Current and potential future impacts of food- and water-borne parasites in a changing world: A Norwegian perspective.

In 2021, the Norwegian Scientific Committee for Food and Environment published a multi-criteria risk ranking of 20 potentially food-borne pathogens in Norway. The pathogens ranked included five parasite taxa (3 species, one genus, one family): Toxoplasma gondii, Echinococcus multilocularis, Giardia duodenalis, Cryptosporidium spp., and Anisakidae. Two of these, T. gondii and E. multilocularis, scored very highly (1st and 3rd place, respectively), Cryptosporidium was about midway (9th place), and G. duodenalis and Anisakidae ranked relatively low (15th and 20th place, respectively). Parasites were found, on average, more likely to present an increasing food-borne disease burden in the future than the other pathogens. Here, we review the current impact of these five potentially food-borne parasites in Norway, and factors of potential importance in increasing their future food-borne disease burden. Climate change may affect the contamination of water and fresh produce with transmission stages of the first four parasites, potentially leading to increased infection risk. Alterations in host distribution (potentially due to climate change, but also other factors) may affect the occurrence and distribution of Toxoplasma, Echinococcus, and Anisakidae, and these, coupled with changes in food consumption patterns, could also affect infection likelihood. Transmission of food-borne pathogens is complex, and the relative importance of different pathogens is affected by many factors and will not remain static. Further investigation in, for example, ten-years' time, could provide a different picture of the relative importance of different pathogens. Nevertheless, there is clearly the potential for parasites to exert a greater risk to public health in Norway than currently occurs.

Benzimidazole-resistance associated mutation in Haemonchus contortus in Norwegian sheep, as detected by droplet digital PCR.

The aim of this study was to investigate the occurrence of benzimidazole-resistant Haemonchus contortus in Norwegian sheep flocks. Screening was based on detection of one of the resistance-conferring mutations in the ß tubulin isotype 1 gene (F200Y, TAC) in larvae (L3) cultivated from H. contortus eggs from naturally infected sheep. Faecal samples were collected in 2021/2022 from flocks in the northern (n = 34), central (n = 7), eastern (n = 40), southern (n = 1), and western (n = 87) areas of Norway. In total, samples were taken from 169 flocks (spring-ewes samples: 167, autumn-lambs samples: 134). Individual faecal samples were collected from 10 randomly selected ewes (spring) and 10 randomly selected lambs (autumn) in each flock. Faecal samples collected from each flock on each occasion were pooled (lamb and ewe samples pooled separately) and cultured for L3 development. After harvest of larvae (Baermann method), DNA was extracted and then analysed using droplet digital PCR with primer/probe sets targeting the BZ-associated F200Y (TAC) mutation. Haemonchus was found in 60% (80/134) of samples from lambs, and in 63% (106/167) from ewes. Among these, the F200Y mutation was detected in 73% (58/80) of larval samples from lambs and 69% (73/106) of larval samples from ewes, respectively. Although regional differences were evident, the mutation was detected in all areas indicating a widespread distribution and a strong potential for an increasing problem with treatment-resistant haemonchosis in Norwegian sheep flocks.

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR.

BACKGROUND: Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA. METHODS: We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106-1); (ii) diluted template DNA from pure oocysts (×10-×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105-1 and 5 × 104-50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10-×1000). RESULTS: Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations. CONCLUSIONS: For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.