Cryptosporidium and Giardia infections in humans in Tigray, Northern Ethiopia: an unexpectedly low occurrence of anthropozoonotic transmission.

Enteric protozoans Cryptosporidium spp. and Giardia duodenalis are among the leading causes of diarrhoea in children. These parasites have particular impact in low- and middle-income countries. In these countries, people often live in close contact with their animals, highlighting the potential role of zoonotic routes of transmission in disease spread. The occurrence and species/genotypes of Cryptosporidium and Giardia duodenalis infecting humans in Tigray, Ethiopia were investigated, along with the risk associated with infection. Stool samples from 249 asymptomatic people (4-80 years of age) in four rural districts in Tigray and 58 from symptomatic young children (1-33 months) attending health centres in Mekelle, Tigray's main city, were analysed for Cryptosporidium oocysts and Giardia cysts. Participants in the rural areas completed questionnaires regarding potential risk factors, with emphasis on livestock contact and sources of water. The occurrence of Cryptosporidium infection was 6% and 5% in people in the rural districts and young children from Mekelle, respectively; equivalent figures for Giardia infection were 29% and 14%. Molecular characterization of Cryptosporidium isolates revealed C. ubiquitum, subtype XIIa in a sample from rural districts, and C. hominis subtype IdA17 (1 sample) and IbA9G3 (2 samples) in infants from Mekelle with diarrhoea. For Giardia, Assemblage B predominated (22/25; 88%), but we also identified three samples with Assemblage A (AII). Our major finding was that, despite the close contact between people and livestock in our rural study sites, transmission of Cryptosporidium and Giardia between humans and their animals seems to be surprisingly uncommon. Our results are discussed in relation to other relevant studies, and also draws attention to the possibility that introduction of zoonotic species and/or subtypes, such as C. parvum, could have serious consequences for both human and animal health. As our study was conducted in Tigray, further investigation in different settings in Ethiopia could provide relevant information on transmission and zoonotic potential, and the potential for spread of zoonotic transmission. In addition, given the importance of these two parasites in causing diarrhoea in children, this information is vital for developing effective appropriate interventions against transmission that can be applied not only in Tigray or Ethiopia, but throughout Africa and beyond.

Gastrointestinal nematodes and Fasciola hepatica in Norwegian cattle herds: a questionnaire to investigate farmers' perceptions and control strategies.

BACKGROUND: Pasture management influences the prevalence and impact of the pasture parasites (PP) in cattle herds, which cause production-limiting disease worldwide. Evaluating farmer management strategies is vital when considering sustainable PP control practices. The aim of this questionnaire-based study was to describe the pasture management and control strategies regarding PP in Norwegian beef cattle (BC) and dairy cattle (DC) production systems with a focus on gastrointestinal nematodes (GIN) and Fasciola hepatica. RESULTS: A total of 745 responses from BC (return rate 20.5%) and 1347 responses from DC farmers (30.7%) were included. The mean total pasture time for DC was 4.2 months for first-season grazers and 4.3 months for second-season grazers and cows, while the corresponding finding in BC was 5.4 months. Home pasture was used for most of the pasture period, particularly for first-season grazer dairy heifers (81%), which were also commonly grazed on the same pasture every year (79%). For most farmers it was necessary for grazing areas to be used for cattle for more than one season (77% of BC farmers and 89% of DC farmers). However, changing the pasture during the season was common in both DC (67%) and BC (70%) herds. The majority of DC farmers (60%) stated that they did not consider that they had a problem with PP. Of the remaining 40%, few respondents could specify whether their herds had a problem due to infection by GIN (11%) or liver flukes (12%). Treatment for GIN was performed by 52% of DC and 34% of BC farmers. Diagnostic faecal samples were collected upon suspicion of parasitic disease by 5% of DC and 16% of BC farmers. Veterinarians were stated as a central source of information about parasite management and treatment. CONCLUSIONS: Potential risks for exposure to PP were identified, such as use of the same pasture every year for first-season grazers and frequent use of home pasture. The perception of problems related to PP appeared low. Regular anthelmintic treatment without concurrent use of diagnostic faecal samples seems to be common practice.

