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Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL-1) and IVM at 1.1 μm (1 μg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Background: Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors., Methods: Live adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS)., Results: Proteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations., Conclusions: These findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection., (© 2024. The Author(s).)

Introduction: Following the approval of tocilizumab (TCZ) for giant cell arteritis (GCA), recent studies have shown a high relapse frequency after abrupt discontinuation of TCZ. However, a thorough exploration of TCZ tapering compared to abrupt discontinuation has never been undertaken. Likewise, adverse events have only been scarcely investigated in routine care. This study aimed to compare the incidence of relapses in GCA patients undergoing TCZ tapering compared to abrupt discontinuation., Methods: We performed a single-center retrospective cohort study from 2012 to 2022. Data from GCA patients treated with TCZ was obtained from the Electronic Patients Record. Relapse-free survival is reported in Kaplan-Meier plots and tapering versus abrupt discontinuation were compared using a Wilcoxon-Brewlos-Gehan test., Results: We included 155 patients receiving TCZ treatment for GCA, of which 104 discontinued TCZ. Among the 104 patients discontinuing TCZ, 42 (40 %) experienced a relapse within the first year. A total of 57 patients underwent taper with 6/38 (16 %) and 2/19 (11 %) relapsing while receiving TCZ every second or third week, respectively. In comparison, 59 patients underwent abrupt discontinuation with 27 (46 %) relapsing during follow-up. The patients undergoing abrupt TCZ discontinuation demonstrated a significantly shorter time to relapse compared to all tapered patients (p = 0.02) as well as patients tapered from weekly TCZ treatment to every second week (p < 0.01). Furthermore, 15 % of patients discontinued TCZ due to adverse events., Conclusion: This is the first study indicating that TCZ taper induced longer relapse-free survival than abrupt discontinuation, implying that taper may be favored over discontinuation in patients with GCA., Competing Interests: Declaration of competing interest BDN has received speaking fees unrelated to this manuscript from Novartis an AbbVie. EMH has received fees unrelated to this manuscript for speaking and/or consulting from Novo, Novartis, AbbVie, Sanofi, Sobi; research funding to Aarhus University Hospital from Independent Research Fund Denmark, Novo Nordic Foundation, Danish Rheumatism Association, Aarhus University, Danish Regions Medicine Grants, Galapagos, AbbVie; travel expenses from Pfizer, Sobi, AbbVie. EMH has been the principal investigator in trials by SynACT Pharma and involved as site principal investigator in trials by AbbVie, Novartis, Novo Nordisk, Sanofi. All other authors have nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Caring for a family member can be demanding, particularly when caregivers experience profound distress. Supportive interventions may help prevent mental and physical illness in the caregiver. General practice plays a key role by regularly engaging with patients and caregivers, thereby being able to identify their support needs, offer talk therapy and refer to care initiatives., Objective: This study aimed to develop and pilot-test the Caregiver Care Model to mitigate grief reactions among caregivers in general practice., Design: A participatory intervention development study., Methods: A prototype was developed in a workshop with healthcare professionals. The prototype was refined based on a pilot-test among 40 caregivers from 5 general practice clinics and a workshop with general practitioners. The data were obtained from workshops, feedback questionnaires completed by healthcare professionals, and interviews with caregivers and general practitioners. The analysis focused on model development and mechanisms of impact., Results: The prototype was refined by focusing the dialogue questionnaire, minimising the grief facilitation tools and expanding the target group. The prototype seemed to accommodate the needs among caregivers by acknowledging their situation. The final model includes up to seven caregiver consultations in general practice. A dialogue questionnaire filled in by the caregiver serves as a fixed starting point and preparation for the first consultation. If needed, talk therapy in general practice or referrals to other services are used., Conclusion: The model offers promising support for caregivers. Its flexible structure allows for customisation. The viability of the model should be further tested., Competing Interests: The authors declare that there is no conflict of interest., (© The Author(s), 2024.)

Competing Interests: The authors have declared that no competing interests exist.

Recently, semi-synthetic cannabinoids have entered the illegal market and are produced to mimic the psychoactive effects of tetrahydrocannabinol (THC). This is a case report of a 19-year-old man, who was hospitalized due to severe sedation, hypotension and tachy- and bradycardia after ingestion of the semi-synthetic cannabinoids hexahydrocannabinol (HHC) and hexahydrocannabiphorol (HHC-P) mixed in food. HHC-P, HHC and metabolites were identified in blood samples. The amount of semi-synthetic cannabinoids in illegal products can be high, which can explain the described prolonged clinical effects., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

In Argentina, as in the rest of the world, cyathostomins are the most common nematodes parasitizing horses. Control is based almost exclusively on the administration of benzimidazoles, pyrimidines, and macrocyclic lactones. However, intensive use of these drugs is resulting in the development of anthelmintic resistance (AR). For example, AR to benzimidazoles is currently distributed throughout Argentina, while incipient AR to pyrimidines (pyrantel embonate) is appearing in areas where this drug is used. Macrocyclic lactones and especially ivermectin, are by far the most used drugs by the vast majority of equine premises in the country. Although ivermectin has been used since 1982, its efficacy against equine strongylid parasites has remained very high until the present. In this study we report for the first time, the presence of a cyathostomin population with resistance to ivermectin in adult horses belonging to an equine premise located in central Argentina. Fecal egg count reduction tests (FECRT) were performed following the most recent guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for the diagnosis of anthelmintic resistance (research protocol) and resistance was considered when the Upper 90% Credible Interval fell below the expected efficacy threshold of 99.9%. Calculations were carried out using two different online calculation interfaces suggested by WAAVP. For the 14-day post-treatment interval, ivermectin efficacy was 79.5% (90% Credible Interval: 68.1-88.8) and 79.3% (74.2-83.6.3%) with the two methods, respectively. At 19 days post treatment, fecal egg count reductions were 68.6% (50.5-83.1) and 68.4% (61.9-74.1), respectively. At both intervals, this cyathostomin population fullfilled the criteria for AR. These findings suggest dispersion of ivermectin resistant cyathostomins in Argentina. Given the widespread use of macrocyclic lactones, it is important that veterinarians and the equine industry promote a more selective and evidence-based use of these drugs and establish routine monitoring to determine anthelmintic field efficacy to detect treatment failures as early as possible and avoid potential health problems as well as further spread of resistant genes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anthelmintic performance against equine cyathostomins can be evaluated by two different non-terminal measures; the Fecal Egg Count Reduction Test (FECRT) and the Egg Reappearance Period (ERP). Most available FECRT and ERP data have been determined in populations of young horses, and very little information is available from mature and senior horses. Furthermore, it is unknown how commonly occurring equine endocrine disorders such as Insulin dysregulation (ID) and Pituitary pars intermedia dysfunction (PPID) may interfere with these measurements, but it has been suggested that horses with these conditions could be more susceptible to parasitic infections. A research population of senior horses and horses with or without PPID, ID, or both were enrolled in this study. All strongylid egg count positive horses were included in an ivermectin (200 μg/kg) efficacy study. These were distributed among the following groups: ID: six, PPID: three, PPID and ID: seven, and healthy controls: three. Strongylid fecal egg counts were determined on the day of ivermectin administration, at two weeks post deworming, and on weekly intervals until eight weeks post treatment. Determination of FECRT and ERP were carried out following World Association for the Advancement of Veterinary Parasitology guidelines. Results revealed high ivermectin efficacy with mean egg count reduction at 99.7% or above in all groups at two weeks post treatment. Egg reappearance was documented at six and seven weeks in the ID and PPID/ID groups, respectively, whereas the PPID and healthy control groups both had ERP at 8 weeks. Statistical analysis found no significant differences in egg count levels between groups during the study. The expected ERP for ivermectin is 8-10 weeks, meaning that two of the groups displayed shortened ERPs. However, due to the small group sizes, these data should be interpreted with caution. Nonetheless, results do indicate a need for further investigation of the possible influence of endocrine disorders on anthelmintic performance in horses., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cancer patients with pre-existing severe mental disorders (SMDs) less frequently receive guideline recommended cancer treatment and have a higher cancer mortality. However, knowledge is needed concerning end-of-life care in this patient group. The aim of this systematic review was to provide an overview of the literature concerning end-of-life care in cancer patients with pre-existing SMD., Methods: A systematic search was conducted in the following databases: PubMed, Embase and Science Direct and all results were downloaded to Endnote on 1st of September 2023. The review was registered on International Prospective Register of Systematic Reviews (PROSPERO) (ID: CRD42023468571). The quality of the studies was assessed according to the Newcastle-Ottawa Scale., Results: Ten studies fulfilling the inclusion criteria were included. There was a recurring pattern indicating a difference between the end-of-life care received by cancer patients with SMD, compared to those without. Cancer patients with pre-existing SMD received more palliative end-of-life care but less high-intensive-end-of-life (HIEOL) care, e.g., less hospitalisations and chemotherapy at the end of life, and died less frequently at hospital., Conclusions: The study indicates that patients with pre-existing SMD and cancer more often received palliative end-of-life care and less HIEOL care compared to controls. Further research regarding the difference in end-of-life care is lacking, including the consequences of less intense HIEOL care for this patient group. Thus, further studies are needed to identify reasons for less intense HIEOL among cancer patients with pre-existing SMD.

