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Fascioliasis, an emerging parasitic infection, impacts significantly on both veterinary and human health worldwide. Endemic foci are not limited only to areas of extensive livestock farming, but owing to the parasite's abilities to colonise new intermediate hosts and adapt to new environments, also occur in other places, including Cuba. In Cuba, despite a high prevalence of fascioliasis in livestock, and the widespread occurrence of two potential intermediate hosts, human infection has decreased steadily over the past 10 years. In other parts of the world, human fascioliasis is apparently becoming more frequent. Problems in counteracting the spread of fascioliasis, and approaches used in Cuba to limit zoonotic transmission are discussed, with emphasis on diagnostic and treatment problems, malacological initiatives, and the importance of an integrated control programme. Such programmes may be of benefit in other countries where the prevalence of human fascioliasis is increasing, and lessons may perhaps be learned from the Cuban approach.

Genotyping of Giardia duodenalis cysts in faecal samples has become a regularly employed tool by researchers investigating different aspects of the epidemiology and pathology of Giardia infection in human and animal populations. However, such investigations are often limited to some extent by lack of PCR amplification from a proportion of the samples, and this often seems to be associated with the storage medium used for the samples. Various different storage media have been used in different studies, but investigation of which storage media are most appropriate and which may compromise subsequent PCR investigations has not been systematically explored to date. In this study, 4 different, commonly used storage media were investigated for their effects over time on subsequent PCR amplification of DNA from Giardia cysts in stool samples. Microscopic examination of the samples and real-time PCR were used to investigate 7 different samples over a period of 3 months. Our findings indicate that storage in ethanol or potassium dichromate at 4 °C gave the best results and, that if immunomagnetic separation was used prior to PCR (as may be appropriate for samples with low cyst numbers), then storage in potassium dichromate gave the best results.

SUMMARYThe 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 ofCryptosporidiuminfections 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.

Stool samples containing Giardia duodenalis cysts were collected from 95 primary-school children in central Cuba, and preserved by storing at -20 degrees 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 parasite's beta-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.

Investigation of shellfish for Cryptosporidium oocysts and Giardia cysts is of public health interest because shellfish may concentrate these pathogens in their bodies, and because shellfish are frequently eaten raw or lightly cooked. To date, the methods used for the analysis of shellfish for these parasites are based on those originally designed for water concentrates or fecal samples; the reported recovery efficiencies are frequently relatively low and the amount of sample examined is small. Here, we describe the development and use of a pepsin digestion method for analyzing shellfish samples for these parasites. The conditions of the isolation method did not affect subsequent parasite detection by immunofluorescent antibody test, and allowed examination of 3-g samples of shellfish homogenate, with recovery efficiencies from blue mussel homogenates of between 70 and 80%, and similar recoveries from horse mussel and oyster homogenates. Although exposure of the parasites to the conditions used in the technique affected their viability, as assessed by vital dyes, the maximum reduction in viability after 1-h incubation in digestion solution was 20%. In a preliminary survey of shellfish collected from the Norwegian coast, Cryptosporidium oocysts were detected in blue mussel homogenates in 6 (43%) of 14 batches and Giardia cysts in 7 (50%) of these batches. However, this relatively high occurrence, compared with other surveys, may be due to the higher recovery efficiency of the new method, and the relatively large sample size analyzed. A more comprehensive study of the occurrence of these parasites in shellfish would be of pertinence to the Norwegian shellfish industry.

Summary 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.

Aims: In autumn/winter 2004, a large outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1 year later, the concentrations and genotypes of Giardia cysts occurring in sewage influent were studied to investigate the impact of the outbreak event on Giardia infections in the community. Methods and Results: Sewage influent samples from four sewage treatment works (STW) serving Bergen were analysed for Giardia cysts on four occasions between 15 and 23 months after the outbreak. Cysts genotypes were investigated at one to three genes. Data from influent analysis from one of the STW before the outbreak, and from patient faecal samples analysed during the outbreak, provided baseline comparative data. Relatively high concentrations of Giardia cysts of diverse genotypes, both from Assemblages A and B, were detected at all STW. Conclusions: Comparison of data suggests that although Giardia cyst concentrations in sewage influent returned to pre-outbreak levels within 18 months after the outbreak peak, the genetic composition of the isolates remained significantly influenced by the Assemblage B isolate associated with the outbreak. Significance and Impact of the Study: Genotypes associated with an extensive outbreak of giardiasis continued to occur in Giardia infections in Bergen’s population many months after the outbreak was considered to be over.