Treatment against helminths in Norwegian sheep: a questionnaire-based survey.

A questionnaire was distributed to 5487 farmers throughout Norway in order to obtain information about management practices regarding helminth infections in sheep. In addition, the farmers' perceptions of helminths and anthelmintic efficacy were investigated. Most farmers (80%) treated prophylactically against nematodes, and 24% also used prophylactic treatment against Fasciola hepatica. Overall, few farmers (11%) used parasitological analysis as a tool to assess the timing of treatment, but rather based it on other factors such as previous experience (70%). In the surveyed sheep flocks, the use of benzimidazoles was reduced from 2018 (52%) to 2019 (47%) (p < 0.01), whereas the use of macrocyclic lactones increased from 2017 (23%) to 2019 (36%) (p < 0.001). Poor anthelmintic efficacy was suspected by 10% of the farmers, and 11% reported that helminths were an increasing problem in their flocks. The majority of farmers (72%) considered their veterinarian as the most important advisor for treatment of parasites, but reported a high level of uncertainty regarding which parasites were present in their flocks, with unknown status most frequently reported for Haemonchus contortus (71.5%). This is probably related to the fact that very few farmers (15%) regularly test their animals for parasites. The present study provides up-to-date information on treatment practices for helminths in Norwegian sheep flocks.

TITLE: Traitement contre les helminthes chez les moutons norvégiens : une enquête par questionnaire. ABSTRACT: Un questionnaire a été distribué à 5487 éleveurs dans l'ensemble de la Norvège afin d'obtenir des informations sur la gestion des helminthiases chez les ovins. Le questionnaire a porté, en outre, sur la perception des éleveurs concernant les helminthiases et l'efficacité des anthelminthiques. La plupart des éleveurs (80 %) réalisent des traitements prophylactiques contre les nématodes et 24 % font de même vis-à-vis de Fasciola hepatica. Globalement, peu d'éleveurs (11 %) recourent aux analyses parasitologiques pour déterminer les dates de traitement, s'appuyant plutôt sur d'autres éléments tels que leur propre expérience (70 %). Dans les troupeaux enquêtés, l'utilisation des benzimidazoles a diminué de 2018 (52 %) à 2019 (47 %) (p < 0,01) tandis que celle des lactones macrocycliques a augmenté entre 2017 (23 %) et 2019 (36 %) (p < 0,001). Une faible efficacité des anthelminthiques est suspectée par 10 % des éleveurs tandis que 11 % des éleveurs signalent que les helminthiases sont un problème croissant dans leurs troupeaux. La majorité des éleveurs (72 %) considèrent leur vétérinaire comme le conseiller le plus important en matière de traitement antiparasitaire mais rapportent un haut niveau d'incertitude concernant le type de parasites présents dans leur troupeau, en particulier en ce qui concerne la présence d'Haemonchus contortus (71,5 %). Ceci est probablement à relier au fait que très peu d'éleveurs (15 %) testent régulièrement leurs animaux vis-à-vis des parasites. La présente étude fournit des informations actualisées sur les pratiques de traitement anthelminthique des troupeaux ovins en Norvège.

Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts.

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Occurrence of faecal endoparasites in reindeer (Rangifer tarandus) in two grazing areas in northern Norway.

