From field to genetics : anthelmintic resistance in the equine roundworm Parascaris univalens

The equine roundworm Parascaris univalens is a common parasite of foals. Most foals show mild clinical symptoms, but large worm burdens can lead to severe colic and even death. Regular treatment with anthelmintic drugs has resulted in resistance development, mainly to ivermectin but also to pyrantel and fenbendazole in sporadic cases. In Sweden, resistance to ivermectin is considered to be widespread. The aim of the thesis was to examine the efficacy of anthelmintic drugs on stud farms in Sweden and Iceland, develop novel models for research and study genetic mechanisms potentially involved in drug metabolism and anthelmintic resistance. Faecal egg count reduction tests showed that resistance to both pyrantel and fenbendazole has emerged on Swedish stud farms, and that ivermectin resistance is common on Icelandic farms. Due to the potentially lethal consequences of infection, this is a serious situation. We developed a novel method to hatch P. univalens eggs in order to use larvae in in vitro experiments to study resistance mechanisms. Quantitative PCR, RNA sequencing and amplicon sequencing were used to study genetic and transcriptomic mechanisms behind anthelmintic resistance in P. univalens. Several genes coding for drug metabolising enzymes, transport proteins and a possible drug target for ivermectin were found to be differentially expressed in P. univalens after exposure to anthelmintic drugs. However, mutations in β-tubulin genes responsible for benzimidazole resistance in many other parasitic nematodes were not present in a fenbendazole-resistant P. univalens population. In conclusion, the current level of resistance in P. univalens has been updated in this thesis, a novel research method has been developed and novel candidate genes for future research have been identified

Osmotic pressure: resisting or promoting DNA ejection from phage

Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I on the course of ejection. We argue in this work by combination of experimental techniques (osmotic suppression without DNaseI monitored by UV absorbance, pulse-field electrophoresis, and cryo-EM visualization) and simple scaling modeling that intact genome (i.e. undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resisting it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed

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

Gene co-expression network analysis reveal core responsive genes in Parascaris univalens tissues following ivermectin exposure

Anthelmintic resistance in equine parasite Parascaris univalens, compromises ivermectin (IVM) effectiveness and necessitates an in-depth understanding of its resistance mechanisms. Most research, primarily focused on holistic gene expression analyses, may overlook vital tissue-specific responses and often limit the scope of novel genes. This study leveraged gene co-expression network analysis to elucidate tissue-specific transcriptional responses and to identify core genes implicated in the IVM response in P. univalens. Adult worms (n = 28) were exposed to 10(-11) M and 10(-9) M IVM in vitro for 24 hours. RNA-sequencing examined transcriptional changes in the anterior end and intestine. Differential expression analysis revealed pronounced tissue differences, with the intestine exhibiting substantially more IVM-induced transcriptional activity. Gene co-expression network analysis identified seven modules significantly associated with the response to IVM. Within these, 219 core genes were detected, largely expressed in the intestinal tissue and spanning diverse biological processes with unspecific patterns. After 10(-11) M IVM, intestinal tissue core genes showed transcriptional suppression, cell cycle inhibition, and ribosomal alterations. Interestingly, genes PgR028_g047 (sorb-1), PgB01_g200 (gmap-1) and PgR046_g017 (col-37 & col-102) switched from downregulation at 10(-11 )M to upregulation at 10(-9 )M IVM. The 10(-9 )M concentration induced expression of cuticle and membrane integrity core genes in the intestinal tissue. No clear core gene patterns were visible in the anterior end after 10(-11) M IVM. However, after 10(-9) M IVM, the anterior end mostly displayed downregulation, indicating disrupted transcriptional regulation. One interesting finding was the non-modular calcium-signaling gene, PgR047_g066 (gegf-1), which uniquely connected 71 genes across four modules. These genes were enriched for transmembrane signaling activity, suggesting that PgR047_g066 (gegf-1) could have a key signaling role. By unveiling tissue-specific expression patterns and highlighting biological processes through unbiased core gene detection, this study reveals intricate IVM responses in P. univalens. These findings suggest alternative drug uptake of IVM and can guide functional validations to further IVM resistance mechanism understanding

Exploring the beta-tubulin gene family in a benzimidazole-resistant Parascaris univalens population

Benzimidazole (BZ) drugs are frequently used to treat infections with the equine ascarid Parascaris univalens due to increasing resistance to macrocyclic lactones and pyrantel. Benzimidazole resistance is rare in ascarids in contrast to strongyle parasites where this resistance is widespread. In strongyles, single nucleotide polymorphisms (SNPs) at codons 167, 198 and 200 in a 13-tubulin gene have been correlated to BZ resistance, but little is known about the 13-tubulin genes and their possible involvement in BZ resistance in P. univalens and other ascarids. Previously two 13-tubulin genes have been identified in P. univalens. In this study, we present five additional 13-tubulin genes as well as the phylogenetic relationship of all seven genes to 13-tubulins of other clade III and V nematodes. In addition, the efficacy of fenbendazole for treatment of P. univalens on a Swedish stud farm was studied in 2019 and 2020 using faecal egg count reduction test. Reductions varied from 73% to 88%, indicating the presence of a resistant P. univalens population on the farm. The emergence of BZ resistance emphasizes the need for development of molecular markers for rapid and more sensitive detection of resistant populations. We therefore investigated whether possible SNPs at positions 167, 198 or 200 in any of the 13-tubulin genes could be used to distinguish between resistant and susceptible P. univalens populations. Amplicon sequencing covering the mutation sites 167, 198 and 200 in all seven 13-tubulin genes revealed an absence of SNPs in both resistant and susceptible populations, suggesting that the mechanism behind BZ resistance in ascarids is different from that in strongyle nematodes and the search for a molecular marker for BZ resistance in P. univalens needs to continue

Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa

Kungsbackafjorden (Västerhavet) 2011

Totalt fångades 22 arter av fisk och sju arter av kräftdjur under provfisket i Kungsbackafjorden i augusti 2011. De vanligaste fiskarterna i fångsten var ål, skärsnultra och stensnultra. Strandkrabban var den vanligaste kräftdjursarten i fångsten. • Antalet arter i fångsten och den totala medelfångsten av fisk i Kungsbackafjorden skiljer sig inte nämnvärt från fångster i övriga områden längs med västkusten som provfiskades med samma metodik under 2011. • Medelfångsten av rovfisk i Kungsbackafjorden är något lägre än i jämförda områden. Detta beror främst på lägre fångster av torskfiskar (torsk, vitling och gråsej) vilket kan vara en effekt av förekomsten av sötare vatten, där torskfiskarna inte trivs, i delar av fjorden. Fångsten av rovfisk är dock överlag låg i samtliga områden. • Medelfångsten av mesopredatorer i Kungsbackafjorden skiljer sig inte mot jämförda områden. De vanligaste mesopredatorerna i Kungsbackafjorden är skärsnultra, stensnultra, skrubbskädda, svart smörbult och tånglake. • Stora fiskar (≥ 30 cm) är, bortsett från ål (gulål), ovanliga i fångsterna från samtliga provfiskade områden 2011. Medelfångsten av större torskar och skrubbskäddor skiljer sig heller inte nämnvärt i jämförelse med övriga områden. • Fisksamhället i Kungsbackafjorden är mångfasetterat, dels på grund av att arternas förekomst skiljer sig i djupled beroende på varmare och kallare vatten men även på grund av att det finns en tydlig gradient i salthalt från inre till yttre delarna av fjorden som påverkar artsammansättningen. • Torskfiskarna (torsk, sej, vitling och lyrtorsk) förekommer i högre antal i fjordens yttre och djupare delar där salthalten är högre medan de limniska arterna (abborre, id, mört och löja) förekommer längre in i fjorden där salthalten är lägre. Förekomsten av skärsnultra och stensnultra, två arter med likartade livsstrategier, skiljer sig åt i fjorden med mer skärsnultra i de västra delarna av områdets yttre del medan stensnultran förkommer i större antal i den östra delen men också lite mer skyddat och längre in i fjorde

Demonstration of reduced efficacy against cyathostomins without change in species composition after pyrantel embonate treatment in Swedish equine establishments

Consisting of approximately 50 different species, the cyathostomin parasites are ubiquitous in grazing horses. Coinfection with several species is common, and large burdens can cause the fatal disease of larval cyathostominosis. Due to intense anthelmintic drug use, cyathostomin resistance has developed to all available anthelmintic drug groups. Resistance to the anthelmintic drug pyrantel (PYR) has been documented in over 90% of studies published over the past two decades. In Sweden, a study performed in the early 2000s only confirmed resistance in 4.5% of farms. Further, prescription-only administration of equine anthelmintic drugs was enforced in Sweden in 2007. However, it is unknown if this conservative drug use has maintained PYR efficacy in cyathostomins. The aim of the present study was to investigate the effect of PYR on cyathostomin infection in Sweden using fecal egg count reduction tests (FECRTs). Further, the effect of PYR treatment on cyathostomin species composition was studied using metabarcoding. Sixteen farms with at least six horses excreting a minimum of 100 eggs per gram feces were included. Using the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines, PYR resistance was demonstrated in nine of farms, with seven farms showing full susceptibility. Farms with low biosecurity measures had significantly lower efficacy of PYR treatment. The most common cyathostomin species were Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cys. calicatus, Cys. goldi, Cys. minutus, Coronocyclus coronatus and Cya. pateratum, accounting for 97% of all sequence reads prior to treatment. Of these, Cyc. nassatus and Cya. catinatum had the highest occurrence, accounting for 68% of all sequence reads prior to PYR treatment. Treatment did not significantly affect the species composition. The results highlight the importance of drug efficacy testing when using PYR to treat cyathostomin infection, even when selective anthelmintic treatment and thus low treatment intensity, is used on the farm

Experimental study on the thermal plume from a surgeon in an operating room with mixing ventilation during COVID-19 pandemic

Following the outbreak of COVID-19 (SARS-CoV-2) in 2019, studies show positive results in protecting the surgical staff from patients infected by COVID-19 in operating rooms (ORs) with negative pressure. A negative pressure environment inside the operating room (OR) reduces the virus's circulation outside the OR (Chen et al., 2020). Nevertheless, it is unclear whether the surgeon's thermal plume can impact the transport of contaminants up to the breathing zone and thus cause infection in ORs with various pressure differences compared to adjacent rooms. The results show that a gap between the surgical manikin and the operating table greatly affects the development of the thermal plume from the head surgeon. A plate between the surgical manikin and the operating table may significantly influence the airflow distribution in front of the head surgeon more than the pressure difference inside the operating room.publishedVersio