Most marine molluscan bivalve shellfish feed on suspended phytoplankton which are trapped from water pumped across the gills by ciliary action. Pathogenic microorganisms in the water may be filtered by the gills during feeding, and become concentrated in the digestive glands/tract. If these pathogens are not excreted or inactivated by the shellfish, or in subsequent preparatory processes, they may be ingested by consumers, the shellfish thereby acting as vehicles of infection. The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii have the potential to be transmitted in this way, and here we review the accumulating knowledge on the occurrence and survival of the transmission stages of these parasites in shellfish, whilst also emphasising the considerable gaps in our knowledge. Relevant information is particularly lacking for T. gondii, which, in comparison with Cryptosporidium spp. and G. duodenalis, has been relatively under-researched in this context. Although it seems evident that these shellfish can accumulate and concentrate all three of these parasites from the surrounding water, whether Giardia cysts remain viable and infectious is unknown, and some evidence suggests that they may be inactivated by the shellfish. Although both Toxoplasma and Cryptosporidium apparently retain their infectivity for prolonged periods in shellfish, the actual public health threat posed by these parasites via these shellfish is unclear, largely because there is minimal evidence of infection transmission. Reasons for this apparent lack of infection transmission are discussed and it is recommended that the potential for transmission via shellfish consumption is recognised by those concerned with investigating transmission of these infections.

A recent review by Brendon King and Paul Monis once again puts Cryptosporidium oocysts under the spotlight. Why is this tough transmission stage so troublesome now? And are future environments likely to assist or hinder its apparent ubiquity? Here, we explore further the fascination and challenge engendered by this parasite transmission stage.

The aim of this study was to investigate local opioid effects in the inflamed skin of healthy human volunteers. To induce inflammation, the circular tip of a 10-mm-diameter probe was heated to 48 degrees C and applied for 120 seconds to a site on each forearm of 24 healthy participants. Thirty minutes later, 0.2 mL of normal saline was injected subcutaneously into 1 inflamed site, and the opioid antagonist naloxone hydrochloride (80 microg in 0.2 mL) was injected subcutaneously into the other inflamed site. Participants completed tests of pain sensitivity (heat pain thresholds, heat pain ratings, and mechanical pain ratings) before and after the injections. Fentanyl citrate (10 microg in 0.2 mL) was then injected into the pretreated sites, and pain sensitivity was measured again. The thermal injuries produced thermal and mechanical hyperalgesia that did not differ between the saline and naloxone sites. After the fentanyl injections, decreases in thermal and mechanical hyperalgesia were greater at the saline site than the naloxone site. These findings demonstrate that pretreatment with naloxone blocks local opioid effects produced by the subcutaneous injection of a low dose of fentanyl in the inflamed skin of healthy humans. Thus, peripheral opioid receptors could be a therapeutic target for painful cutaneous disorders.This article demonstrates that activation of opioid receptors in the skin inhibits sensitivity to painful mechanical and thermal stimuli. Thus, local application of low-dose opioid medications could relieve painful skin disorders.

Faecal samples from 684 litters of suckling piglets from 100 indoor swine herds from all regions of Norway were examined for the presence of Cryptosporidium and Giardia, using sucrose gradient flotation concentration and immunofluorescent staining. Thirty-one (31%) herds and 57 (8.3%) litters tested positive for Cryptosporidium, while 10 (1.5%) litters in 10 different herds (10%) tested positive for Giardia. Molecular characterisation of nine Cryptosporidium isolates demonstrated both C. suis and Cryptosporidium sp. pig genotype II. There was significantly more diarrhoea among the Cryptosporidium positive piglets than among the Cryptosporidium negative piglets. Diarrhoea was not observed amongst litters in which Cryptosporidium sp. pig genotype II was found. There was a significant difference in the Cryptosporidium prevalence between litters from different geographical areas of Norway. This study demonstrates that both Cryptosporidium (C. suis and Cryptosporidium sp. pig genotype II) and Giardia infections are prevalent among suckling piglets in Norway.