BACKGROUND: Semi-domesticated reindeer represent an important livestock industry and livelihood for a proportion of the human population in northern Fennoscandia. Reindeer husbandry is considered an extensive animal husbandry, where the animals are kept mostly on natural pastures, although sometimes kept in fenced areas for shorter periods. These reindeer may harbour a variety of parasites that may affect animal health and production. The relatively limited close contact between herds and owners gives limited opportunities for diagnosis and treatment of diseases in general. Furthermore, the effects of subclinical parasitism in livestock are commonly expressed as a reduction in productivity rather than clinical disease and mortality. Thus, specific knowledge of endoparasites and parasitic infections in these herds is scarce. This study investigated the occurrence of various endoparasites in reindeer by analysis of a total of 114 faecal samples from winter-slaughtered reindeer from two different grazing areas in Troms and Finnmark, Norway. RESULTS: Using a McMaster method, a Baermann technique, and a direct immunofluorescent antibody test, the following parasites were identified in the faecal samples with the occurrence data given as percentages: Strongylid eggs (62%), Nematodirinae spp. eggs (24%), Capillaria sp. eggs (42%) and Moniezia spp. eggs (17%), Dictyocaulus spp. larvae (14%) protostrongylid larvae (40%), Eimera spp. oocysts (23%), and Giardia duodenalis cysts (5%). Cryptosporidium oocysts were not detected. Parasite eggs, oocysts, and cysts were quantified. Molecular analysis revealed G. duodenalis sub-assemblage AI, a potentially zoonotic genotype not previously reported in reindeer from this region. Morphological analyses of Eimeria oocysts identified two species, Eimeria mayeri and Eimeria rangiferis, and molecular analyses of the cytochrome C oxidase I (coi) gene and 18 s rRNA (18SSU) gene of Eimeria confirmed the presence of Eimeria species that are specific to reindeer. CONCLUSIONS: A high prevalence, but low burden, of endoparasites was detected in samples from these semi-domesticated reindeer. The samples were collected during winter, when adult gastrointestinal parasites usually produce only low numbers of transmission stages. Therefore, together with the low number of samples, detailed and definitive conclusions regarding parasite status of semi-domesticated reindeer are avoided. Nevertheless, these data provide a snapshot overview of parasite occurrence in a semi-domesticated animal group vulnerable to the various environmental changes to which they are exposed.

Removal of Parasite Transmission Stages from Berries Using Washing Procedures Suitable for Consumers.

Due to the delicate nature of berries and the reduced shelf-life once washed, producers usually do not wash berries. Therefore, consumers are expected to wash the berries prior to consumption, and this might be a more effective way of infection prevention. However, the efficacy of consumer berry-washing procedures in removing the parasite contaminants from the berries surface has not been investigated. The aim of the present study was, therefore, to compare the efficacy of three different washing techniques in removing parasite contaminants. Three alternatives to washing berries before consumption were compared on berries artificially contaminated with oo/cysts of Cyclospora cayetanensis, Cryptosporidium parvum, and Giardia duodenalis. The results show that simple washing of berries under the cold tap for 1 min could remove on average at least 80% of the parasites, except for C. cayetanensis, which seems to be stickier than both G. duodenalis and C. parvum. The percent removal was slightly lower for raspberries as compared to blueberries. Although the differences are expected, a relevant result of the study is that washing contaminated berries prior to consumption by the consumer removes a considerable proportion of parasites and thereby lowers the risk of ingesting parasites' transmission stages.

Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries.

The potential public health impact of foodborne parasites (FBP) transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower Cq values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method.

Pre- and Post-Race Intestinal Microbiota in Long-Distance Sled Dogs and Associations with Performance.

Although our understanding of the role of the gut microbiota in different diseases is improving, our knowledge regarding how the gut microbiota affects functioning in healthy individuals is still limited. Here, we hypothesize that the gut microbiota could be associated with sled dog endurance-race performance. We investigated the gut microbiota in 166 fecal samples from 96 Alaskan Huskies, representing 16 teams participating in the 2016 Femund Race (400 km) in Norway, relating the microbiota composition to performance and metadata derived from questionnaires. For 16S rRNA gene sequencing-derived compositional data, we found a strong negative association between Enterobacteriaceae (dysbiosis-associated) and Clostridium hiranonis (normobiosis-associated). The teams with the best performances showed both the lowest levels of dysbiosis-associated bacteria prior to the race and the lowest change (decrease) in these bacteria after the race. Taken together, our results support the hypothesis that normobiosis-associated bacteria are involved in resilience mechanisms, potentially preventing growth of Enterobacteriaceae during the race.

Surface waters as a potential source of Giardia and Cryptosporidium in Serbia.

