mung bean seed were analysed for Cryptosporidium oocysts and Giardia cysts. One or both of these parasites were detected in six of the unsprouted seed samples at concentrations of between 1 and 5 oocysts/cysts per 100 g unsprouted seed. Whilst our results support spent irrigation water as the most suitable matrix for testing for bacteria, unsprouted seed is considered the more useful matrix for analysing for parasite contaminants.

Occurrence of Cryptosporidium oocysts and Giardia cysts in Sewage in Norway

ABSTRACT Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of >20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and >80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.

Application of Genotyping during an Extensive Outbreak of Waterborne Giardiasis in Bergen, Norway, during Autumn and Winter 2004†

ABSTRACT During the autumn and winter of 2004 and 2005, an extensive outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1,500 patients were diagnosed with giardiasis. Analysis of water from the implicated source revealed low numbers of Giardia cysts, but the initial contamination event probably occurred up to 10 weeks previously. While sewage leakage from a residential area is now considered to be the probable source of contamination, during the episode waste from one particular septic tank was thought to be a possible source. Genotyping of cysts from the septic tank demonstrated that they were assemblage A cysts, although the sequences were not identical to any previously published sequences. For the β-giardin gene, the closest published subgenotype was subgenotype A3; for the gdh gene, the closest published subgenotype was subgenotype A2. Genotyping of cysts from 21 patient samples revealed that they were assemblage B cysts; thus, the septic tank was unlikely to be the contamination source. Sequencing of the β-giardin and gdh genes from patient samples and a comparison of the sequences gave complex results. For the β-giardin gene, three isolates had sequences identical to subgenotype B3 sequences. However, other isolates had between one and four single-nucleotide polymorphisms (SNPs). For the gdh gene, none of the sequences were identical to the sequence published for subgenotype B3, and the sequences had between one and three SNPs. One isolate, which was identical to subgenotype B3 at the β-giardin gene, was more similar to subgenotype B2 at the gdh gene. Grouping the isolates on the basis of SNPs resulted in different groups for the two genes. The results are discussed in relation to giardiasis in Norway and to other Giardia genotyping studies.

Phylogenetic relationships among Sarcocystis species in cervids, cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene ☆

Coccidian parasites in the genus Sarcocystis have a two-host life cycle, and have traditionally been identified on the basis of morphological features of the sarcocyst stage in their intermediate hosts. Additional molecular species identification, delimitation and phylogeny of Sarcocystis spp. have been based mainly on the nuclear ssrRNA gene. This gene is well suited for discrimination between more distant species but less so for closely related species. The objective of this study was therefore to establish the mitochondrial cytochrome c oxidase subunit I gene (cox1) as a novel genetic marker for Sarcocystis spp. and assess its utility for species identification and delimitation. New primers were developed and 1,020-1,095 bp long cox1 sequences were obtained from 155 isolates of 22 Sarcocystis spp. from cattle, sheep, red deer, reindeer, roe deer and moose, and used for phylogenetic reconstructions. For 18 species, the intraspecific and interspecific sequence identities were 98.5-100% and 58-92%, respectively. The four other species had previously been regarded as two species (Sarcocystis rangiferi, Sarcocystis tarandi), each infecting both reindeer and red deer. From cox1 data, each of those appeared to be two separate species, with S. rangiferi and S. tarandi being restricted to reindeer. Thus, cox1 sequences seem to perform better than ssrRNA gene sequences for delimitation of closely related species. The 22 species were distributed in three major clades according to their definitive hosts as in phylogenetic trees obtained from the ssrRNA gene. There were only minor differences in the branching order of different taxa between the trees obtained from either gene. This study has successfully established cox1 as a novel genetic marker for future research on Sarcocystis spp. It has also provided the first published molecular identification of Sarcocystis gigantea and Sarcocystis tenella in Norwegian sheep, and of Sarcocystis hirsuta and Sarcocystis sinensis in Argentinean cattle.