Equine strongylid parasites are ubiquitous around the world and are main targets of parasite control programmes. In recent years, automated fecal egg counting systems based on image analysis have become available allowing for collection and analysis of large-scale egg count data. This study aimed to evaluate equine strongylid fecal egg count (FEC) data generated with an automated system over three years in the US with specific attention to seasonal and regional trends in egg count magnitude and sampling activity. Five US regions were defined; North East, South East, North Central, South Central and West. The data set included state, region and zip code for each FEC. The number of FECs falling in each of the following categories were recorded: (1) 0 eggs per gram (EPG), (2) 1 ⩽ 200 EPG, (3) 201 ⩽ 500 EPG and (4) >500 EPG. The data included 58 329 FECs. A fixed effects model was constructed fitting the number of samples analysed per month, year and region, and a mixed effects model was constructed to fit the number of FECs falling in each of the 4 egg count categories defined above. The overall proportion of horses responsible for 80% of the total FEC output was 18.1%, and this was consistent across years, months and all regions except West, where the proportion was closer to 12%. Statistical analyses showed significant seasonal trends and regional differences of sampling frequency and FEC category. The data demonstrated that veterinarians tended to follow a biphasic pattern when monitoring strongylid FECs in horses, regardless of location.

Tapeworm infection in horses can cause serious health concerns, and recent data have documented treatment failures in the most common species, Anoplocephala perfoliata. The threat of anthelmintic resistance in A. perfoliata is of particular concern because of poor diagnostic performance of standard egg counting techniques for detecting this parasite. This study compared the performance of three diagnostic techniques 1) Mini-FLOTAC, 2) Cornell-Wisconsin, and 3) Proudman and Edwards used to detect and quantify A. perfoliata eggs in naturally infected horses. Eighteen adult female horses from the University of Kentucky's historic parasitology herd were included in this study. Fecal samples were collected from all horses at five collection time points two weeks apart and analyzed with the three techniques. A total of 90 samples were collected and 270 counts determined in the study. The proportions of positive samples determined by the three techniques were significantly different from each other (p<0.05): Mini-FLOTAC (16%), Cornell-Wisconsin (47%), and Proudman and Edwards (70%). The Proudman and Edwards technique counted consistently higher numbers of tapeworm eggs compared to the other two techniques throughout the study [p < 0.05]. Total raw counts of tapeworm eggs across the study for each technique were 16, 88, and 410 for the Mini-FLOTAC, Cornell-Wisconsin, and Proudman and Edwards, respectively. This study demonstrated that the Proudman and Edwards technique was superior in diagnosing A. perfoliata infection. Future work needs to assess this technique's potential for Fecal Egg Count Reduction Testing (FECRT)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study quantified the extent of anthelmintic resistance (AR) in ascarid and strongylid nematodes against commonly used anthelmintics in Australian Thoroughbred horses. Faecal egg count reduction tests (FECRTs, n = 86) and egg reappearance period (ERP) tests were conducted on 22 farms across Australia. Faecal egg counts (FECs) were determined using the modified McMaster technique, and percent faecal egg count reduction (%FECR) was calculated using the Bayesian hierarchical model and hybrid Frequentist/Bayesian analysis method. The results were interpreted using old (published in 1992) and new (2023) research guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP). The species composition of strongylid nematodes was detected utilising a DNA-metabarcoding method using pre- and post-treatment samples. Resistance was observed in strongylid nematodes to commonly used single-active and combination anthelmintics, including ivermectin (IVM %FECR range: 82%-92%; 95% lower credible interval (LCI) range: 80%-90%), abamectin (ABM: 73%-92%; 65%-88%), moxidectin (MOX: 89%-91%; 84%-89%), oxfendazole (OFZ: 0%-56%; 0%-31%) and its combination with pyrantel (OFZ + PYR: 0%-82%; 0%-78%). Resistance in Parascaris spp. was observed to IVM (10%-43%; 0%-36%), ABM (0%; 0%) and MOX (0%; 0%). When the new thresholds recommended by the WAAVP were used, AR was detected in six additional FECRTs for strongylids and three more tests for Parascaris spp., introducing resistance to OFZ and OFZ + PYR in the latter. Shortened ERPs (4-6 weeks) of strongylids were observed in 31 FECRTs in which AR was not detected at 2 weeks post-treatment for all the anthelmintics tested. Among cyathostomins, Cylicocyclus nassatus, Cylicostephanus longibursatus and Coronocyclus coronatus were the most prevalent species at 2 weeks post-treatment, whereas the main species appearing at five weeks following treatments with macrocyclic lactones were Cylicocyclus nassatus, Cylicostephanus longibursatus and Cylicocyclus ashworthi. After treatment with OFZ + PYR, the latter three, plus Coronocyclus coronatus and Cyathostomum catinatum, were detected at 5 weeks post-treatment. Overall, the study highlights the prevalence of AR in both ascarids and strongylid nematodes against commonly used anthelmintic products to control worms in Australian horses. The results indicate that ML combination products provided acceptable efficacy at 2 weeks. However, ERP calculations suggest that products work less effectively than previously measured. It is suggested to regularly monitor the efficacy of the anthelmintics and consider changing the worm control practices to better manage worms and AR in Australian horses., Competing Interests: Declaration of competing interest The authors of this manuscript are members of the Australian Equine Parasitology Advisory Panel (AEPAP), including Abdul Jabbar, Ghazanfar Abbas, Jenni Bauquier, Charles El-Hage, Abdul Ghafar and Ian Beveridge (The University of Melbourne), Anne Beasley (University of Queensland), Kristopher Hughes (Charles Sturt University), Caroline Jacobson and Emma McConnell (Murdoch University), Edwina Wilkes (Racing Victoria), Peter Carrigan and Lucy Cudmore (Scone Equine Hospital) and John Hurley (Swettenham Stud). Boehringer Ingelheim supported the panel. