Lucy J. Robertson

In vitro excystation of Cryptosporidium parvum

Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

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.

The impact of globalisation on the distribution of Echinococcus multilocularis.

In the past three decades, Echinococcus multilocularis, the cause of human alveolar echinococcosis, has been reported in several new countries both in definitive hosts (canids) as well as in people. Unless treated, infection with this cestode in people is fatal. In previously endemic countries throughout the Northern Hemisphere, geographic ranges and human and animal prevalence levels seem to be increasing. Anthropogenic influences, including increased globalisation of animals and animal products, and altered human/animal interfaces are thought to play a vital role in the global emergence of this pathogenic cestode. Molecular epidemiological techniques are a useful tool for detecting and tracing introductions, and differentiating these from range expansions.

Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination.

The public health significance of giardiasis and cryptosporidiosis in sheep is currently unclear. Some research suggests that they are probably not an important zoonotic reservoir, whilst other research indicates this potential exists, and some outbreaks have also been associated with infections in sheep. Actions to limit water supply contamination by sheep have sometimes been severe, occasionally creating problems between farming and public health communities. Here our knowledge on these parasites in both sheep and goats is reviewed; although direct evidence of transmission to humans via water supply contamination is limited, the data accrued indicate that this is a real possibility. As cryptosporidiosis in sheep is generally more prevalent than giardiasis, and species/genotypes of Cryptosporidium infections in sheep are likely to be infectious to humans, this parasite may be considered the greater threat. Nevertheless, geographical variation in prevalence and genotypic distribution is extensive and as measures to limit sheep grazing can have a highly negative impact, it is important that cases are judged individually. If water contamination from a particular population of sheep/goats is suspected, then suitable investigations should be instigated, investigating both prevalence and species/genotype, before precautionary measures are imposed.

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.

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.

Giardiasis: The Ever-Present Threat of a Neglected Disease

Although giardiasis has been a threat to mankind for thousands of years, this protozoan infection was, until recently, relatively neglected. Giardia duodenalis is recognised as a major cause of parasite-induced diarrhoea in humans and animals, and is currently an important public health problem, placing a heavy burden on both diagnostic and treatment services at health care institutions, mostly in developing countries, but also in highly developed countries. Steady progress in recent years, using a combination of molecular, immunological, and clinical approaches, has substantially increased our understanding of Giardia and important aspects of the clinical manifestations that it causes. The purpose of this review is to provide an overview of the extent of Giardia infection, the implications of water and food in Giardia transmission, new aspects regarding clinical diagnosis and environmental detection, treatment, and some approaches towards prevention and control. A number of future research priorities are also presented.

Treatment-ladder and genetic characterisation of parasites in refractory giardiasis after an outbreak in Norway

OBJECTIVES To evaluate the efficacy of a treatment ladder in metronidazole-refractory giardiasis, and to compare genetic characteristics of the parasites. METHODS A clinical observational study was carried out in 38 adult patients with metronidazole-refractory giardiasis, during an outbreak in Norway with more than 1200 cases. All patients were treated with albendazole in combination with metronidazole. Those who failed were treated with paromomycin. Those who failed on paromomycin were treated with quinacrine in combination with metronidazole. Giardia isolates from 17 patients were characterised by PCR and sequencing at two separate genes. RESULTS Metronidazole in combination with albendazole was effective in 30 (79%) out of 38 patients. Paromomycin was effective in three out of six patients. Quinacrine in combination with metronidazole was effective in 3 patients. Molecular characterisation of the Giardia isolates revealed that these parasites were identical at two different gene segments, while sequence profiles from isolates at the peak of the outbreak were more heterogenous. CONCLUSIONS Albendazole and quinacrine both in combination with metronidazole were effective in treating metronidazole-refractory giardiasis in this cohort. Paromomycin was less effective. Particular Giardia sub-genotypes may have been associated with the treatment-refractory giardiasis in these patients, although other undefined factors are probably also of importance.