Faecal samples from 269 Norwegian wild red foxes (Vulpes vulpes) shot during the hunting season (October-April) in 2002-2004 were examined for the presence of Giardia and Cryptosporidium. Cryptosporidium oocysts were detected in samples from 6 (2.2%) of the foxes, and Giardia cysts in 13 (4.8%) of the foxes. The prevalence of Giardia infection was significantly higher in juvenile male foxes than in adult male foxes, but no other significant differences between age and sex were found. No significant differences in prevalence related to geographical origin of animals were found. Insufficient nucleated Cryptosporidium oocysts were isolated for successful PCR, but genotyping of Giardia duodenalis isolates from seven foxes demonstrated a high degree of heterogeneity amongst them, with all isolates belonging to the zoonotic Assemblages A and B.

Methods for analysing water for viruses are known to have variable and relatively poor recovery efficiencies. Quantitative method recovery data are needed to correct virus enumeration results so that estimates of virus concentrations in surface waters for QMRA are not too low. Obtaining quantitative data representing method recoveries for different pathogenic viruses is a significant challenge. In this study, we investigated the use of mengovirus process control data for quantifying recovery efficiency of human adenovirus (AdV) and noroviruses GI (NoVGI) and GII (NoVGII) from surface waters. Samples were collected from the inlet to a drinking water treatment plant on the Glomma River, Norway. Performance of the sample concentration procedure was quantified by comparing the virus concentrations found in concentrated and unconcentrated samples. The mean recovery of viruses (1.2%, 0.31%, 0.15% and 0.053% for mengovirus (n = 86), AdV (n = 20), NoVGI (n = 33) and NoVGII (n = 21) respectively) estimated in this study were lower than expected, and the between sample variability in estimated recovery was very high, spanning around 6 orders of magnitude for mengovirus. Within-sample correlation between the estimated recovery of mengovirus and human viruses was poor, and therefore sample specific mengovirus data could not be used to correct all human virus concentrations. Instead beta distributions were fitted to human virus-specific recovery estimates. The magnitude and variability of virus concentration when corrected for the variable recovery efficiency was orders of magnitude higher than the uncorrected concentration. Better estimates of virus concentration could be achieved if a sample-specific spiking control could be developed that mimicked closely the behaviour of human viruses in environmental samples.

In order to minimize the likelihood of foodborne infection with Chagas disease the whole food-chain, from production to consumption, needs to be considered using a Hazard Analysis and Critical Control Point (HACCP) approach in order to identify where control measures can be most usefully implemented that will have the greatest effect at reducing risk. Legislation regarding food control in general is in place, and application of the steps of microbial risk assessment is also possible, but, nevertheless, it is clear that foodborne outbreaks of Chagas disease continue to occur in endemic regions. Although the international perspective on outbreaks of orally transmitted Chagas disease is, and unfortunately continues to be, that this is a local and limited problem and without any global impact, it is nevertheless clear that acai, sugarcane, and fruit juices are ideal food matrices for foodborne transmission; vital risk factors include the crops for these drinks often being grown, harvested, and pressed locally, (with the pressing often occurring at night-time under artificial light), in rural or periurban areas where sylvatic triatomines may be abundant. In this chapter we discuss not only the legislation and tools available to reduce the likelihood of foodborne transmission, but also practical measures that could be implemented at all stages along the food-chain, not forgetting educational measures for the vendors and consumers of the final products.

Although first identified over a century ago, Trypanosoma cruzi continues to infect millions of people in the Americas. The disease, as well as being potentially fatal, is chronic, disabling, and debilitating, and has a huge impact on the societies in which it occurs. Despite a reduction in numbers of cases in recent years, probably as a result of initiatives targeting the vectors, it has spread geographically as people and vectors also move. Nevertheless, treatment options remain limited and ineffectual. Furthermore, as vectorborne transmission has reduced, an upsurge of outbreaks associated with contaminated food has occurred. Here, as a lead in to the following chapters, we introduce historical and geographical factors associated with this protozoan parasite, and also how an infection that is generally considered vectorborne, should also be considered as one of the many pathogens that may be transmitted via food; its human and public health impact as a foodborne parasite in terms of probably exceeds those of other more “traditional” foodborne parasites that remain in focus, such as Trichinella spp. or Taenia saginata.