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study assessed worm control practices used by Australian Thoroughbred farm managers with an online questionnaire survey. The questionnaire comprised 52 questions (close-ended: 44; open-ended: 8) about farm demography and general husbandry practices, farm managers' knowledge of gastrointestinal nematodes (GIN) and their importance, diagnosis, worm control strategies and anthelmintics, anthelmintic resistance (AR) and grazing management. Following the pilot survey, the link for the questionnaire survey was sent to all (n = 657) registered members of the Thoroughbred Breeders Australia on 12th April 2020. The response rate for the questionnaire was 18.5% (122 of 675). The farm managers reported a good understanding of GIN and their importance in different age groups of horses as most respondents (70% of 122) perceived worm-related illness to be more important in young (i.e., foals, weanlings and yearlings) than adult (> 3 years old) horses. Although most respondents (93%, 113 of 122) used anthelmintics prophylactically to control GIN, only 15% (18 of 122) observed worm-related illness in their horses. Just under 40% of respondents were performing faecal egg counts, with less than 20% using the results of faecal egg counts to guide deworming decisions. The interval-based deworming strategy was the most common method (≥55% of 122 respondents) to control GIN in all age groups of horses. Macrocyclic lactones were the first choice of anthelmintics for all age groups of horses. Although the majority of respondents (88%, 107 of 122) perceived resistance in GIN against commonly used anthelmintics as an important issue in managing worms in horses, only 29% assessed the efficacy of anthelmintics and 91% (111 of 122) were unaware of AR on their properties. Grazing management practices, such as manure removal, were more frequently performed on smaller paddocks (<0.20 ha: 58%) than on larger paddocks (>0.20 ha: 18%). Multiple correspondence analyses showed that the likelihood of suboptimal worm control practices on small farms (n = ≤50 horses) was greater than that of medium (n = 51-100) and large (n = >100) farms. This study provides insights into the demography of Thoroughbred farms in Australia, husbandry practices used by stud managers and their knowledge about worms, control options and AR concerns, thereby paving the way for taking any initiatives to address the problem of AR in GIN of Australian Thoroughbred horses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work stated in this report., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study reports the spatial and temporal distribution of ascarid and strongylid nematodes in Thoroughbred horses by age category across different climatic zones in Australia over an 18-month period. Faecal samples (n = 2046) from individual horses were analysed using the modified McMaster technique for faecal egg counts (FECs). Strongylids were identified using PCR-directed next-generation sequencing of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA. Yearlings had the highest prevalence (82%) of strongyle eggs followed by weanlings (79%), foals (58%), wet mares (49%) and dry mares (46%). For Parascaris spp., foals had the highest prevalence (35%) followed by weanlings (21%) and yearlings (10%). The highest mean FECs for Parascaris spp. were observed in foals (525 eggs per gram [EPG] of faeces) while those for strongyles were in yearlings (962 EPG). Among horses that were classified as adults at the time of sampling, 77% (860 of 1119) of mares were low (i.e., <250 EPG) strongyle egg-shedders. Mean strongyle FEC counts were highest in the Mediterranean (818 EPG) followed by summer (599 EPG), winter (442 EPG), and non-seasonal (413 EPG) rainfall zones. Twenty-six nematode species were detected, with Cylicostephanus longibursatus (26.5%), Cylicocyclus nassatus (23.7%) and Coronocyclus coronatus (20.5%) being the most frequently detected species. Their richness and relative abundance varied with horse age, season and climatic zone. In addition, Strongylus equinus and Triodontophorus spp. (T. brevicauda and T. serratus) were also detected. This comprehensive study elucidates spatial (climatic zone) and temporal (i.e., seasonal) trends in prevalence and burdens of intestinal nematodes in Australian horses using non-invasive conventional and molecular methods. The information presented in this study is crucial for developing integrated management strategies to control horse parasites in farmed horses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work stated in this report., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The microbiome plays an important role in health, where changes in microbiota composition can have significant downstream effects within the host, and host-microbiota relationships can be exploited to affect health outcomes. Parasitic helminths affect animals globally, but an exploration of their microbiota has been limited, despite the development of anti-Wolbachia drugs to help control infections with some filarial nematodes. The equine ascarids, Parascaris spp., are considered the most pathogenic nematodes affecting juvenile horses and are also the only ascarid parasite to have developed widespread anthelmintic resistance. The aim of this study was to characterize the microbiota of this helminth, focusing on the female gonad, determine a core microbiota for this organ, identify bacterial species, and show bacterial localization to the female gonad via in situ hybridization (ISH). A total of 22 gonads were isolated from female Parascaris spp. collected from three foals, and 9 female parasites were formalin-fixed and paraffin-embedded for ISH. Next-generation sequencing was performed using V3-V4 primers as well as the Swift Amplicon™ 16S+ ITS Panel. Overall, ten genera were identified as members of the Parascaris spp. female gonad and twelve bacterial species were identified. The most prevalent genus was Mycoplasma, followed by Reyranella, and there were no differences in alpha diversity between parasites from different horses. Specific eubacteria staining was identified in both the intestine and within the gonad using ISH. Overall, this study provided in-depth information regarding the female Parascaris spp. microbiota and was the first to identify the core microbiota within a specific parasite organ., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Age-related macular degeneration (AMD) is a common cause of visual loss among the elderly. Genetic variants in the gene encoding complement factor H (CFH) have been identified as an AMD susceptibility gene, however, the mechanistic link is debated. Here, we investigated the link between the CFH Y402H genotype and low-grade inflammation. We recruited 153 healthy individuals, 84 participants with dry stages of AMD, and 148 participants with neovascular AMD. All participants were subjected to detailed retinal examination, and interview regarding comorbidities and lifestyle. Blood samples were analyzed for level of C-Reactive Protein (CRP), white blood cell differential count, and stained with fluorescent antibodies to differentiate CD4 + and CD8 + T cells. CFH Y402H genotyping was performed using an allele-specific polymerase chain reaction genotyping assay. Splenocytes from young and aged wild type and Cfh null mutant C57BL/6J mice were examined for CD4 + and CD8 + T cells. Healthy individuals with the CFH Y402H at-risk polymorphism HH had higher levels of CRP and lower proportions of CD4 + T cells compared to persons with the YH or YY polymorphism (P = 0.037, Chi-square). Healthy individuals with the HH polymorphism displayed lower proportions of CD4 + T cells with ageing (P < 0.01, one-way ANOVA), whereas both young and aged Cfh null mutant mice displayed lower proportions of CD4 + T cells (P < 0.001 and P < 0.05; unpaired t test). Participants with dry AMD and the HH polymorphism had similarly lower proportions of CD4 + T cells (P = 0.024, one-way ANOVA), but no difference in CRP-levels. In the neovascular stage of AMD, there was no difference in proportion of CD4 + cells or CRP levels according to genotype. The risk-associated CFH genotype is associated with an age-related decrease in proportion of CD4 + T cells and increased levels of CRP in healthy individuals. This indicates that decreased complement regulation results in extensive changes in innate and adaptive immune compartments that precede development of AMD., (© 2023. The Author(s).)