The zoonotic potential of Giardia and Cryptosporidium in Norwegian sheep: a longitudinal investigation of 6 flocks of lambs.

Faecal samples collected from lambs on 6 Norwegian farms on 2 separate occasions during spring/summer 2008 (approximately 550 samples collected at each occasion) were examined for Cryptosporidium oocysts and Giardia cysts by immunofluorescent antibody test. Overall prevalence at the first sampling was approximately 23% for Giardia and 15% for Cryptosporidium, and at the second sampling approximately 31% for Giardia and 24% for Cryptosporidium, with substantial between-farm variation on each sampling occasion. To assess the potential public health significance of these infections, molecular analyses were conducted on 42 Giardia isolates and 42 Cryptosporidium isolates, with PCR targeted at one or both of two genes (Giardia: glutamate dehydrogenase and beta-giardin genes; Cryptosporidium: SSU rRNA and actin genes) for each parasite. Of the Giardia isolates, 41 were Assemblage E (non-zoonotic) and 1 was Assemblage B (zoonotic). Of the Cryptosporidium isolates, 35 were cervine genotype (potentially zoonotic) and 7 C. xiaoi (non-zoonotic). These results suggest that sheep in Norway are unlikely to be an important reservoir of zoonotic Giardia in Norway, but might have some public health significance with respect to Cryptosporidium.

Occurrence of Cryptosporidium oocysts and Giardia cysts in raw waters in Norway

Aims: This paper reports the first investigation into the occurrence of Cryptosporidium and Giardia in Norwegian raw water sources. Methods: Between June 1998 and October 1999, 408 raw water samples, collected from 147 different sites across Norway, were analysed for these parasites. Analysis was based upon US EPA Method 1623. Results: In 305 samples (75%), parasites were not detected. In 55 samples (13.5%), Cryptosporidium only was detected. In 38 samples (9%), Giardia only was detected. In 10 samples (2.5%) both Cryptosporidium and Giardia were detected. Of the sites sampled, parasites were not detected at 100 (68%) of them, Cryptosporidium only was detected at 20 (13.5%), Giardia only was detected at 11 (7.5%), and both Cryptosporidium and Giardia were detected at 16 (11%). Concentrations of parasites were low; usually one cyst/ oocyst detected per 10 litres of water. Conclusions: Significant associations were demonstrated for these samples between the detection of these parasites and (a) turbidity ≥2.0 NTU, and (b) high numbers of domestic animals within the catchment area. No association between seasonality and the occurrence of these parasites could be detected. The results are discussed in relation to other studies and the potential public health implications for Norway.

GIARDIA DUODENALIS CYSTS ISOLATED FROM WILD MOOSE AND REINDEER IN NORWAY : GENETIC CHARACTERIZATION BY PCR-RFLP AND SEQUENCE ANALYSIS AT TWO GENES

There are few genotyping studies of Giardia duodenalis isolates from cervid hosts, although a previous study suggested that cervids may be a source of infection for humans and cattle. Giardia duodenalis isolates collected from wild moose (Alces alces) and reindeer (Rangifer tarandus) in Norway during 2002 and 2003 were characterized by polymerase chain reaction–restriction fraction length polymorphism (PCR-RFLP) at the β-giardin gene, and sequence analysis at both the β-giardin and glutamate dehydrogenase (gdh) genes. All results suggested that these isolates (n=25) belonged to assemblage A. Three different restriction patterns were obtained with PCR-RFLP, one of which has previously been associated with assemblage A. At the β-giardin gene, sequences from six reindeer isolates and one moose isolate were identical to a previously published assemblage A sequence from G. duodenalis cysts isolated from dairy calves. The other 10 moose isolates could be divided into five groups, with between two and 14 single nucleotide polymorphisms (SNPs) from the published genotype A2. At the gdh gene, three different sequences were obtained, differing from each other by between one and 15 SNPs and which have all been previously published as genotype A1, but with different specific hosts. Grouping of the isolates based on the sequences from both genes gave complex results; whereas all the G. duodenalis isolates from reindeer grouped together, two moose isolates, which had identical sequences at the β-giardin gene, had sequences that differed from each other by 15 SNPs at the gdh gene. The results of these studies, together with the large Norwegian populations of these cervids and the amount of fecal matter they produce, indicate that moose and reindeer may be significant reservoirs of G. duodenalis infection in Norway, which may be of importance to veterinary and public health.

