The complete ITS2 barcoding region for Strongylus vulgaris and Strongylus edentatus

Gastrointestinal nematode parasites are of major concern for horses, where Strongylus vulgaris is considered the most pathogenic among the Strongylus species. Diagnosis of S. vulgaris infections can be determined with next generation sequencing techniques, which are inherently dependent on reference sequences. The best marker for parasitic nematodes is internal transcribed spacer 2 (ITS2) and we provide the first complete ITS2 sequences from five morphologically identified S. vulgaris and additional sequences from two S. edentatus. These sequences have high similarity to already published partial sequences and amplicon sequence variants (ASV) based on next generation sequencing (NGS). The ITS2 sequences from S. vulgaris matched available partial ITS2 sequences and the full ASVs, whereas the S. edentatus sequence matched another complete sequence. We also compare Sanger sequencing and NGS methods and conclude that the ITS2 variation is better represented with NGS methods. Based on this, we recommend that further sequencing of morphologically identified specimens of various species should be performed with NGS cover the intraspecific variation in the ITS2

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

Evaluation of Strategies to Reduce Equine Strongyle Infective Larvae on Pasture and Study of Larval Migration and Overwintering in a Nordic Climate

Simple Summary All grazing horses are exposed to parasites that when ingested have the potential to cause disease. Drugs designed to kill parasites in horses have been used extensively since the 1960s, but this intensive use has led to the development of drug resistance, emphasising the need for more sustainable methods to lessen parasite burdens. The efficacy and suitability of pasture-management methods aiming to reduce the level of parasitic larvae in the herbage are, however, dependent on the prevailing weather conditions. The aim of the present study was therefore to generate data on the effect of regular faecal removal and harrowing of the pasture on reducing the number of parasites in herbage in a Nordic climate. Furthermore, the ability of parasite larvae to migrate from faeces to the herbage and to survive the winter season in Sweden's cold temperate climate was investigated. Twice-weekly faecal removal significantly reduced the number of larvae on the pasture, whereas harrowing on a single occasion in the summer under dry weather conditions did not. Parasite larvae migrated as far as 150 cm from faecal pats and were able to survive one winter season, with two years of rest from grazing horses required to achieve parasite-free pasture. Horses, as grazing animals, are inadvertently exposed to intestinal parasites that, if not controlled, may cause disease. However, the indiscriminate use of anthelmintic drugs has led to drug resistance, highlighting the need for pasture-management practices to reduce the level of parasitic exposure and lessen reliance on drugs. The efficacy of such methods depends both on the epidemiology of the parasites and the prevailing weather conditions. The aim of the study was to investigate the effect of faecal removal and harrowing on reducing the number of parasite larvae in herbage. Moreover, the migratory and survival ability of strongyle larvae in a Nordic climate was studied. Faeces from horses naturally infected with strongyle nematodes were used to contaminate pastures and grass samples were collected to harvest larvae. Twice-weekly faecal removal significantly reduced larval yields, whereas harrowing on a single occasion under dry weather conditions in the summer did not. Strongyle larvae were able to migrate 150 cm from the faecal pats, but most larvae were found within 50 cm. Both Cyathostominae and S. vulgaris survived the winter months with larvae harvested up to 17-18 months after faecal placement. Resting of pastures for one year greatly reduced the parasite level, but two years of rest were required for parasite-free pasture

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

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

Parasite Occurrence and Parasite Management in Swedish Horses Presenting with Gastrointestinal Disease - A Case-Control Study

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

Transcriptomics of ivermectin response in Caenorhabditis elegans: Integrating abamectin quantitative trait loci and comparison to the Ivermectin-exposed DA1316 strain

Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10(-7) and 10(-8) M) for 4 hours at 20 degrees C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL

Retained efficacy of ivermectin against cyathostomins in Swedish horse establishments practicing selective anthelmintic treatment