This study aimed to assess Australian veterinarians' knowledge, perceptions and treatment strategies for worm control in horses with an online questionnaire. The questionnaire comprised 64 questions covering various aspects of: (i) veterinary practice; (ii) the veterinarian's knowledge of gastrointestinal nematodes (GINs) and the importance of parasites in different age groups of horses; (iii) the diagnosis and control of worms; (iv) anthelmintics and anthelmintic resistance (AR); (v) grazing management; and (vi) the means of communication and the discussion between veterinarians and their clients regarding worm control. Following a pilot survey, a link for the questionnaire survey was sent to all ( n = 1,148) registered members of Equine Veterinarians Australia in April 2020. The response rate for the questionnaire was 10% (118 of 1,148). The findings of this study illustrate veterinarians' good understanding of aspects of equine parasites, including control. However, respondents mainly recommended frequent, interval-based prophylactic deworming in young horses, and only 40% (96 of 239) diagnosed GIN infections based on faecal egg count (FEC) results in all age groups of horses. Furthermore, only 27% (88 of 330) of the respondents made deworming decisions based on FECs. Most of the respondents recommended macrocyclic lactones (MLs) for all age groups of horses (71%, 481 of 677), and the most frequently used method to calculate the dose of anthelmintics was by estimating the weight of animals visually (53%, 63 of 118). Although the majority of respondents (97%, 115 of 118) perceived AR to be a critical issue in managing worms in horses, 58% (67 of 118) of them were unaware of the status of AR on their clients' properties. Forty-two percent (50 of 118) of the respondents perceived the presence of AR in worms, including pinworms (16%), strongylins (15%), species of Draschia and Habronema (6%), Strongyloides westeri (2%) and tapeworms (1%). Twenty-seven percent (32 of 118) of the respondents rarely discussed equine worm control practices with their clients. This study provides insights into the perception and worm control practices recommended by Australian veterinarians to manage equine parasites. The findings highlight the importance of continued education and awareness of AR, and the use of non-chemical methods as well as consideration of the legislation of prescription-only use of anthelmintics based on FECs to achieve sustainable control of GINs in Australian horses., Competing Interests: The authors of this manuscript are members of the Australian Equine Parasitology Advisory Panel (AEPAP), including Abdul Jabbar, Ghazanfar Abbas, Jenni Bauquier, Charles El-Hage, Abdul Ghafar and Ian Beveridge (The University of Melbourne), Anne Beasley (University of Queensland), Kristopher Hughes (Charles Sturt University), Caroline Jacobson and Emma McConnell (Murdoch University), Edwina Wilkes (Racing Victoria), Peter Carrigan and Lucy Cudmore (Scone Equine Hospital) and John Hurley (Swettenham Stud). Boehringer Ingelheim supported the panel. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Abbas, Stevenson, Bauquier, Beasley, Jacobson, El-Hage, Wilkes, Carrigan, Cudmore, Hurley, Beveridge, Nielsen, Hughes and Jabbar.)

Long-term intensive use of anthelmintics for parasite control of livestock, companion animals, and humans has resulted in widespread anthelmintic resistance, a problem of great socioeconomic significance. But anthelmintic therapy may also select for other biological traits, which could have implications for anthelmintic performance. Here, we highlight recent examples of changing parasite dynamics following anthelmintic administration, which do not fit the definition of anthelmintic resistance. We also consider other possible examples in which anthelmintic resistance has clearly established, but where coselection for other biological traits may have also occurred. We offer suggestions for collecting more information and gaining a better understanding of these phenomena. Finally, we propose research questions that require further investigation and make suggestions to help address these knowledge gaps., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10-14%) and 72% (70-74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251-500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work stated in this report., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In drug-facilitated sexual assault (DFSA), the victim is unable to provide consent or resists sexual activity due to substance intoxication by voluntary or covert consumption. Obtaining forensic evidence of the assault is challenged by rapid drug metabolism and late sample collection. The objective of this study was to present toxicological findings and associated demographics from police reported sexual assault cases in Eastern Denmark from 2015 to 2022. A total of 369 sexual assault cases were submitted for analysis and a subgroup of 268 cases were categorized as suspected DFSA cases. The majority of the total sexual assault victims were women at the age 15-25 and the perpetrators were often unknown or an acquaintance. Time from assault to sample collection was slightly longer for suspected DFSA cases (12-24 h) compared to non-DFSA (<12 h). Positive toxicology was observed in 86 % of cases and the most common drug groups included alcohol (45 %), drugs of abuse (38 %), antidepressants (14 %), antihistamines (12 %), and benzodiazepines (11 %). Hypnotics were detected to a smaller extent (7 %). A total of 77 drugs were detected and the most commonly observed were cocaine, tetrahydrocannabinol (THC), cetirizine, amphetamine, diazepam and sertraline. The high level of observed alcohol and drugs of abuse indicated that most DFSA cases in Eastern Denmark were of an opportunistic approach rather than proactive., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Parascaris spp. infect foals worldwide and foals typically shed eggs in the feces from about three to six months of age, upon which natural immunity is incurred. High levels of anthelmintic resistance of Parascaris spp. are a global concern, and further understanding egg shedding patterns and fecal egg counting (FEC) data variability is of high importance. The aims of this study were to monitor Parascaris spp. egg shedding in untreated foals during 12-23 weeks of age, estimate sources of data variability, and assess precision of two ascarid FEC techniques. Fecal samples were collected weekly from 11 foals born in 2022, from May through November (29 weeks). Six subsamples were extracted from each weekly sample to determine 30 FECs between two techniques: a McMaster technique and an Automated Egg Counting System (AECS). Mixed linear modeling was carried out with age, sex, birth month, seasonality, spring- or summer-born foals, and egg counting technique as explanatory variables. Ascarid FECs were associated with age (p < 0.001), seasonality (p < 0.001), and technique (p < 0.001). The McMaster technique was more precise with a mean coefficient of variation (CV) of 34.57% and a 95% confidence interval (CI) of 30.80%- 38.30% compared to the CV for the AECS, which was 42.22% (CI: 37.70%-46.70%). Seasonality accounted for the highest proportion of variance (PV) of all covariates, but differences in PVs for covariates existed between techniques with foal age and subsample contributing more variance to the McMaster, and individual foal and seasonality contributing more to the AECS. Subsamples and replicate counts accounted for less than 1% of the total data variance. The results highlighted substantial differences in PVs between the two techniques at the subsample (AECS: 57.14%; McMaster: 77.51%) and replicate count levels (AECS: 42.86%; McMaster: 22.49%). While differences in precision were observed between the two FEC techniques, they were negligible in the data set, as the overwhelming majority of the data variability in ascarid FECs was attributed to individual foal, seasonality, and foal age., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Injuries to the cartilaginous structures of the nose can arise after minor trauma and without a concomitant nasal fracture. They are diagnosed with a thorough clinical examination and can, if not diagnosed and treated in the acute phase, result in nasal/septal deformation, saddle nose or abscess formation with subsequent intracranial spreading of the infection. Even with proper treatment in the acute phase, the cartilage often heals with deviation. This can result in functional and cosmetic problems which may require later reconstructive surgery, as argued in this review., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

