Kristoffer Relling Tysnes

A cross-sectional study of Tritrichomonas foetus infection among healthy cats at shows in Norway.

BackgroundIn recent years, the protozoan Tritrichomonas foetus has been recognised as an important cause of chronic large-bowel diarrhoea in purebred cats in many countries, including Norway. The aim of this cross-sectional study was to determine the proportion of animals with T. foetus infection among clinically healthy cats in Norway and to assess different risk factors for T. foetus infection, such as age, sex, former history of gastrointestinal symptoms and concurrent infections with Giardia duodenalis and Cryptosporidium sp.MethodsThe sample population consisted of 52 cats participating in three cat shows in Norway in 2009. Samples were examined for motile T. foetus by microscopy, after culturing and for T. foetus-DNA by species-specific nested PCR, as well as for Giardia cysts and Cryptosporidium oocysts by immunofluorescent antibody test (IFAT).ResultsBy PCR, T. foetus-DNA was demonstrated in the faeces of 11 (21%) of the 52 cats tested. DNA-sequencing of five positive samples yielded 100% identity with previous isolates of T. foetus from cats. Only one sample was positive for T. foetus by microscopy. By IFAT, four samples were positive for Giardia cysts and one for Cryptosporidium oocysts, none of which was co-infected with T. foetus. No significant associations were found between the presence of T. foetus and the various risk factors examined.ConclusionsT. foetus was found to be a common parasite in clinically healthy cats in Norway.

Subclinical Giardia in dogs: a veterinary conundrum relevant to human infection.

Human infection with Giardia duodenalis tends to be associated with diarrheal disease requiring treatment - despite our awareness that often it is asymptomatic and sometimes, perhaps, even protective. We discuss here whether canine giardiasis can serve as a model to help to understand why Giardia is pathogenic. We discuss factors that should be considered when Giardia is identified in dogs, challenging the assumption that infection necessarily means disease that requires chemotherapeutic treatment. To make the best treatment decision for canine Giardia infection we need to think about zoonotic risks, transmission possibilities, and risk factors for disease development. In addition, in both humans and dogs, Giardia sometimes may be considered as a harmless passenger, or even as a beneficial friend.

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.

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.

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.

Establishment of Canine-Derived Giardia duodenalis Isolates in Culture

Abstract 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.

Treatment of feline giardiasis during an outbreak of diarrhoea in a cattery: potential effects on faecal Escherichia coli resistance patterns

Objectives An outbreak of diarrhoea involving 16 cats at a cattery in Norway was investigated. Treatment and control of the outbreak were the primary objectives, but the effects of treatment on the antimicrobial resistance profiles of Escherichia coli isolated from faeces were also investigated. Methods Faecal samples were investigated for Giardia cysts by immunofluorescence microscopy, and multi-locus genotyping was performed to determine the Giardia genotype. Faecal E coli were assessed, before and after treatment for giardiasis, for antimicrobial resistance. Results The outbreak was probably caused by Giardia duodenalis, Assemblage F. Although infection was eliminated in most cats following treatment with fenbendazole, over 30% of the infected cats required a second treatment round (combined fenbendazole and metronidazole). Investigation of sensitivity to antibacterial drugs of E coli that had been isolated both prior to and following treatment demonstrated that fenbendazole treatment may select for resistant bacteria. Conclusions and relevance Controlling Giardia infections in dense cat populations can be challenging, and requires strict hygiene measures. In cases where fenbendazole alone does not result in treatment success, a combination treatment with fenbendazole and metronidazole may be effective. Although this study did not include untreated controls, we suggest that the potential for changes in gut microbiota and antimicrobial resistance development should be considered when choosing antiprotozoal drugs, particularly in cases of treatment failure and where repeat treatment is required.

Giardia duodenalis in primates: Classification and host specificity based on phylogenetic analysis of sequence data

Giardia duodenalis colonizes the gastrointestinal tract of a wide range of hosts, including humans and other primates. It is grouped into eight different Assemblages and, beyond that, into a number of sub‐Assemblages, defined ad hoc on the basis of genetic differences; these various groups are often considered to be associated with a specific restricted host range. The aim of this study was to use publicly available genotyping data to investigate the relatedness of human and non‐human primate (NHP) Giardia isolates in order to evaluate the usefulness of current taxonomic classification and to assess whether there is potential for zoonotic transmission between humans and NHP. Our final data set consisted of sequence data from 165 isolates, 111 from NHP and 54 from humans. Assemblages were well defined, but sub‐Assemblages across Assemblage B were not resolved. Although sub‐Assemblages AI and AII were resolved, the terms were not found to capture any useful molecular or host/deme properties. In the phylogenetic tree, NHP isolates were scattered among human isolates across Assemblages A and B, and were even found in Assemblage E. We conclude that there does not appear to be significant molecular distinction between human and NHP Giardia isolates across these four molecular markers. Thus, on the basis of these markers, we cannot exclude a risk for zoonotic and anthropozoonotic transmission of Assemblages A and B isolates, irrespective of sub‐Assemblage classification. We further evaluated the relative merit of the four genes used in genotyping studies. The tpi, gdh and bg genes gave relatively congruent tree topologies, but the SSU gene did not resolve Assemblages according to the current classification. Future genotyping efforts should aim for multilocus or whole‐genome approaches and, in particular, use of the SSU gene as the sole marker should be avoided when possible.

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.

Occurrence of Giardia in Swedish Red Foxes (Vulpes vulpes)

Abstract 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.