Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10 −9 M, 10 −11 M, 10 −13 M), pyrantel citrate (10 −6 M, 10 −8 M, 10 −10 M), thiabendazole (10 −5 M, 10 −7 M, 10 −9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ − 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10 −9 M of ivermectin. Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.
Simple Summary: Abdominal pain, colic, is a common clinical sign in horses, sometimes reflecting life-threatening disease. One cause of colic is parasitic infection of the gut. Various drugs, anthelmintics, can be used to reduce or eliminate such parasites. However, frequent use has led to problems of drug resistance, whereby many countries now allow anthelmintics to be used on a prescription-only basis. In Sweden, this has led to a concern that parasitic-related colic in horses is increasing. This study aimed to investigate whether horses with colic differed in parasitological status compared to horses without colic. A secondary aim was to collect information regarding current parasite control measures used by horse owners. Exposure to S. vulgaris, a parasite with the potential to cause life-threatening disease, appeared high as determined by the presence of antibodies in the blood. Horses with inflammation in the abdominal cavity had higher antibody levels than other causes of colic. Despite new legislation, 29% of owners did not use fecal analyses for parasites and the use of extended methods to diagnose specific parasites was low. Also, owners rarely used alternative methods to reduce the pasture parasite burden. The study suggests a need for education in the use of both fecal analyses and pasture management.Abstract: All grazing horses are exposed to intestinal parasites, which have the potential to cause gastrointestinal disease. In Sweden, there is a concern about an increase in parasite-related equine gastrointestinal disease, in particular Strongylus vulgaris, since the implementation of prescription-only anthelmintics approximately 10 years ago. In a prospective case-control study, parasitological status, using fecal analyses for strongyle egg counts, the presence of Anoplocephala perfoliata eggs and S. vulgaris Polymerase chain reaction (PCR) as well as serology for S. vulgaris, were compared between horses presenting with or without gastrointestinal disease at a University hospital during a one-year period. Information regarding anthelmintic routines and pasture management was gathered with an owner-filled questionnaire. Although the prevalence of S. vulgaris PCR was 5.5%, 62% of horses were positive in the enzyme-linked immunosorbent assay (ELISA) test and horses with peritonitis showed higher antibody levels for S. vulgaris, as compared to other diagnoses or controls. Overall, 36% of the horse owners used only fecal egg counts (FEC), 32% used FEC combined with specific diagnostics for S. vulgaris or A. perfoliata, and 29% dewormed routinely without prior parasite diagnostics. Effective management methods to reduce the parasitic burden on pastures were rare and considering exposure to S. vulgaris appears high; the study indicates a need for education in specific fecal diagnostics and pasture management.
P-glycoprotein (P-gp) is an important drug transporter, which is expressed in a variety of cells, such as the intestinal enterocytes, the hepatocytes, the renal tubular cells and the intestinal and peripheral blood lymphocytes. We have studied the localization and the gene and protein expression of P-gp in these cells in horse. In addition we have compared the protein sequence of P-gp in horse with the protein sequences of P-gp in several other species. Real time RT-PCR and Western blot showed gene and protein expression of horse P-gp in all parts of the intestines, but there was no strict correlation between these parameters. Immunohistochemistry showed localization of P-gp in the apical cell membranes of the enterocytes and, in addition, staining was observed in the intestinal intraepithelial and lamina propria lymphocytes. Peripheral blood lymphocytes also stained for P-gp, and gene and protein expression of P-gp were observed in these cells. There was a high gene and protein expression of P-gp in the liver, with P-gp-immunoreactivity in the bile canalicular membranes of the hepatocytes. Gene and protein expression of P-gp were found in the kidney with localization of the protein in different parts of the nephrons. Protein sequence alignment showed that horse P-gp has two amino acid insertions at the N-terminal region of the protein, which are not present in several other species examined. One of these is a 99 amino acid long sequence inserted at amino acid positions 23-121 from the N-terminal. The other is a six amino acid long sequence present at the amino acid positions 140-145 from the N-terminal. The results of the present study indicate that P-gp has an important function for oral bioavailability, distribution and excretion of substrate compounds in horse.
Susceptability of Ascaridia galli to benzimidazole (BZ) was investigated using faecal egg count reduction test (FECRT), in ovo larval development test (LDT) and genetic markers (mutations at codons 167, 198 and 200 of β-tubulin gene). Six flocks (F1-F6) of a commercial laying hen farm with different number of exposure to BZ were recruited. The FECR was calculated by analyzing individual faeces (F1, F2, F4 and F5) before and 10 days after treatment. The LDT was performed on parasite eggs from pooled samples from F1 to F6 and LC50 and LC99 were calculated. DNA was extracted from 120 worms and sequenced for β-tubulin gene. In all flocks, the FECRs were above 95% (lower CI above 90%). No significant difference was observed (p > 0·05) among obtained LC50 (F1/F4 and F2/F5 vs F3/F6) in the LDT. However, LC50 and LC99 were higher than suggested values for declaration of resistance in other nematode species. No variation was observed in codon positions involved in BZ resistance. Overall, our results indicated lack of evidence of resistance to BZ in A. galli. More research is needed to confirm these results and to further optimize the existing tools for detection and monitoring of anthelmintic resistance in A. galli.
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