Purpose: The participation rate in the Danish National Screening Program for Colorectal Cancer is around 60%. Since early detection of cancer through the colorectal cancer screening program (CRCS) reduces mortality rates, it is important to understand why people do not participate. The aim of this study was therefore to examine in depth why some people do not participate in the Danish CRCS program., Methods: Individual semi-structured interviews were conducted with 18 people who had not participated in the national CRCS program. Data was analyzed using an inductive hermeneutic approach., Results: The analysis uncovered three main themes describing barriers to screening: (a) the accountability for ensuring personal health covered the following sub-themes: (i) when invitation is out of sight it is out of mind, (ii) aversion against own stool sample collection, and (iii) the perceived risk of getting colorectal cancer (CRC); (b) attitudes towards the healthcare system and its organization covered the following sub-themes: (i) positivity towards CRCS, (ii) trust in the healthcare system, (iii) lack of internal communication in the healthcare system, and (iv) wish for involvement of the general practitioner (GP); (c) knowledge about disease and screening covered the following sub-themes: (i) knowledge about CRC and CRCS, (ii) wish for information via new channels, and (iii) CRC is rarely something you talk about., Conclusion: Among the informants included in this study, lack of participation in the CRCS program was not due to an active opt-out, as most informants had intentions of participating. The informants thought it would motivate them to participate if their GP would remind them of the screening program., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Anoplocephalid tapeworms are commonly occurring in grazing horses around the world. Two currently available anthelmintics have documented high efficacy against Anoplocephala perfoliata; praziquantel in various dosages ranging from 1.0 to 2.5 mg/kg and pyrantel pamoate administered at 13.2 mg base/kg. Anthelmintic resistance has not been reported in A. perfoliata, but anecdotal reports made during 2022 have suggested a possible loss of efficacy for both actives. This paper reports fecal egg count data from a Thoroughbred operation in Central Kentucky in 2023. Fifty-six yearlings were first dewormed with a combination of ivermectin (200 μg/kg) and praziquantel (1.5 mg/kg) and subsequently treated with pyrantel pamoate (13.2 mg base/kg). Fecal egg counts were determined at the day of treatment and again 14 days post-treatment. Two groups of mares (n = 39 and 45) were also treated with ivermectin/praziquantel and examined pre- and post-treatment. Low efficacy of ivermectin and pyrantel pamoate was demonstrated against strongylid parasites in the yearlings with mean Fecal Egg Count Reductions (FECRs) at 75.6% or below and upper 95% credible interval (CI) limits below 90% in all cases. Overall anti-cestodal FECR levels in the yearlings were 23.5% (95% CI: 11.2-48.0) for praziquantel and 50.9% (20.5-72.0) for pyrantel pamoate. Praziquantel eliminated anoplocephalid eggs from three of 17 yearlings, but another 5 yearlings went from negative to positive status following treatment. Pyrantel pamoate failed to eliminate anoplocephalid eggs from any of 14 treated tapeworm-positive yearlings. Nine of 84 mares tested positive for anoplocephalid eggs, and seven of these were still positive post praziquantel treatment. These findings sharply contrast data from historic field efficacy studies conducted for both actives and raise concern about anthelmintic resistance having possibly developed. This emphasizes the need for developing and refining antemortem methodologies for evaluating anti-cestodal treatment efficacy and for searching for possible alternative treatment options., Competing Interests: Declaration of competing interest The author declares no conflicts of interest., (Copyright © 2023 The Author. Published by Elsevier Ltd.. All rights reserved.)

Over the past three decades, equine strongylid egg reappearance periods (ERPs) have shortened substantially for macrocyclic lactone anthelmintics. The ERPs of ivermectin and moxidectin were originally reported in the 8-10 and 12-16 week ranges, respectively, but several recent studies have found them to be around 4-5 weeks for both actives. This loss of several weeks of suppressed strongylid egg output could have substantial implications for parasite control. This study made use of a computer simulation model to evaluate the impact of shortened ERPs on the anthelmintic performance of ivermectin and moxidectin against equine cyathostomins. The original ERPs were set to 7.1 and 15.4 weeks for ivermectin and moxidectin, respectively, while the reduced ERP was set to 4.6 weeks for both actives. Simulations were set to compare model outputs between original and reduced ERP scenarios and results expressed as percent increase in strongylid egg output, infective third stage larvae on herbage (L3h), and encysted early third stage larvae (EL3). For each drug, simulations were evaluated for two different treatment scenarios (2 and 4 treatments annually), two different age groups (yearlings and adults), and for four different climates (cold humid continental, temperate oceanic, humid subtropical, and hot/cold semi-arid). Across all simulations, there was a substantial increase of the three evaluated parameters. With the ivermectin simulations, all three parameters increased in the 100-300% range across climates, age groups and treatment intensities. The moxidectin simulations displayed a wider range of results with parameters increasing from a few hundred to several thousand percent. The increases were most pronounced for L3h in the two cooler climates, reaching as high as 6727%. Overall, the loss of anthelmintic performance was at a magnitude of 10 times larger for moxidectin compared to ivermectin. This performance loss was climate dependent, and was also affected by treatment intensity, but not by horse age. This is the first study to evaluate consequences of shortened ERPs in horses and demonstrated a substantial loss in anthelmintic performance resulting from this development. The results illustrate that anthelmintic efficacy is more than the percent reduction of fecal egg counts at 14 days post treatment, and that substantial anthelmintic performance can be lost despite FECRTs remaining at 100%., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The faecal egg count reduction test (FECRT) remains the method of choice for establishing the efficacy of anthelmintic compounds in the field, including the diagnosis of anthelmintic resistance. We present a guideline for improving the standardization and performance of the FECRT that has four sections. In the first section, we address the major issues relevant to experimental design, choice of faecal egg count (FEC) method, statistical analysis, and interpretation of the FECRT results. In the second section, we make a series of general recommendations that are applicable across all animals addressed in this guideline. In the third section, we provide separate guidance details for cattle, small ruminants (sheep and goats), horses and pigs to address the issues that are specific to the different animal types. Finally, we provide overviews of the specific details required to conduct an FECRT for each of the different host species. To address the issues of statistical power vs. practicality, we also provide two separate options for each animal species; (i) a version designed to detect small changes in efficacy that is intended for use in scientific studies, and (ii) a less resource-intensive version intended for routine use by veterinarians and livestock owners to detect larger changes in efficacy. Compared to the previous FECRT recommendations, four important differences are noted. First, it is now generally recommended to perform the FECRT based on pre- and post-treatment FEC of the same animals (paired study design), rather than on post-treatment FEC of both treated and untreated (control) animals (unpaired study design). Second, instead of requiring a minimum mean FEC (expressed in eggs per gram (EPG)) of the group to be tested, the new requirement is for a minimum total number of eggs to be counted under the microscope (cumulative number of eggs counted before the application of a conversion factor). Third, we provide flexibility in the required size of the treatment group by presenting three separate options that depend on the (expected) number of eggs counted. Finally, these guidelines address all major livestock species, and the thresholds for defining reduced efficacy are adapted and aligned to host species, anthelmintic drug and parasite species. In conclusion, these new guidelines provide improved methodology and standardization of the FECRT for all major livestock species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Widespread overuse of anthelmintics has produced a growing population of intestinal parasites resistant to control measures. A paradigm shift in equine parasite control is warranted to prevent continued resistance development and maintain equine health. Small strongyles, which are ubiquitous in horses, are currently the most important intestinal parasites of adult horses. Sustainable management programs consider the variation in egg shedding by individual horses, and varied risks associated with age, use, density, climate, and environment. To develop regional recommendations for Prince Edward Island (PEI), Canada, it is important to first characterize strongyle egg shedding patterns and parasite management practices in use. Study objectives were to conduct a cross-sectional observational survey and risk factor analysis of parasite control programs, strongyle egg shedding and Strongylus vulgaris serology. A total of 339 horses from 40 PEI farms were sampled. Mean farm size was 8 horses and ranged from 2 to 30. Mean horse age was 11.6 years (Std. Dev. =7.2) and ranged from 2 months to 32 years. Mean fecal egg count (FEC) was 322 eggs per gram (EPG) (Std. Dev. =648). On average, 32% (Std. Dev. =16%) of horses shed 80% of strongyle eggs across 32 eligible farms. When considering all horses (n = 313) as one large herd, 18.7% of horses shed 80% of strongyle eggs. Use of FEC was identified in 4.6% of horses at 15% (n = 6) of PEI farms. Reported deworming intervals included 37.4% (n = 123) every 2-3 months and 58% (n = 191) every 3-4 months. Positive S. vulgaris titers were identified in 60% of horses (n = 200). Univariate analysis revealed that months since last deworming, age, and body condition score (BCS) were associated with strongyle shedding. The estimated odds of being in the high FEC category (>500 EPG) was 1.4 (95% CI, 1.1-1.8) times higher when last deworming increased by one month. Under-conditioned (BCS <4.5) horses had 3.6 (95% CI, 1.2-10.6) times odds of being in the higher FEC category than over-conditioned horses. Non-racing horses had 5.4 times odds of having a positive S. vulgaris titer than racehorses. This cross-sectional observational study is the first to report on the occurrence, risk factors and control of equine strongyle nematode infections in PEI, Canada. We conclude that the 80:20 rule can be used to develop control recommendations in PEI. Very few farms in PEI currently use FEC to guide parasite management. These findings provide a basis for future client education and investigations aimed at providing region specific recommendations., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Background: The atrophic late stage of age-related macular degeneration (AMD) is termed geographic atrophy (GA), and affects visual acuity (VA) as well as quality of life (QoL). Previous studies have found that best-corrected VA (BCVA), the standard vision assessment often underrepresents functional deficits. Therefore, the purpose of this study was to evaluate the correlation between atrophic lesion size, VA and QoL measured with the National Eye Institute Visual Function Questionnaire (VFQ-39) in a Danish population. Moreover, we wanted to evaluate the correlation between comorbidities, behavioural factors, and QoL., Methods: This was prospective clinical study of 51 patients with GA in one or both eyes, of these 45 patients had bilateral GA. Patients were consecutively included between April 2021 and February 2022. All patients filled in the VFQ-39 questionnaire except the subscales "ocular pain" and "peripheral vision." Lesion size was measured from fundus autoflourescense images, and BCVA was assessed by the Early Treatment Diabetic Retinopathy Study (ETDRS) protocol., Results: We found an overall low score in each VFQ-39 subscale scores reflected by GA. Lesion size and VA were both significantly associated with all VFQ-39 subscale scores except for "general health." VA showed a larger effect on QoL than lesion size. Chronic obstructive pulmonary disease (COPD) was associated with a lower score in the subscale score "general health" but none of the other subscale scores were affected. Cardiovascular disease (CVD) was associated with a lower BCVA as well as in QoL reflected in the subscale scores "poor general vision," "near activities," and "dependency" of VFQ-39., Conclusion: Both atrophic lesion size and visual acuity affects QoL in Danish patients with GA, who reports an overall poor QoL. CVD seems to have a negative effect on disease, as well as in VFQ-39 in several subscales, whereas COPD did not affect disease severity or vision-related subscales in VFQ-39., (© 2023. The Author(s).)