A longitudinal study on the occurrence of Cryptosporidium and Giardia in dogs during their first year of life.

BackgroundThe primary aim of this study was to obtain more knowledge about the occurrence of Cryptosporidium and Giardia in young dogs in Norway.The occurrence of these parasites was investigated in a longitudinal study by repeated faecal sampling of dogs between 1 and 12 months of age (litter samples and individual samples). The dogs were privately owned and from four large breeds. Individual faecal samples were collected from 290 dogs from 57 litters when the dogs were approximately 3, 4, 6, and 12 months old. In addition, pooled samples were collected from 43 of the litters, and from 42 of the mother bitches, when the puppies were approximately 1 and/or 2 months old.MethodsThe samples were purified by sucrose gradient flotation concentration and examined by immunofluorescent staining.Results128 (44.1%) of the young dogs had one or more Cryptosporidium positive samples, whilst 60 (20.7%) dogs had one or more Giardia positive samples. The prevalence of the parasites varied with age. For Cryptosporidium, the individual prevalence was between 5.1% and 22.5%, with the highest level in dogs < 6 months old, and declining with age. For Giardia, the individual prevalence was between 6.0% and 11.4%, with the highest level in dogs > 6 months old, but the differences between age groups were not statistically significant. Significant differences in prevalences were found in relation to geographic location of the dogs. Both parasites occurred at low prevalences in Northern Norway.ConclusionBoth Cryptosporidium and Giardia are common in Norwegian dogs, with Cryptosporidium more prevalent than Giardia. Prevalences of the parasites were found to be influenced by age, geographical location, and infection status before weaning.

Giardia infections in Cuban children: the genotypes circulating in a rural population

Abstract Stool samples containing Giardia duodenalis cysts were collected from 95 primary-school children in central Cuba, and preserved by storing at −20°C in 70% ethanol. Clinical data were collected for each child. Although 57% of the children were asymptomatic, the remaining 43% each reported between one and three symptoms. Following cyst quantification and isolation, molecular analyses were attempted on all cyst isolates, with the focus on the parasites β-giardin and glutamate-dehydrogenase (gdh) genes. Unfortunately, the cyst-preservation procedure appeared to have a deleterious effect on the cysts, since genotyping data could only be obtained for 20 of the 95 isolates. These data indicated, however, an approximately equal distribution between assemblage A (nine isolates) and assemblage B (11 isolates). Children found to be excreting relatively large numbers of cysts were more likely to be symptomatic than children who were excreting fewer cysts, and children with Giardia isolates from assemblage B were more likely to have symptomatic infections than children with isolates from assemblage A. Although considerable sequence variability was seen in the assemblage-B isolates, the assemblage-A isolates were relatively genetically homogeneous. This is the first publication from the Caribbean in which the Giardia genotypes circulating within the population have been identified, the first from the Americas providing information on associations between clinical presentation and the assemblage of the infecting Giardia, and the first to indicate that levels of cyst excretion may have clinical significance.

Cryptosporidium parvum Infections in Bergen, Norway, during an Extensive Outbreak of Waterborne Giardiasis in Autumn and Winter 2004

ABSTRACT During a large waterborne giardiasis outbreak in Norway, many diarrheic patients were found to have Cryptosporidium infections. Gene sequencing identified these infections as Cryptosporidium parvum infections, although they were not identical. Whether these infections were due to a simultaneous outbreak of waterborne cryptosporidiosis or reflected background levels not normally detected is discussed.