Cyathostominae are ubiquitous to grazing horses and regarded the most prevalent internal parasite in the horse. Unfortunately, decades of indiscriminate use of anthelmintic drugs have resulted in the development of resistance in cyathostomins to all currently available drug groups, the most recent being a documented lack of efficacy to the macrocyclic lactones (ML). In vivo determination of anthelmintic resistance in horses most often utilises the faecal egg count reduction test (FECRT). Further, a shortened egg reappearance period (ERP) can indicate a change in response to the applied treatment and suggest an upcoming reduction of efficacy. Although both true resistance as demonstrated by the FECRT and shorter ERPs after ML treatment have now been shown in cyathostomins worldwide, the efficacy of ML as regards to cyathostomins in Sweden is currently unknown. The aim of the present study was therefore to determine FECRTs and ERPs after ivermectin (IVM) treatment in Swedish horses. Sixteen equestrian establishments with a minimum of six horses excreting at least 150 eggs per gram faeces (EPG) at screening were selected. For each establishment, FECRTs and ERPs were determined by collecting faecal samples prior to and 14 days after IVM treatment (200 mu g/kg), and thereafter at weekly intervals for a total of eight weeks. All participants responded to a questionnaire detailing pasture management methods and anthelmintic routines.Questionnaire results showed that the majority of establishments (69%) only treated horses with anthelmintic drugs if indicated by faecal diagnostics and all of the establishments had a mean FECRT exceeding 99.0% and ERPs ranging from six to over eight weeks. The ERP was shown to increase with age as young individuals were shown to excrete cyathostomin eggs earlier after treatment compared with older horses (R = 0.21, p = 0.015). Riding schools, stud farms and those declaring not to use separate summer and winter paddocks had significantly shorter ERPs (p <0.01).In conclusion, retained ERPs and no confirmed resistance to IVM were found in Swedish equine establishments practising selective anthelmintic treatment, and supports the use of selective deworming regimens as a means of reducing the risk of anthelmintic resistance development

Differential expression of beta-tubulin isotypes in different life stages of Parascaris spp after exposure to thiabendazole

Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to beta-tubulin, which prevents polymerisation of microtubules. In this study, beta-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study beta-tubulin gene expression of both isotypes in parasacaris eggs after exposure to different concentrations of thiabendazole (TBZ) for five days at 25 degrees C. A strong pattern of differential expression of beta-tubulin and isotype 1 was observed in all stages, while isotype 2 expression was mainly found at an early phase of the embryogenesis. For isotype 1, a 5-fold increase was observed during the first 48 h, but gene expression gradually decreased after 72, 96 and 120 h. Isotype 2 was only expressed during the first 24 h, followed by a 130-fold decrease at (time points) 72, 96 and 120 h. The in ovo larval developmental test, in which we exposed initially unembryonated eggs to increased concentrations of TBZ, did affect isotype I gene expression but not isotype 2. This assumes that each isotype has specific functions in different life stages. This is in agreement with the 'multi-tubulin' hypothesis, which states that different tubulin isotypes are required for specialised microtubule functions. Isotype I is the most likely drug target for BZs, as isotype 2 was only expressed at very low levels later in development. Increasing concentrations of TBZ altered beta-tubulin isotype 1 gene expression after exposure of the eggs for five days, but this was not seen for isotype 2

Clinical features and treatment response to differentiate idiopathic peritonitis from non-strangulating intestinal infarction of the pelvic flexure associated with Strongylus vulgaris infection in the horse

Background Peritonitis in horses secondary to non-strangulating infarction (NSII) has a guarded prognosis, even after intestinal resection. In contrast, horses with idiopathic peritonitis respond well to medical treatment. Affected horses in both cases often show signs of both colic and systemic inflammation, but early diagnosis is crucial for optimal treatment and an accurate prognosis. One cause of NSII is thrombus formation secondary to Strongylus vulgaris larval migration. There has been a documented increase in S. vulgaris prevalence in Sweden since the implementation of selective anthelmintic treatment in 2007, which subsequently could result in a rise in NSII cases. In a retrospective clinical study, medical records from cases diagnosed with NSII of the pelvic flexure or idiopathic peritonitis from three equine referral hospitals in Sweden during 2017-2020 were reviewed. Information including demographic data, relevant medical history, and clinical- and laboratory parameters were obtained from patient records. To facilitate the differentiation between cases of idiopathic peritonitis and cases with confirmed NSII of the pelvic flexure, the aim of the study was to compare clinical and laboratory parameters, clinical progression and initial response to antimicrobial treatment. A secondary aim was to compare survival-rates. Results Horses with NSII (n = 20) were significantly more likely to present during the winter months with a poorer response to medical treatment within 48 h. Cases of idiopathic peritonitis (n = 107) had a 100% survival rate with medical treatment, although one case required surgical correction of a colon displacement. In comparison, all confirmed NSII cases were non-responsive to antimicrobial treatment, with a survival rate to discharge of 50% after colon resection. Specific rectal findings and peripheral blood neutropenia were strongly associated with NSII. Conclusions In Sweden, idiopathic peritonitis cases still predominate over S. vulgaris associated NSII cases and have an excellent survival rate with antimicrobial treatment. However, horses presenting with septic peritonitis during the winter months with a palpable rectal mass and displaying fever and colic signs beyond 48 h of medical treatment are likely to suffer from NSII of the pelvic flexure and should be considered for abdominal surgery