Cyathostomins are the most common and highly prevalent parasites of horses worldwide. Historically, the control of cyathostomins has mainly relied on the routine use of anthelmintic products. Increasing reports on anthelmintic resistance (AR) in cyathostomins are concerning. A potential method proposed for detecting emerging AR in cyathostomins has been estimating the egg reappearance period (ERP). This paper reviews the data available for the ERP of cyathostomins against the three major classes of anthelmintics, macrocyclic lactones, tetrahydropyrimidines, and benzimidazoles. Published peer-reviewed original research articles were obtained from three databases (PubMed, CAB Direct and Web of Science) and were evaluated for their inclusion in a systematic review. Subsets of articles were then subjected to a review of ERP data. A total of 54 (of 134) studies published between 1972 and 2022 met the criteria for inclusion in the systematic review. Until the beginning of 2022, there was no agreed definition of the ERP; eight definitions of ERP were identified in the literature, complicating the comparison between studies. Additionally, potential risk factors for the shortening of the ERP, including previous anthelmintic use and climate, were frequently not described. Reports of shortened ERP for moxidectin and ivermectin are frequent: 20 studies that used comparable ERP definitions reported shortened moxidectin and ivermectin ERPs of 35 and 28 days, respectively. It is unclear whether the ERPs of these anthelmintics reduced to such levels are due to the development of AR or some biological factors related to horses, cyathostomin species, and/or the environment. The ERPs for other anthelmintics, such as fenbendazole and pyrantel, were frequently not reported due to established resistance against these drugs. Future research in horses is required to understand the mechanism(s) behind the shortening of ERP for cyathostomins. Based on this systematic review, we propose recommendations for future ERP studies., Competing Interests: Declaration of competing interest The authors are members of the Australian Equine Parasitology Advisory Panel supported by AgriFutures Australia and Boehringer Ingelheim Animal Health Australia. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The faecal egg count reduction test (FECRT) is the primary diagnostic tool used for detecting anthelmintic resistance at the farm level. It is therefore extremely important that the experimental design of a FECRT and the susceptibility classification of the result use standardised and statistically rigorous methods. Several different approaches for improving the analysis of FECRT data have been proposed, but little work has been published on how to address the issue of prospective sample size calculations. Here, we provide a complete and detailed overview of the quantitative issues relevant to a FECRT starting from basic statistical principles. We then present a new approach for determining sample size requirements for the FECRT that is built on a solid statistical framework, and provide a rigorous anthelminthic drug efficacy classification system for use with FECRT in livestock. Our approach uses two separate statistical tests, a one-sided inferiority test for resistance and a one-sided non-inferiority test for susceptibility, and determines a classification of resistant, susceptible or inconclusive based on the combined result. Since this approach is based on two independent one-sided tests, we recommend that a 90 % CI be used in place of the historically used 95 % CI. This maintains the desired Type I error rate of 5 %, and simultaneously reduces the required sample size. We demonstrate the use of this framework to provide sample size calculations that are rooted in the well-understood concept of statistical power. Tailoring to specific host/parasite systems is possible using typical values for expected pre-treatment and post-treatment variability in egg counts as well as within-animal correlation in egg counts. We provide estimates for these parameters for ruminants, horses and swine based on a re-examination of datasets that were available to us from a combination of published data and other sources. An illustrative example is provided to demonstrate the use of the framework, and parameter estimates are presented to estimate the required sample size for a hypothetical FECRT using ivermectin in cattle. The sample size calculation method and classification framework presented here underpin the sample size recommendations provided in the upcoming FECRT WAAVP guidelines for detection of anthelmintic resistance in ruminants, horses, and swine, and have also been made freely available as open-source software via our website (https://www.fecrt.com)., Competing Interests: Conflict of interest The authors declare that they have no direct conflict of interest with this work., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This review is aimed to (i) appraise the literature on the use of molecular techniques for the detection, quantification and differentiation of gastrointestinal helminths (GIH) of equids, (ii) identify the knowledge gaps and, (iii) discuss diagnostic prospects in equine parasitology. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we retrieved 54 studies (horses: 50/54; donkeys and zebras: 4/54) from four databases. Polymerase chain reaction (PCR) was employed in all of the studies whereas PCR amplicons were sequenced in only 18 of them. Other techniques used (including modifications of PCR) were reverse line blot, quantitative (q)PCR, restriction fragment length polymorphism, nested-PCR, PCR-directed next-generation sequencing, Southern blotting, single strand conformation polymorphism, PCR-enzyme linked immunosorbent assay, matrix-assisted laser desorption/ionisation-time of flight and random amplification of polymorphic DNA. Most of the studies (53/54) used nuclear ribosomal RNA (including the internal transcribed spacers, intergenic spacer, 5.8 S, 18 S, 28 S and 12 S) as target loci while cytochrome c oxidase subunit 1 and random genomic regions were targeted in only three and one studies, respectively. Overall, to date, the majority of molecular studies have focused on the diagnosis and identification of GIHs of equids (i.e. species of Anoplocephala, Craterostomum, cyathostomins, Oesophagodontus, Parascaris, Strongylus, Strongyloides and Triodontophorus), with a recent shift towards investigations on anthelmintic resistance and the use of high-throughput nemabiome metabarcoding. With the increasing reports of anthelmintic resistance in equid GIHs, it is crucial to develop and apply techniques such as advanced metabarcoding for surveillance of parasite populations in order to gain detailed insights into their diversity and sustainable control. To the best of our knowledge, this is the first systematic review that evaluates molecular investigations published on the diagnosis and quantification of equid GIHs and provides useful insights into important knowledge gaps and future research directions in equid molecular parasitology., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Boehringer Ingelheim did not have any role in the design or content of this manuscript., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Context: The Danish health care system provides palliative care for terminally ill patients and their family caregivers. However, initiatives to support family caregivers are not systematically organized., Objectives: We aimed to examine the association between self-reported experience of missing contact to health care professionals involved in palliative care, and symptoms of grief and depression three years post-loss., Methods: We conducted a prospective population-based survey of 3635 family caregivers to terminally ill patients. At six months follow-up, the caregivers reported whether they missed contact to the general practitioner, home care nurse, hospital staff, and/or palliative care team. Associations between missing contact and symptoms of prolonged grief (Prolonged-Grief-13) and depression (Beck Depression Inventory-II) three years after bereavement were analyzed with multivariable logistic regression analysis., Results: We found that an experience of missing contact with health care professionals six months after bereavement was significantly associated with symptoms indicative of prolonged grief disorder and depression after three years. The strongest association was found for missing contact with the general practitioner with an adjusted OR = 4.0 (95%CI: 1.9;8.3) for prolonged grief and an adjusted OR = 5.2 (95% CI: 3.4;7.9) for depression., Conclusion: Experiencing missing contact with health care professionals shortly after bereavement was associated with adverse psychological reactions. Family caregivers may benefit from bereavement support to prevent further complications. A proactive approach with assessment of support needs and risk of complications early during the patient's illness trajectory may target support at those who needs it., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Anthelmintic resistance is reported in equine nematodes with increasing frequency in recent years, and no new anthelmintic classes have been introduced during the past 40 years. This manuscript reviews published literature describing anthelmintic resistance in cyathostomins, Parascaris spp., and Oxyuris equi with special emphasis on larvicidal efficacy against encysted cyathostomin larvae and strongylid egg reappearance periods (ERP). Resistance to benzimidazoles and pyrimidines is highly prevalent in cyathostomin populations around the world, and macrocyclic lactone resistance has been documented in cyathostomins in recent years as well. Two recent studies have documented resistance to the larvicidal regimen of fenbendazole, whereas the larvicidal efficacy of moxidectin is variable, but with no evidence of a reduction from historic levels. In the 1990s, ERP estimates were 8-10 and 12-16 weeks for ivermectin and moxidectin, respectively, while several studies published after year 2000 found ERPs to be 5 weeks for both compounds. This is a clear change in anthelmintic performance, but it remains unclear if this is due to development of anthelmintic resistance or selection for other biological traits leading to a quicker resumption of strongylid egg shedding following anthelmintic treatment. Macrocyclic lactone resistance is common in Parascaris spp. around the world, but recent reports suggests that resistance to the two other classes should be monitored as well. Finally, O. equi has been reported resistant to ivermectin and moxidectin in countries representing four continents. In conclusion, multi-drug resistance is becoming the norm in managed cyathostomin populations around the world, and a similar pattern may be emerging in Parascaris spp. More work is required to understand the mechanisms behind the shortened ERPs, and researchers and veterinarians around the world are encouraged to routinely monitor anthelmintic efficacy against equine nematodes., (Copyright © 2022 The Author. Published by Elsevier Ltd.. All rights reserved.)