Giardiasis and cryptosporidiosis are recognized by the WHO as important emerging diseases of the 21st century. Symptoms are similar and include diarrhoea and vomiting, which may be severe, even life-threatening, for the immunocompromised and children under five years of age. Between 2013 and 2017, the Institute for Public Health in Serbia recorded 10 waterborne epidemics that manifested as gastrointestinal disease. Routine testing for enteropathogenic bacteria and viruses did not identify the aetiological agents of these outbreaks. As water is not examined for the presence of protozoa in Serbia, we performed a pilot study to analyse samples from four major rivers and their tributaries using a newly implemented methodology for detection of Giardia and Cryptosporidium, based on the ISO 15553:2006 standard. Using immunofluorescence microscopy, Giardia was detected in 10 out of the 31 samples, Cryptosporidium in five, while two samples were positive for both. Presence of G. duodenalis gDNA was confirmed by amplification of the ß-giardin gene in eight samples, of which one and two, respectively, were identified by RFLP as potentially zoonotic assemblages A and B. The results suggest that surface water in Serbia may be a potential source of infection and call for more in-depth studies using sophisticated molecular tools.

Toxoplasma gondii infection in two captive Patagonian maras.

Toxoplasma gondii infection was diagnosed in 2 captive Patagonian maras (Dolichotis patagonum). One animal developed fatal systemic toxoplasmosis and had concurrent localized bacterial and fungal infections; its daughter remained clinically healthy. Microscopic findings included acute, coagulative necrosis, lymphohistiocytic inflammatory infiltrates, and extra- and intracellular parasites in the liver, myocardium, urinary bladder, and adrenal glands of the diseased animal. PCR and subsequent genotyping of parasites from fresh tissue from both cases revealed infection with T. gondii genotype II. Direct agglutination testing of blood from the healthy individual revealed high levels of T. gondii IgG antibodies. T. gondii is a potential cause of disease and lethality in captive and wild Patagonian maras, and toxoplasmosis should be considered when managing and providing veterinary care for this species.

A New Protocol for Molecular Detection of Cyclospora cayetanensis as Contaminants of Berry Fruits.

Cyclospora cayetanensis is a coccidian parasite that is associated with foodborne outbreaks of gastrointestinal illnesses. Raspberries have been implicated as a vehicle of infection in some of these outbreaks. Most of the molecular techniques used for the detection of parasites commonly use the 18s rRNA as a target gene, which is highly conserved. The conserved nature of the 18s rRNA gene among coccidia means that there is potential for cross-reactivity from primers intended to target this gene in C. cayetanensis with the same gene in related coccidia. This provides an additional challenge in developing a specific detection method. The aim of this study is to develop a new, more specific assay to detect C. cayetanensis in berry fruits. This new assay, targeting the internal transcribed spacer 1 (ITS-1) region, was tested on three different berry matrices: raspberries, blueberries, and strawberries. The new assay showed good efficiency (102%), linearity (r 2 = 0.999), repeatability (standard deviation of Cq 0.2 (95% CI: 0.2, 0.3) and specificity for Cyclospora, with no cross-reactivity with related coccidia (Toxoplasma gondii, Eimeria mitis, Cystoisospora canis, and Cryptosporidium parvum) when tested in vitro. The method development was initially conducted using Cyclospora DNA only. After it was confirmed to have an acceptable performance, the method was evaluated using the oocysts of C. cayetanensis. The method was also improved by incorporating an internal control as a duplex in order to monitor PCR inhibition due to sample matrix components. The duplex assay also showed a good efficiency (100%) and linearity (r 2 = 0.99). The results showed that the new assay has potential for standard use in food testing laboratories. Furthermore, results regarding important factors related to assay robustness are discussed.

A novel multiplex real-time PCR for the detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis on berries.

