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.

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.

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.

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.

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.

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.

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.

Establishment of Canine-Derived Giardia duodenalis Isolates in Culture.

Researchers continue to rely on axenic cultivation of Giardia duodenalis trophozoites in vitro to study the life cycle and host-parasite interactions of G. duodenalis and to develop vaccines and drugs to prevent and treat giardiasis. The majority of in vitro studies of G. duodenalis have used a small subset of isolates, mostly of assemblage A, and these isolates are usually originally isolated from humans. The most commonly used isolate for lab studies is known as WB. Canine giardiasis is a disease of veterinary importance, but it may also be of relevance in zoonotic transmission. Few G. duodenalis isolates from dogs have been adapted to in vitro culture, probably because the methods used are not suitable for the canine-specific genotypes that tend to dominate in most dog populations. In the current study, an experimental approach to cultivating canine-derived isolates of G. duodenalis was attempted by modification of the standard protocol based on physiological differences between the human and canine digestive system. An adapted method is described for improving the rate of in vitro excystation of cysts isolated from dogs by chemically weakening the cyst wall. A new canine-derived assemblage A G. duodenalis isolate was successfully adapted to axenic culture by using this method; the dog apparently had a mixed infection of assemblages A and D, but the assemblage A successfully outcompeted the assemblage D under conditions of in vitro culture. Based on the results, reasons regarding why humans do not seem to be suitable hosts for G. duodenalis in assemblages C and D are discussed.

Investigation of effects of Giardia duodenalis on transcellular and paracellular transport in enterocytes using in vitro Ussing chamber experiments.

The mechanisms by which different genotypes of Giardia duodenalis result in different symptoms remain unresolved. In particular, we lack detailed knowledge on which transport mechanisms (transcellular or paracellular) are affected by different Giardia isolates. Using horse radish peroxidase (HRP) and creatinine as transcellular and paracellular probes, respectively, we developed a robust assay that can be used with an Ussing chamber to investigate epithelial transport, as well as short-circuit current as an indicator of net ion transport. We investigated 2 Giardia isolates, both Assemblage A, one a lab-adapted strain and the other a field isolate. Results indicate that products from sonicated Giardia trophozoites increase both transcellular and paracellular transport. A non-significant increase in transepithelial electrical resistance (TEER) and short-circuit current were also noted. The paracellular transport was increased significantly more in the field isolate than in the lab-adapted strain. Our results indicate that while both transcellular and paracellular transport mechanisms may be increased following exposure of cells to Giardia trophozoite sonicate, perhaps by inducing non-specific increases in cellular traffic, it is important that in vitro studies of Giardia pathophysiology are conducted with different Giardia isolates, not just lab-attenuated strains.

Preliminary experiments on use of zebrafish as a laboratory model for Giardia duodenalis infection.

Although Giardia duodenalis is considered a parasite of mammals, different genotypes have been identified as infecting several species of freshwater and marine fish in Australia. Establishment of G. duodenalis infection in common laboratory zebrafish (Danio rerio), could provide an excellent tool for a range of studies on Giardia. We conducted preliminary experiments to investigate this possibility. Zebrafish were inoculated with viable G. duodenalis cysts from two different Assemblages (A and D) using a modified oro-gastric tube. Direct microscopy and immunofluorescent antibody test were used to check for Giardia cysts/trophozoites in the intestine, and histology was performed on intestinal mucosa to evaluate possible pathological changes. Giardia cysts were successfully deposited in the zebrafish alimentary tract using a modified oro-gastric tube, and were maintained in the fish gut for at least 8 days. Although a single trophozoite was observed in one fish three days post-exposure, we were unable to demonstrate established, propagative infection under the conditions tested.