In this final section we try to pull together some of the key points made in previous sections and emphasise that whilst we may be coming to grips with vectorborne transmission of Chagas disease, epidemiological changes are likely to exacerbate the rising threat of foodborne transmission. In addition, the threat of exporting this disease beyond the confines of Latin America should be considered—globalization of people and insects, and the adaptation of the versatile Trypanosoma cruzi to potential new vectors, reservoirs and transmission routes mean that this is not just idle speculation. Investment in prevention, detection, treatment, and education is fundamental for meeting these challenges.

The increasing number of cases, clusters and outbreaks of foodborne Chagas disease are likely to be due to a number of factors that have resulted in conditions for oral transmission becoming more favourable. This is, in part, emphasised by vector-control initiatives reducing the spread of vectorborne transmission. In this chapter we explore a range of epidemiological factors that have facilitated the foodborne transmission of Chagas disease. These factors include human migration and urban development, both of which have, in their turn, impacted upon important facets in the lifecycle of T. cruzi, such as the invasion and domiciliation of triatomine species, and alterations in the population dynamics of triatomines and wild animal reservoirs. It should be remembered that although transmission routes may be understood, they seldom remain static and different factors may alter and tweak different aspects. For the future, factors such as climate change (resulting in wider distribution of some vectors), the emergence of new vectors, the migration of individuals infected with T. cruzi to non-endemic areas, and globalization of the food-chain may be suggested as such as factors that may ensure that oral transmission of T. cruzi continues and expands, both in endemic areas, but also in other regions beyond the traditionally endemic areas.

Although several parasites are transmitted by contaminated food, most of these are environmental transmission stages with a robust outer wall, and this is one of the respects by which Trypanosoma cruzi differs from other parasites. Although some food commodities, specifically fruit juices, are now a well-recognized transmission vehicle for T. cruzi, there is currently a lack of survey data on how often juices are contaminated, and the association of cases or outbreaks with particular vehicles of infection is generally epidemiological rather than demonstrative. This means that compared with other parasites no surveillance data are available, and there is no tested tool for examining juices during outbreak situations. Surveillance data are also useful for risk assessment and for identifying areas in the food-chain of greater risk.

We investigated the survival of Cryptosporidium oocysts and Giardia cysts during winter in an aquatic environment (approximate temperature measurements between 1 and 7°C) in Norway, using morphology and uptake of dyes as indicators of viability. Previous research has shown that in the terrestrial environment, shear forces caused by freeze and thaw cycles probably cause the parasites to be inactivated. Such forces occurred infrequently in the aquatic environment, as freezing of the water around the parasites was not observed during the study period (although freezing of the water surface did occur). The rate of decline in viability (log10 N t/N 0) was similar in control and experimental environments for both parasites; no Cryptosporidium oocysts with viable morphology were detected after approximately 20 weeks and no Giardia cysts with apparently viable morphology could be detected after 1 month. These results suggest that infection with these parasites in Norway is not usually from transmission stages that have overwintered in the Norwegian environment.

Faecal samples were collected from 1,190 wild cervids in Norway and analyzed for cysts/oocysts of the protozoan parasites Giardia and Cryptosporidium. Samples were from calves, yearlings and adults of moose (Alces alces), red deer (Cervus elaphus), roe deer (Capreolus capreolus) and reindeer (Rangifer tarandus) shot during the hunting season. Cryptosporidium was found in 15 (3.3%) of 455 moose, 1 (0.3%) of 289 red deer, 18 (6.2%) of 291 roe deer, but was not found in any of 155 reindeer. Giardia was found in 56 (12.3%) moose, 5 (1.7%) red deer, 45 (15.5%) roe deer and 11 (7.1%) reindeer. The calves had the highest prevalence of infection, but this was only statistically significant for Giardia in moose and for Cryptosporidium and Giardia in roe deer. Calves generally had the highest intensity of infection, but this difference was only statistically significant for calves with Giarda and the highest intensity of infection. Both Giardia and Cryptosporidium were found in samples from several geographical areas, indicating that these parasites are distributed among the cervid population in all parts of Norway, especially in moose and roe deer. This is the first published report of Cryptosporidium in moose and of Giardia in reindeer.

Cryptosporidiosis is rarely diagnosed in Norway. This is the first report of a human outbreak in Norway and involved 3 calves and 5 people. Sequencing studies were performed. Although 4 of the people acquired their infections from the calves, the other probably became infected from other calves or contaminated pens.