Foodborne parasites (FBP) are of major public health importance and warrant appropriate detection and control strategies. Most of the FBP considered for risk-ranking by a panel of experts are potentially transmitted via consumption of contaminated fresh produce, including berries. In this study we focused on the potential of three FBP, namely Echinococcus multilocularis, Toxoplamsa gondii, and Cyclospora cayetanensis, as contaminants of berries. Surveys to assess these parasites as contaminants of fresh produce in general, and berries in particular, are scanty or non-existent mainly due to the lack of optimized laboratory methods for detection. The aim of the present study was to develop and evaluate a novel multiplex qPCR for the simultaneous detection of E. multilocularis, T. gondii, and C. cayetanensis from berry fruits. The efficiency and linearity of each channel in the multiplex qPCR were within the acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.2 Cq) and intermediate precision (pooled standard deviation of 0.3-0.6 Cq). The limit of detection was estimated to 10 oocysts for Toxoplasma and Cyclospora, and 5 eggs for Echinococcus per 30 g of raspberries or blueberries. In conclusion, evaluation of the present method showed that the newly developed multiplex qPCR is highly specific, precise, and robust method that has potential for application in food-testing laboratories.

Anoplocephala perfoliata Infection in Horses in Iceland: Investigation of Associations Between Intensity of Infection and Lesions.

In Iceland, there is at least 1 horse for every 5 human inhabitants, mostly kept on uncultivated rangelands. Although the Icelandic horse is considered robust compared with other breeds, it is nevertheless susceptible to disease. Few studies have investigated the prevalence of intestinal parasites in horses in Iceland. The purpose of this study was to determine the prevalence of the tapeworm, Anoplocephala perfoliata, in horses in Iceland and to explore associations between intensity of infection and the severity of macroscopic pathological lesions in this population. In addition, the relationship between infection and geographical location in Iceland, horse age, and gender were investigated. The utility of a modified McMaster flotation method for identifying infected horses from fecal samples was also studied. The study sample consisted of 104 horses (aged 1-30 yr) slaughtered in 3 abattoirs in North and South Iceland during June and July 2016. The prevalence of A. perfoliata in the horses was 64.4%, with between 1-131 worms found in infected horses. The pathological changes in the horses associated with infection varied from mild (hyperemia in small areas) to severe (large ulcers and necrotic lesions coated with fibrin), and intensity of infection correlated with the size and type of pathological lesions in the intestines. No statistically significant associations between infection with A. perfoliata and geographical location, horse age, or horse gender were detected. The McMaster egg counting method used here was of very low sensitivity (7.4%) and was concluded to be inadequate for detection of A. perfoliata infection in horses. The prevalence of A. perfoliata in horses in Iceland in this study was found to be relatively high compared with results from many studies performed elsewhere in Europe. This may reflect the lack of treatment of horses in Iceland for this parasite, the environmental suitability for the intermediate mite host, and grazing management practices in Iceland. The high prevalence and association with pathology suggest that diagnosis of infection using a sensitive method, along with appropriate treatment, should be considered for horses in Iceland.

Parasite contamination of berries: Risk, occurrence, and approaches for mitigation.

Fresh fruits and vegetables, including berries, are essential components of a healthy diet and are relevant in the prevention of chronic non-communicable diseases such as cancer and heart disease. Associations between diet and health are becoming an increasing focus of consumers, and, in response, consumption of fresh berries has been increasing rapidly in recent decades. However, increased consumption of berries may be associated with an increased risk of acquiring foodborne infections, including parasites. In this review, we describe how parasite contamination of berries may occur at several points on the farm-to-fork pathway, starting from the use of contaminated water for irrigation and pesticide application, and contact with animal and human faeces during cultivation, through contaminated harvesting equipment, and including unhygienic practices of berry pickers in the production field or others handling berries prior to consumption. Parasite transmission stages tend to be robust and therefore likely to survive from contamination in the field, through the various stages of harvesting, packaging, and sale, until consumption. We describe outbreaks of parasitic disease associated with consumption of berries - so far only described for Cyclospora and Trypanosoma cruzi, both of which are briefly introduced - but also show from survey data summarised in this review that sporadic infections or undetected outbreaks associated with contaminated berries may also occur. In addition, we describe methods for assessing whether berries are contaminated with parasite transmission stages, with emphasis on the challenges associated with analysing this particular matrix. Emphasis on current possibilities for mitigation and control are addressed; avoidance of contamination and implementation of good management practices and a hazard analysis and critical control points (HACCP) approach are essential.

Not just a walk in the park: Occurrence of intestinal parasites in dogs roaming recreational parks in Chandigarh, Northern India.

