Constitutive and differential expression of transport protein genes in Parascaris univalens larvae and adult tissues after in vitro exposure to anthelmintic drugs

The equine roundworm Parascaris univalens has developed resistance to the three anthelmintic substances most commonly used in horses. The mechanisms responsible for resistance are believed to be multi-genic, and transport proteins such as the P-glycopmtein (Pgp) family have been suggested to be involved in resistance in several parasites including P. univlaens. To facilitate further research into the mechanisms behind drug metabolism and resistance development in P. univalens we aimed to develop an in vitro model based on larvae. We developed a fast and easy protocol for hatching P. univalens larvae for in vitro studies, resulting in a hatching rate of 92 %. The expression of transport protein genes pgp-2, pgp-9, pgp-11.1, pgp-16.1 and major facilitator superfamily (MFS) genes PgR006_g137 and PgR015_g078 were studied in hatched larvae exposed to the anthelmintic drugs ivermecin (IVM) 10(-9) M, pyrantel citrate (PYR) 10(-6) M and thiabendazole (TBZ) 10(-5) M for 24 h. In comparison, the expression of these transport protein genes was studied in the anterior end and intestinal tissues of adult worms in vitro exposed to IVM, TBZ and PYR, at the same concentrations as larvae, for 3 h, 10 h and 24 h. Larval exposure to sub-lethal doses of IVM for 24 h did not affect the expression levels of any of the investigated genes, however larvae exposed to PYR and TBZ for 24 h showed significantly increased expression of pgp-9. In vitro drug exposure of adult worms did not result in any significant increases in expression of transport protein genes. Comparisons of constitutive expression between larvae and adult worm tissues showed that pgp-9, pgp11.1, pgp-16.1 and MFS gene PgR015_g078 were expressed at lower levels in larvae than in adult tissues, while pgp-2 and MFS gene PgR006_g137 had similar expression levels in larvae and adult worms. All investigated transport protein genes were expressed at higher rates in the intestine than in the anterior end of adult worms, except pgp-11.1 where the expression was similar between the two tissues. This high constitutive expression in the intestine suggests that this is an important site for xenobiotic efflux in P. univalens. Despite the fact that the results of this study show differences in expression of transport protein genes between larvae and adult tissues, we believe that the larval assay system described here will be an important tool for further research into the molecular mechanisms behind anthelmintic resistance development and for other in vitro studies

Molecular identification and transmission studies of X-cell parasites from Atlantic cod Gadus morhua (Gadiformes: Gadidae) and the northern black flounder Pseudopleuronectes obscurus (Pleuronectiformes: Pleuronectidae)

Background: Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata. Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan. Results: Phylogenetic analyses of small subunit ribosomal DNA (SSU rDNA) sequence data from Atlantic cod X-cells show that they are a related parasite that occupies a basal position to the clade containing other X-cell parasites. The X-cell parasite causing epidermal pseudotumours in P. obscurus is the same parasite that causes pseudotumours in H. dubius. Direct, fish to fish, transmission of the X-cell parasites used in this study, via oral feeding or injection, was not achieved. Non-amoeboid X-cells are contained within discrete sac-like structures that are loosely attached to epidermal pseudotumours in flatfish; these X-cells are able to tolerate exposure to seawater. A sensitive nested PCR assay was developed for the sub clinical detection of both parasites and to assist in future life cycle studies. PCR revealed that the parasite in P. obscurus was detectable in non-pseudotumourous areas of fish that had pseudotumours present in other areas of the body. Conclusions: The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites. Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum. Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish

Surveillance of Ixodes ricinus ticks (Acari: Ixodidae) in Iceland

Background: Ixodes ricinus is a three-host tick, a principal vector of Borrelia burgdorferi (s.l.) and one of the main vectors of tick-borne encephalitis (TBE) virus. Iceland is located in the North Atlantic Ocean with subpolar oceanic climate. During the past 3–4 decades, average temperature has increased, supporting more favourable conditions for ticks. Reports of I. ricinus have increased in recent years. If these ticks were able to establish in a changing climate, Iceland may face new threats posed by tick-borne diseases. Methods: Active field surveillance by tick flagging was conducted at 111 sites around Iceland from August 2015 to September 2016. Longworth mammal traps were used to trap Apodemus sylvaticus in southwestern and southern Iceland. Surveillance on tick importation by migratory birds was conducted in southeastern Iceland, using bird nets and a Heligoland trap. Vulpes lagopus carcasses from all regions of the country were inspected for ticks. In addition, existing and new passive surveillance data from two institutes have been merged and are presented. Continental probability of presence models were produced. Boosted Regression Trees spatial modelling methods and its predictions were assessed against reported presence. Results: By field sampling 26 questing I. ricinus ticks (7 males, 3 females and 16 nymphs) were collected from vegetation from three locations in southern and southeastern Iceland. Four ticks were found on migratory birds at their arrival in May 2016. A total of 52 A. sylvaticus were live-trapped but no ticks were found nor on 315 V. lagopus carcasses. Passive surveillance data collected since 1976, reports further 214 I. ricinus ticks from 202 records, with an increase of submissions in recent years. The continental probability of presence model correctly predicts approximately 75% of the recorded presences, but fails to predict a fairly specific category of recorded presence in areas where the records are probably opportunistic and not likely to lead to establishment. Conclusions: To the best of our knowledge, this study represents the first finding of questing I. ricinus ticks in Iceland. The species could possibly be established locally in Iceland in low abundance, although no questing larvae have yet been detected to confirm established populations. Submitted tick records have increased recently, which may reflect an increase in exposure, or in interest in ticks. Furthermore, the amount of records on dogs, cats and humans indicate that ticks were acquired locally, presenting a local biting risk. Tick findings on migratory birds highlight a possible route of importation. Obtaining questing larvae is now a priority to confirm that I. ricinus populations are established in Iceland. Further surveys on wild mammals (e.g. Rangifer tarandus), livestock and migratory birds are recommended to better understand their role as potential hosts for I. ricinus.Work was carried out under VectorNet, a European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents (framework contract OC/EFSA/AHAW/2013/02-FWC1) funded by the European Food Safety Authority (EFSA) and the European Centre for Disease prevention and Control (ECDC). JM is also partly funded by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Environmental Change and Health at the London School of Hygiene & Tropical Medicine in partnership with Public Health England (PHE), and in collaboration with the University of Exeter, University College London, and the Met Office; and partly funded by the NIHR HPRU on Emerging Infections and Zoonoses at the University of Liverpool in partnership with PHE and Liverpool School of Tropical Medicine.Peer Reviewe

Endoparasites in humans in Iceland found in studies during 1973-1988

Neðst á síðunni er hægt að nálgast greinina í heild sinni með því að smella á hlekkinn Skoða/Opna(view/open)During 1973-1988 altogether 3922 samples from 2528 individuals suspected of carrying endoparasites were examined at the Institute for Experimental Pathology at Keldur, University of Iceland (Figures 1 and 2). Approximately 99% of the samples were faecal samples. In these samples at least 24 species of endoparasites were found (Table). Altogether 234 patients (9.3%) were found to harbour one or more species of endoparasites.Meðferð sjúklinga með kransæðastíflu er eitt af meginverkefnum lyflækningadeilda með bráðaþjónustu. Lyflækningadeildir þriggja stærstu sjúkrahúsa landsins hafa á undanförnum árum látið frá sér fara greinargerðir um kransæðasjúklinga, sem þar hafa vistast (1-7). Tilgangur þessarar könnunar var að athuga hvernig staðið hefði verið að greiningu, rannsóknum og meðferð sjúklinga með bráða kransæðastíflu sem vistuðust á lyflækningadeild FSA á árunum 1984-1986. Einnig voru kannaðir helstu áhættuþættir og fylgikvillar og hvaða áhrif þeir hefðu á afdrif sjúklinganna, en þeim var fylgt eftir til 1. desember 198

First Report of Resistance to Ivermectin in Parascaris univalens in Iceland

Horses in Iceland have been isolated for more than 1,000 yr but still harbor a similar range of gastrointestinal parasites as do horses across the world. The long isolation of the horses and their parasites presumably means that no resistance genes have been introduced into the Parascaris spp. population. It is therefore of particular interest to investigate the efficacy of ivermectin on Parascaris spp. infecting Icelandic foals. Potential treatment failure of ivermectin in Iceland will add substantial new information on how resistance can arise independently. This study aimed to determine the efficacy of subcutaneous injection of ivermectin for the treatment of Parascaris spp. infection in foals and to identify the Parascaris species present in the west and north of Iceland. A fecal egg count reduction (FECR) test (FECRT) was performed on 50 foals from 8 farms, including an untreated control group of 6 foals, from September to November 2019. The foals were between 3 and 5 mo of age at the start of the study and had not previously been treated with anthelmintic drugs. Each foal was treated subcutaneously with off-label use of Ivomec (R) injection 10 mg/ml or Noromectin (R) 1% at a dose of 0.2 mg/kg. The FECR for each farm was calculated in 2 ways, by the eggCounts package in R and by the Presidente formula (FECRT). Both calculation methods resulted in efficacy levels between 0% and 80.78%, indicating ivermectin resistance on all farms. We also confirmed, by karyotyping, that the species of equine ascarid present in the west and north of Iceland is Parascaris univalens. This study provides evidence for treatment failure of ivermectin against P. univalens infection in foals. Since Icelandic horses have been isolated on the island for more than 1,000 yr, this implies that resistance alleles have developed independently in the Icelandic Parascaris population. The actual clinical impact of ivermectin resistance is unknown but another drug of choice should be considered to treat Parascaris infection in foals in Iceland