Cryptosporidiosis usually manifests as gastrointestinal infection and is associated with considerable morbidity and, in some circumstances, mortality. Effective treatment that is suitable for all patients, including those that are particularly affected by infection, children and the immunocompromised, is lacking.

The current work evolved from a microbial survey of fruits and vegetables conducted in Norway between 1999 and 2001. This survey found that mung bean sprouts were more likely to be contaminated with Cryptosporidium and Giardia than the other produce included in the survey. To support this observation and to demonstrate to public health officials that this might be a risk warranting further attention, a simple risk assessment was initiated. Assuming that 60,000 people in Norway consume a single serving of bean sprouts per week, and contamination levels are similar to those found in the survey, it was calculated that there could be in the order of 20 cases of Giardia or Cryptosporidium infection per 100,000 population attributable to consumption of mung bean sprouts. A number of assumptions were made for the calculations, including parasite factors (e.g. viability, genotype), product factors (e.g. extent of product contamination) and host factors (e.g. composition and extent of consumer group). These assumptions and areas of uncertainty, where further data would improve the risk assessment, are highlighted throughout. Not only does the risk assessment identify new areas of research, but it also demonstrates how risk assessment can be used as a tool to try to influence public health surveillance.

The report on the ranking of the foodborne parasites is the result of an international effort, coordinated and organized by FAO and WHO. The following experts participated in the ranking exercise: Pascal Boireau, Laboratory for Animal Health Maisons Alfort, France; Jorge E. Bolpe, Ministerio de Salud de la Provincia de Buenos Aires, Argentina; Allal Dakkak, Institut Agronomique et Veterinaire Hassan II, Morocco; Brent Dixon (co-chair of meeting), Health Canada, Canada; Ronald Fayer, United States Department of Agriculture, USA; Jorge E. Gomez Marin, Centro de Investigaciones Biomedicas de la Universidad del Quindio, Colombia; Erastus Kang’ethe, University of Nairobi, Kenya; Malcolm Kennedy, University of Glasgow, UK; Samson Mukaratirwa, University of KwaZulu-Natal, South Africa; K. Darwin Murrell, University of Copenhagen, Denmark; Tomoyoshi Nozaki, National Institute of Infectious Diseases, Japan; Ynes Ortega, University of Georgia, USA; Subhash C. Parija, Jawaharlal Institute of Postgraduate Medical Education and Research, India; Lucy Robertson, Norwegian School of Veterinary Science, Norway; Mohammad Bagher Rokni, Tehran University of Medical Sciences, Iran; Patrizia Rossi, Instituto Superiore di Sanita, Italy; Said Shalaby, National Research Center, Egypt; Paiboon Sithithaworn, Khon Kaen University, Thailand; Rebecca Traub (rapporteur of meeting), University of Queensland, Australia; Nguyen van De, Hanoi Medical University, Vietnam; Joke W.B. van der Giessen (Chair of meeting), National Institute of Public Health and the Environment, The Netherlands.Resources persons for this exercise were: Michael Batz, University of Florida, USA; Annamaria Bruno, Joint FAO/WHO Food Standards Programme; Verna Carolissen, Joint FAO/WHO Food Standards Programme; Steve Hathaway, Science and Risk Assessment Standards Branch, New Zealand; Iddya Karunasagar, FAO; Gillian Mylrea, World Organisation for Animal Health (OIE); Patrick Otto, FAO; Edoardo Pozio, Instituto Superiore di Sanita, Italy; and Andrijana Rajic, FAO.The secretariat was composed of: Sarah Cahill, FAO; Marisa Caipo, FAO; Mina Kojima, WHO; Simone Magnino, WHO; and Kaye Wachsmuth, International Public Health Consultant, USA.

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.

Enteric viruses transmitted via the faecal-oral route occur in high concentrations in wastewater and may contaminate drinking water sources and cause disease. In order to quantify enteric adenovirus and norovirus genotypes I and II (GI and GII) impacting a drinking source in Norway, samples of surface water (52), wastewater inlet (64) and outlet (59) were collected between January 2011 and April 2012. Samples were concentrated in two steps, using an electropositive disc filter and polyethylene glycol precipitation, followed by nucleic acid extraction and analysis by quantitative polymerase chain reaction. Virus was detected in 47/52 (90.4 %) of surface water, 59/64 (92 %) of wastewater inlet and 55/59 (93 %) of wastewater outlet samples. Norovirus GI occurred in the highest concentrations in surface water (2.51e + 04) and adenovirus in wastewater (2.15e + 07). While adenovirus was the most frequently detected in all matrices, norovirus GI was more frequently detected in surface water and norovirus GII in wastewater. This study is the first in Norway to monitor both sewage and a drinking water source in parallel, and confirms the year-round presence of norovirus and adenovirus in a Norwegian drinking water source.