Chandigarh, a city in North-west India, has numerous parks and recreational areas where stray dogs roam freely and pet dogs are exercised. This allows for extensive human-dog interaction, which may pose a public health threat. The aim of this study was to determine the occurrence of gastrointestinal parasites of public health importance, and their seasonal variation, in canine faecal samples obtained from recreational parks in Chandigarh. A total of 212 samples were collected from 10 parks during the winter (January 2015; N = 107) and monsoon season (September 2015; N = 105), to assess the prevalence of intestinal zoonotic parasites and any seasonal variations. The samples were analysed for helminth eggs using McMaster counting chambers. Immunofluorescent antibody testing was used to analyse samples for Cryptosporidium oocysts and Giardia cysts. The Giardia-positive samples were genotyped by conventional multi-locus PCR to determine their assemblage and zoonotic potential. Among the 212 samples, strongyle-type eggs were found in 34 (16.0%), Toxocara spp. eggs were found in 6 (2.8%), taeniid eggs in 1 (0.5%), Cryptosporidium spp. oocysts in 4 (1.9%) and Giardia duodenalis cysts in 49 (23.1%). Trichuris eggs were not detected. The majority of the successfully amplified Giardia isolates belonged to canid-specific assemblages. The prevalence of Giardia cysts in faecal samples was significantly higher during winter than in the monsoon season, whereas helminth-egg prevalence unaffected by season. The prevalence of strongyle-type eggs and Giardia cysts in dog faeces was lower in more affluent areas of the city than those of less affluence. There was no significant difference in the intensity of infection between the seasons. The results indicate that faeces from dogs contaminating parks in Chandigarh do not usually contain parasite transmission stages that pose a significant risk to human health. However, the importance of minimising contamination of public parks with dog faeces is highlighted.

Use of lectin-magnetic separation (LMS) for detecting Toxoplasma gondii oocysts in environmental water samples.

Proof-of-principle of lectin-magnetic separation (LMS) for isolating Toxoplasma oocysts (pre-treated with 0.5% acidified pepsin (AP)) from water for subsequent detection by microscopy or molecular methods has been shown. However, application of this technique in the routine water-analysis laboratory requires that the method is tested, modified, and optimized. The current study describes attempts to apply the LMS technique on supernatants from water samples previously analyzed for contamination with Cryptosporidium and Giardia using standard methods, and the supernatant following immunomagnetic separation (IMS) retained. Experiments on AP-treatment of Toxoplasma oocysts in situ in such samples demonstrated that overnight incubation at 37 °C was adequate, but excess AP had to be removed before continuing to LMS; neutralization in sodium hydroxide and a single wash step was found to be suitable. Mucilaginous material in post-IMS samples that had been stored at room temperature without washing, which was found to be probably an exudate from bacterial and fungal overgrowth, hampered the isolation of T. gondii oocysts by LMS beads. For detection, microscopy was successful only for clean samples, as debris occluded viewing in dirtier samples. Although qPCR was successful, for some samples non-specific inhibition occurred, as demonstrated by inhibition of an internal amplification control in the qPCR reaction. For some, but not all, samples this could be addressed by dilution. Finally, the optimized methodology was used for a pilot project in which 23 post-IMS water sample concentrates were analyzed. Of these, only 20 provided interpretable results (without qPCR inhibition) of which one sample was positive, and confirmed by sequencing of PCR product, indicating that Toxoplasma oocysts occur in Norwegian drinking water samples. In conclusion, we suggest that post-IMS samples may be suitable for analysis for Toxoplasma oocysts using LMS, only if freshly processed or washed before being refrigerated. In addition, application of AP treatment requires a neutralization step before proceeding to LMS. For detection, qPCR, rather than microscopy, is the most appropriate approach, although some inhibition may still occur, and therefore inclusion of an internal amplification control is important. Our study indicates that, despite some limitations, this approach would be appropriate for further large-scale analysis of samples of raw and treated drinking water.

Occurrence of Giardia in Swedish Red Foxes ( Vulpes vulpes ).