Anoplocephala perfoliata is a common tapeworm in horses causing colic and even mortalities. Current diagnostic tests to detect A. perfoliata infections have their limitations and an improved method is needed. Immunoreactive excretory/secretory proteins (E/S proteome) of this parasite can provide promising candidates for diagnostic tests. We compared E/S proteins produced by small (length < 20 mm, width < 5 mm) and large (length 20 to 40 mm, width 5 to 10 mm) A. perfoliata worms in vitro by label-free quantitative proteomics using a database composed of related Hymenolepis diminuta, Echinococcus multilocularis/granulosus and Taenia aseatica proteins for protein identifications. Altogether, 509 E/S proteins were identified after incubating the worms in vitro for three and eight hours. The greatest E/S proteome changes suggested both worm size- and time-dependent changes in cytoskeleton remodeling, apoptosis, and production of antigens/immunogens. The E/S proteins collected at the three-hour time point represented the natural conditions better than those collected at the eight-hour time point, and thereby contained the most relevant diagnostic targets. Immunoblotting using antibodies from horses tested positive/negative for A. perfoliata indicated strongest antigenicity/immunogenicity with 13-, 30- and 100-kDa proteins, involving a thioredoxin, heat-shock chaperone 90 (Hsp90), dynein light chain component (DYNLL), tubulin-specific chaperone A (TBCA) and signaling pathway modulators (14-3-3 and Sj-Ts4). This is among the first studies identifying new diagnostic targets and A. perfoliata antigens eliciting a IgG-response in horses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hautala, Pursiainen, Näreaho, Nyman, Varmanen, Sukura, Nielsen and Savijoki.)