Giardia duodenalis is a globally-distributed protozoan parasite that infects the small intestine of a range of mammals. Human infection with G. duodenalis (Assemblage A or B) may cause clinical illness, giardiasis. Various factors in the biology of G. duodenalis, including the high excretion rate, low infectious dose, and the robustness of the cyst transmission stage, mean that it is particularly suited to waterborne transmission. As well as addressing basic background on the parasite and the disease, this chapter covers waterborne and foodborne outbreaks, occurrence and detection in environmental samples, removal and inactivation of cysts, regulatory matters and risk, and, finally, future challenges. It is clear that transmission of giardiasis via contaminated food and water remains a real problem, even in industrialized countries, and should be tackled by a cross-disciplinary, collaborative approach.

While recently published techniques for recovering parasites from fruits and vegetables demonstrate a marked increase in efficiency and utility, there is still scope for further improvement in developing a standard method, particularly with difficult, but important, sample matrices such as bean sprouts. Herein, a number of parameters used in published techniques are investigated more closely. While sample size reduction may improve recovery efficiency because of a range of factors, it is important to keep the sample large enough for detection of low-level contamination. Age of sample is also important, and samples should be as fresh as possible. Elution procedures may contribute to losses of Giardia and should be more thoroughly investigated. Improved immunomagnetic separation techniques currently coming onto the market also have the potential to increase recovery efficiency substantially, even with difficult samples such as aged bean sprouts. However, merely increasing magnetic strength of the capturing magnet does not affect recovery efficiency, which must be reliant on a superior bead system, buffering system, or both.

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.

Published techniques for recovering parasites from fruit and vegetables are generally inadequate, with low and variable recovery efficiencies. Herein, we describe an improved method for analyzing fruit and vegetables for Cyclospora oocysts. The technique includes washing procedures, sonication, and separation using lectin-coated paramagnetic beads. Identification is by microscopy (differential interference contrast and fluorescence). Oocyst recovery efficiencies from mushrooms, lettuce, and raspberries were approximately 12%. Recovery efficiencies from bean sprouts were approximately 4%. Although no significant difference in recovery efficiency could be detected between samples processed using the lectin-coated beads and samples processed without this procedure, distinct advantages were apparent when the lectin-coated beads were used. A considerably smaller, cleaner final volume remained for microscopy, which increases the sensitivity of the technique and reduces operator time.

A three year study on Cryptosporidium oocysts and Giardia cysts in sewage was conducted with emphasis upon: (1) development and assessment of techniques for sampling and processing sewage for these parasites; (2) a field study in which six sewage treatment works (STW) were assessed for removal of these parasites from the liquid phase; and (3) assessment of effect of sewage treatment on survival/destruction of Cryptosporidium oocysts. In sampling, grab samples were superior to filtered samples. In concentration and clarification of samples, the most efficient technique was that in which sample manipulation was minimised. Although calculated parasite removal efficiency varied for both parasites and between and within STW, some consistent patterns were observed. First, Giardia cyst removal was significantly greater than Cryptosporidium oocyst removal. Second, most parasite removal occurred during secondary treatments, rather than during primary settlement. Third, no significant difference between efficacy of activated sludge treatment and trickling filters at parasite removal was detected. Also, in one STW which utilised tertiary sewage treatment, most removal from the liquid phase occurred during preceding treatment stages. Finally, one STW had significantly higher removal efficiencies of both parasites than the other five STW investigated. Low numbers of viable oocysts were detected at both influent and effluent. Survival was monitored by introducing oocysts, in containers bounded by semi-permeable membranes, into various environments within one STW. The results demonstrated that, over one week, none of the environments tested had a deleterious effect on oocyst viability, with the exception of the sludge-holding tank. Results are discussed in relation to other published work on Cryptosporidium and Giardia in sewage and recommendations for further areas of research on this subject are made.