Giardia duodenalis is an intestinal protozoan capable of causing gastrointestinal disease in a range of vertebrate hosts. It is transmitted via the fecal-oral route. Understanding the epidemiology of G. duodenalis in animals is important, both for public health and for the health of the animals it infects. We investigated the occurrence of G. duodenalis in wild Swedish red foxes ( Vulpes vulpes ), with the aim of providing preliminary information on how this abundant predator might be involved in the transmission and epidemiology of G. duodenalis . Fecal samples (n=104) were analysed for G. duodenalis using a commercially available direct immunofluorescent antibody test. Giardia duodenalis cysts were found in 44% (46/104) of samples, with foxes excreting 100 to 140,500 cysts per gram of feces (mean, 4,930; median, 600). Molecular analysis, using PCR with sequencing of PCR amplicons, was performed on 14 samples, all containing over 2,000 cysts per gram feces. Amplification only occurred in four samples at the tpi gene, sequencing of which revealed assemblage B in all four samples. This study provides baseline information on the role of red foxes in the transmission dynamics of G. duodenalis in Sweden.

Lectin-magnetic separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR.

Although standard methods for analyzing water samples for the protozoan parasites Cryptosporidium spp. and Giardia duodenalis are available and widely used, equivalent methods for analyzing water samples for Toxoplasma gondii oocysts are lacking. This is partly due to the lack of a readily available, reliable immunomagnetic separation technique (IMS). Here we investigated the use of lectin-magnetic separation (LMS) for isolating T. gondii oocysts from water sample concentrates, with subsequent detection by microscopy or molecular methods. Four different types of magnetic beads coated with wheat germ agglutinin (WGA) were tested for capture of oocysts from clean or dirty water samples. Dynabeads (Myone T1 and M-280) consistently provided mean capture efficiencies from 1 ml clean water in excess of 97%. High recoveries were also found with Tamavidin beads (in excess of 90%) when LMS was used for capture from a small (1 ml) volume. Dissociation (required for detection by microscopy) using 0.1N hydrochloric acid (HCl), as standard in IMS, was not successful, but could be achieved using a combination of acidified pepsin (AP) and N-acetyl d-glucosamine. Although simple centrifugation was as effective as LMS when concentrating high numbers of oocysts from clean water, LMS provided superior results when oocysts numbers were low or the water sample was dirty. Application of LMS integrated with qPCR enabled detection of 10 oocysts per 10 ml dirty water sample concentrate. These findings indicate that LMS with WGA coupled to magnetic beads could be an efficient isolation step in the analysis of water sample concentrates for T. gondii oocysts, with detection either by microscopy or by qPCR.

Occurrence of Giardia, Cryptosporidium, and Entamoeba in wild rhesus macaques (Macaca mulatta) living in urban and semi-rural North-West India.

Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp. are intestinal protozoa capable of infecting a range of host species, and are important causes of human morbidity and mortality. Understanding their epidemiology is important, both for public health and for the health of the animals they infect. This study investigated the occurrence of these protozoans in rhesus macaques (Macaca mulatta) in India, with the aim of providing preliminary information on the potential for transmission of these pathogens between macaques and humans. Faecal samples (n = 170) were collected from rhesus macaques from four districts of North-West India. Samples were analysed for Giardia/Cryptosporidium using a commercially available direct immunofluorescent antibody test after purification via immunomagnetic separation. Positive samples were characterised by sequencing of PCR products. Occurrence of Entamoeba was investigated first by using a genus-specific PCR, and positive samples further investigated via species-specific PCRs for Entamoeba coli, Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii. Giardia cysts were found in 31% of macaque samples, with all isolates belonging to Assemblage B. Cryptosporidium oocysts were found in 1 sample, however this sample did not result in amplification by PCR. Entamoeba spp. were found in 79% of samples, 49% of which were positive for E. coli. Multiplex PCR for E. histolytica, E. dispar and E. moshkovskii, did not result in amplification in any of the samples. Thus in 51% of the samples positive at the genus specific PCR, the Entamoeba species was not identified. This study provides baseline information on the potential for transmission of these zoonotic parasites at the wildlife-human interface.