Background: Parasitic nematodes, including large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarids, Parascaris spp., are important parasites of juvenile horses and the first ascarids to develop widespread anthelmintic resistance. The microbiota has been shown to be an important factor in the fitness of many organisms, including parasitic nematodes, where endosymbiotic Wolbachia have been exploited for treatment of filariasis in humans., Methods: This study used short-read 16S rRNA sequences and Illumina sequencing to characterize and compare microbiota of whole worm small intestinal stages and microbiota of male and female intestines and gonads. Diversity metrics including alpha and beta diversity, and the differential abundance analyses DESeq2, ANCOM-BC, corncob, and metagenomeSeq were used for comparisons., Results: Alpha and beta diversity of whole worm microbiota did not differ significantly between groups, but Simpson alpha diversity was significantly different between female intestine (FI) and male gonad (MG) (P= 0.0018), and Shannon alpha diversity was significantly different between female and male gonads (P = 0.0130), FI and horse jejunum (HJ) (P = 0.0383), and FI and MG (P= 0.0001). Beta diversity (Fig. 2B) was significantly different between female and male gonads (P = 0.0006), male intestine (MI) and FG (P = 0.0093), and MG and FI (P = 0.0041). When comparing organs, Veillonella was differentially abundant for DESeq2 and ANCOM-BC (p < 0.0001), corncob (P = 0.0008), and metagenomeSeq (P = 0.0118), and Sarcina was differentially abundant across four methods (P < 0.0001). Finally, the microbiota of all individual Parascaris spp. specimens were compared to establish shared microbiota between groups., Conclusions: Overall, this study provided important information regarding the Parascaris spp. microbiota and provides a first step towards determining whether the microbiota may be a viable target for future parasite control options., (© 2022. The Author(s).)

Macrocyclic lactones have been the most widely used drugs for equine parasite control during the past four decades. Unlike ivermectin, moxidectin exhibits efficacy against encysted cyathostomin larvae, and is reported to have persistent efficacy with substantially longer egg reappearance periods. However, shortened egg reappearance periods have been reported recently for both macrocyclic lactones, and these findings have raised several questions: (i) are egg reappearance period patterns different after ivermectin or moxidectin treatment? (ii) Are shortened egg reappearance periods associated with certain cyathostomin species or stages? (iii) How does moxidectin's larvicidal efficacy affect egg reappearance period? To address these questions, 36 horses at pasture, aged 2-5 years old, were randomly allocated to three treatment groups: 1, moxidectin; 2, ivermectin; and 3, untreated control. Strongylid fecal egg counts were measured on a weekly basis, and the egg reappearance period was 5 weeks for both compounds. Strongylid worm counts were determined for all horses: 18 were necropsied at 2 weeks post-treatment (PT), and the remaining 18 at 5 weeks PT. Worms were identified to species morphologically and by internal transcribed spacer-2 (ITS-2) rDNA metabarcoding. Moxidectin and ivermectin were 99.9% and 99.7% efficacious against adults at 2 weeks post treatment, whereas the respective efficacies against luminal L4s were 84.3% and 69.7%. At 5 weeks PT, adulticidal efficacy was 88.3% and 57.6% for moxidectin and ivermectin, respectively, while the efficacy against luminal L4s was 0% for both drugs. Moxidectin reduced early L3 counts by 18.1% and 8.0% at 2 or 5 weeks, while the efficacies against late L3s and mucosal L4s were 60.4% and 21.2% at the same intervals, respectively. The luminal L4s surviving ivermectin treatment were predominantly Cylicocyclus (Cyc.) insigne. The ITS-2 rDNA metabarcoding was in good agreement with morphologic species estimates but suggested differential activity between moxidectin and ivermectin for several species, most notably Cyc. insigne and Cylicocyclus nassatus. This study was a comprehensive investigation of current macrocyclic lactone efficacy patterns and provided important insight into potential mechanisms behind shortened egg reappearance periods., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The equine ascarids, Parascaris spp., are important nematode parasites of juvenile horses and were historically model organisms in the field of cell biology, leading to many important discoveries, and are used for the study of chromatin diminution. In veterinary parasitology, Parascaris spp. are important not only because they can cause clinical disease in young horses but also because they are the only ascarid parasites to have developed widespread anthelmintic resistance. Despite this, much of the general biology and mechanisms of anthelmintic resistance are poorly understood. This review condenses known basic biological information and knowledge on the mechanisms of anthelmintic resistance in Parascaris spp., highlighting the importance of foundational research programs. Although two variants of this parasite were recognized based on the number of chromosomes in the 1870s and suggested to be two species in 1890, one of these, P. univalens, appears to have been largely forgotten in the veterinary scientific literature over the past 100 years. We describe how this omission has had a century-long effect on nomenclature and data analysis in the field, highlighting the importance of proper specimen identification in public repositories. A summary of important basic biology, including life cycle, in vitro maintenance, and immunology, is given, and areas of future research for the improvement of knowledge and development of new systems are given. Finally, the limited knowledge regarding anthelmintic resistance in Parascaris spp. is summarized, along with caution regarding assumptions that resistance mechanisms can be applied across clades., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study aimed to collect information on local and systemic inflammatory responses, and goblet cell-associated components, following anthelmintic treatment with moxidectin and ivermectin in horses naturally infected with cyathostomin parasites. Thirty-six horses aged 2-5 years of age were randomly allocated to three groups. Group 1 received ivermectin/praziquantel (0.2 mg/kg), Group 2 received moxidectin/praziquantel (0.4 mg/kg) and Group 3 were untreated controls. Tissue samples from the Cecum, Dorsal and Ventral Colons were used for histopathological evaluation and preserved for RNA isolation and gene expression analysis. Whole blood was collected weekly for gene expression analysis as well. The control group had significantly higher inflammation associated with higher larval scores. The treatment groups displayed no differences in larval counts and inflammatory cell populations (p > .05). Mucosal larval counts were positively correlated with goblet cell hyperplasia scores (p = .047). The moxidectin-treated group had a significantly lower expression of IFN-γ (p < .05). The data suggest that removal of cyathostomins reduced the pro-inflammatory response associated with cyathostomin infections. Pro-inflammatory reactions associated with anthelmintic treatment were minimal, but lowest for moxidectin-treated horses. Results suggested that cecum, ventral and dorsal colons responded differently to cyathostomin larvae, which may have implications in the disease process., (© 2022 John Wiley & Sons Ltd.)

Essentially all grazing horses are infected with cyathostomin parasites. Adult cyathostomins reside in the large intestine of the horse and larval stages encyst within intestinal mucosa. Manual worm collection from aliquots of intestinal content is the current gold standard for retrieval and enumeration of luminal parasites, however, no research has been conducted to standardize specific parameters for processing and storage of samples. The aims of this study were (1) to evaluate the precision of current standard operating procedures for enumeration of luminal adult cyathostomin populations, (2) investigate the influence of chosen fixative, either 70 % ethanol or 10 % buffered formalin, as well as storage duration, immediately post necropsy vs. stored for eight weeks, on the magnitude and precision of worm counts, and (3) compare the luminal count magnitude between the three intestinal segments (cecum, ventral colon, dorsal colon). Ten miniature horses were enrolled in this study for euthanasia and necropsy over a four-week period. Luminal worm counts were conducted for 2 % aliquots of the cecum, ventral colon, and dorsal colon and samples were allocated to the two fixatives and the two storage durations. Precision was evaluated by coefficient of variation (CV) and was 13.04 % for total cyathostomin counts. Mean CV for large intestinal segments ranged from 15.31 % to 52.50 % irrespective of fixative used or storage duration. cecum worm counts were significantly lower compared to the ventral colon (p = 0.008) and dorsal colon (p = 0.01). Fixative and storage duration were not statistically associated with count precision or magnitude. This study demonstrated moderate to high precision estimates for luminal cyathostomin worm counts but did not identify any effects of fixative and storage duration within the framework of the study. This is the first study to determine cyathostomin worm count precision, and results will be useful for power analyses in the future., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro . Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.