Waterborne transmission of Cryptosporidium infection is well known and many outbreaks of waterborne cryptosporidiosis have been extensively documented. In this chapter the focus is on foodborne outbreaks of cryptosporidiosis, of which over 20 are described. The most common vehicles of transmission associated with foodborne outbreaks are salad bar items and salad garnishes, dairy products, particularly unpasteurised milk, and apple cider. Perhaps surprisingly, case–control studies have also indicated that consumption of fresh produce has a protective effect against infection with Cryptosporidium. The species of Cryptosporidium most associated with foodborne outbreaks, contra the species most associated with waterborne outbreaks, is discussed, along with the geographical distribution of these outbreaks and possible contamination pathways for food products, including food handlers.

Transmission of Cryptosporidium infection occurs when a susceptible host ingests a sufficient quantity of infectious oocysts. Various factors in the biology of Cryptosporidium mean that this parasite is particularly suited to waterborne or foodborne transmission. In this chapter these factors, which include the high oocyst excretion rate, the low infectious dose, the robustness of oocysts and their ability to survive in the environment, the small size of oocysts, and the potential for zoonotic transmission, are discussed in greater detail, with emphasis on how food, particularly fresh produce, may become contaminated with infective Cryptosporidium oocysts and provide a vehicle for transmission. Additionally, the results of a risk-ranking exercise, in which the different foodborne parasitoses were tiered in order of comparative importance, are described, with respect to the relative placing of Cryptosporidium.

Cryptosporidium oocysts are known to be extremely robust. They can survive for extended periods in the damp, cool conditions in which fresh produce or shellfish (likely vehicles for the transmission of Cryptosporidium oocysts) are generally stored, and can also survive harsher conditions such as contact with chlorine. However, they are susceptible to desiccation, heating and freeze-thawing, as well as some types of irradiation, including ultra-violet, and some chemical treatments. This resilience of Cryptosporidium oocysts to environmental pressures means that for food products that are intended for eating with minimal processing (no cooking) in order to retain their sensory qualities, such as taste and texture, inactivation or decontamination is probably the wrong approach; it is preferable to avoid the original contamination.

Detection of Cryptosporidium oocyst contamination of food relies upon isolating and identifying either the Cryptosporidium oocysts themselves or their DNA. Analysis of food samples for Cryptosporidium contamination is based largely on the Standard Methods (ISO Method 15553 and US EPA Method 1623) for analysing water samples for Cryptosporidium contamination. Critical steps involve elution or separation from the food matrix, concentration and isolation using immunomagnetic separation (IMS) and identification, for which immunofluorescent antibody test (IFAT) remains the gold standard and the test of choice despite advances in molecular detection techniques. In this chapter, methods and approaches for detection of contaminating Cryptosporidium oocysts in fresh produce (fruit and vegetables), shellfish, meat, beverages and water used in the food industry are considered separately, as there is no ‘one-method-suits-all’, and characteristics of the food matrix being analysed affect the efficacy of the method. For fruits and vegetables, an ISO Method is under development, particularly designed for leafy vegetables and berry fruits, and method development and method standardisation are described, along with further research. For shellfish, pepsin digestion appears to provide the best results for removing Cryptosporidium oocysts from this matrix into a suspension, but different studies have resulted in different recommendations regarding subsequent steps (e.g. the use, or not, of IMS) and the most appropriate detection techniques (molecular methods or IFAT). Only a few studies have been described for analysing meat for Cryptosporidium contamination, but a number of studies have investigated methodologies for analysing both milk and fruit juices for contamination, presumably due to the various outbreaks of beverage-associated outbreaks of cryptosporidiosis, and these methods are described.

The results of surveys for pathogens, such as Cryptosporidium oocysts, in different food matrices provide a snapshot of what was found on a particular food sample, on a particular occasion, under particular conditions, using a particular method, in a particular laboratory, by a particular analyst. Some surveys do not even provide information regarding the recovery efficiency of the method used, or may only provide information on recovery efficiencies produced in the research group that first developed or published the method. As some of the methods are relatively expensive (particularly IMS, if used) often surveys consist of only a small number of samples, and as recovery efficiencies are often superior with smaller sample sizes, only a small quantity of the product is analysed. Thus, results from such surveys can only be considered to give a very diffuse insight into the risks of ingestion of a Cryptosporidium oocyst from a particular product. Furthermore, surveys that do not investigate the species of any Cryptosporidium oocysts detected cannot even determine with certainty whether the oocysts that are found are infectious to humans, and thus of public health significance. And surveys that do not consider the viability or infection potential of the oocysts are similarly hampered. Nevertheless, while it is important to acknowledge and be aware of the limitations of such surveys, it should also be realised that these results are our only available verified, scientific handle on contamination of food matrices with potentially infective Cryptosporidium oocysts, and thus the information that they provide is useful. In addition, investigation of food matrices for Cryptosporidium contamination in an outbreak situation has the potential to identify infection routes, and thus take measures against them. Information obtained from food products analysed during an outbreak event is of greater value if baseline survey data are also already available against which the outbreak-related analyses can be compared. Occurrence data are being improved all the time, as further studies are conducted with better, more efficient methods. The information outlined in the sections below is intended to provide an insight of what we know about the occurrence of Cryptosporidium oocysts on different product types as of today—more up-to-date information should always be sought. When comparing occurrence results from different studies, the method used and the recovery efficiency of that method should always be borne in mind, as surveys using very different methods or similar methods but with different recovery efficiencies cannot be readily compared, or such comparisons may give an erroneous impression. When recovery data are not provided, it is probably most appropriate to assume that the efficiency of the method used is low, and thus any contamination data provided are likely to be conservative.

In this chapter, the principal approaches and legislation for controlling Cryptosporidium contamination in the food chain are described, with particular emphasis on HACCP and risk assessment (RA). An example is described in which quantitative microbial RA (QMRA) was used to estimate the risk of acquiring cryptosporidiosis from fresh produce (tomatoes, cucumbers, bell peppers and lettuce) being cultivated in Mexico. Data input into the model include the concentration of Cryptosporidium oocysts in irrigation water (including the efficiency of the detection method), the retention of irrigation water on the produce and the daily consumption of the various products by consumers in the USA. Worst-case assumptions included in the model (100 % transfer of oocysts to produce and all cysts being infectious to humans) are also noted. Another study is described that investigates the potential for foodborne cryptosporidiosis being acquired in the UK from ingestion of root crops grown in soil that has been augmented with treated sewage sludge. Although it is acknowledged that such exercises are flawed and limited, the utility of the data achieved is also discussed.

Cryptosporidium is an Apicomplexan parasite, of which around 25 species and several genotypes are recognised, several of which are infectious to humans. The majority of human infections are due to the species Cryptosporidium hominis and C. parvum. The latter parasite is also a common pathogen of a range of animals, particularly young calves and small ruminants. Cryptosporidium is globally distributed and is considered to be one of the four pathogens most associated serious childhood diarrhoea in developing countries; effective chemotherapy in the most vulnerable patient groups is lacking.

Cryptosporidiosis was first identified as a disease of veterinary, rather than human medical, importance, and infection of farmed animals with different species of Cryptosporidium continues to be of veterinary clinical concern. This chapter provides insights into Cryptosporidium infection in a range of farmed animals – cattle, sheep, goats, pigs, cervids, camelids, rabbits, water buffalo and poultry – presenting not only an updated overview of the infection in these animals, but also information on clinical disease, infection dynamics and zoonotic potential. Although extensive data have been accrued on, for example, Cryptosporidium parvum infection in calves, and calf cryptosporidiosis continues to be a major veterinary concern especially in temperate regions, there remains a paucity of data for other farmed animals, despite Cryptosporidium infection causing significant clinical disease and also, for some species, with the potential for transmission of infection to people, either directly or indirectly.

Giardia duodenalis is the most common intestinal protozoan parasite of humans globally. A range of other mammals can also be hosts to this parasite, although the zoonotic potential is currently unresolved and parasites that are infectious to humans are not necessarily infective to other mammals and vice versa. The life cycle of G. duodenalis is simple, and contains two morphologically distinct forms: the vegetative trophozoites that inhabit the host intestine and replicate largely asexually by binary fission, sometimes resulting in the mucosal surface of the intestine being covered, and the transmission stage, the environmentally robust cysts that are excreted in the faeces of the infective host. This chapter provides an overview of the parasite, establishment of infection and details of the life cycle, and also describes the disease, giardiasis, that is associated with infection, with focus on symptoms, diagnosis and treatment.