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Anthelmintic treatment may select for biological traits more relevant than anthelmintic resistance. Some of these biological traits may also affect anthelmintic performance, without fulfilling the definition of resistance. Other non-drug ecological selection pressures could potentially affect anthelmintic performance. 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Is the egg reappearance period (ERP) an early indicator of anthelmintic resistance? • A summary of cyathostomins ERP for equine registered anthelmintics from 1972 to 2022. • Recent developments on the definition of ERP. • Proposed recommendations for future ERP studies. [ABSTRACT FROM AUTHOR]

[Display omitted] • Styrongylid egg reappearance periods were 5 weeks following ivermectin or moxidectin treatment. • Efficacy against adult cyathostomins was >99% for both drugs 2 weeks post-treatment. • Efficacy against luminal L4s was <85% for both anthelmintics 2 weeks post-treatment. • Larvicidal efficacy of moxidectin was within historic ranges. • There was good agreement between morphological and metabarcoding identification methods. 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

Climate change is likely to influence livestock production by increasing the prevalence of diseases, including parasites. The traditional practice of controlling nematodes in livestock by the application of anthelmintics is, however, increasingly compromised by the development of resistance to these drugs in parasite populations. This study used a previously developed simulation model of the entire equine cyathostomin lifecycle to investigate the effect a changing climate would have on the development of anthelmintic resistance. Climate data from six General Circulation Models based on four different Representative Concentration Pathways was available for three New Zealand locations. These projections were used to estimate the time resistance will take to develop in the middle (2040–49) and by the end (2090–99) of the century in relation to current (2006–15) conditions under two treatment scenarios of either two or six yearly whole-herd anthelmintic treatments. To facilitate comparison, a scenario without any treatments was included as a baseline. In addition, the size of the infective and parasitic stage nematode population during the third simulation year were estimated. The development of resistance varied between locations, time periods and anthelmintic treatment strategies. In general, the simulations indicated a more rapid development of resistance under future climates coinciding with an increase in the numbers of infective larvae on pasture and encysted parasitic stages. This was especially obvious when climate changes resulted in a longer period suitable for development of free-living parasite stages. A longer period suitable for larval development resulted in an increase in the average size of the parasite population with a larger contribution from eggs passed by resistant worms surviving the anthelmintic treatments. It is projected that climate change will decrease the ability to control livestock parasites by means of anthelmintic treatments and non-drug related strategies will become increasingly important for sustainable parasite control. Image 1 • The development of anthelmintic resistance under climate change was simulated. • Climate can become more suitable for parasite development, increasing population size. • The time resistance took to develop was linked to changes in parasite population size. • Non-drug related strategies will become increasingly important for parasite control. [ABSTRACT FROM AUTHOR]

Selective anthelmintic therapy has been recommended as a sustainable strategy for cyathostomin control in horse populations for several decades. The traditional approach has been to determine strongyle fecal egg counts (FEC) for all horses, with treatment only recommended for those exceeding a predetermined threshold. The aims are to achieve a reduction of overall egg shedding, while leaving a proportion of the herd untreated, which lowers anthelmintic treatment intensity and reduces selection pressure for development of anthelmintic resistance. This study made use of the cyathostomin model to evaluate the influence of treatment strategies with between 1 and 8 yearly treatment occasions, where either 1) all horses were treated, 2) a predetermined proportion of the herd remained untreated, or 3) horses were treated if their FEC exceeded thresholds between 100 and 600 strongyle eggs per gram. Weather data representing four different climatic zones was used and three different herd age structures were compared; 1) all yearlings, 2) all mature horses 10–20 years old, and 3) a mixed age structure of 1–20 years of age. Results indicated a consistent effect of age structure, with anthelmintic resistance developing quickest in the yearling group and slowest among the mature horses. Development of anthelmintic resistance was affected by treatment intensity and selective therapy generally delayed resistance. Importantly, the results suggest that the effects of selective therapy on resistance development are likely to vary between climatic zones and herd age structures. Overall, a substantial delaying of resistance development requires that the average number of treatments administered annually across a herd of horses needs to be about two or less. However, results also indicate that an age-structured prioritisation of treatment to younger horses should still be effective. It appears that a 'one-size-fits-all' approach to the management of anthelmintic resistance in cyathostomins is unlikely to be optimal. Image 1 [ABSTRACT FROM AUTHOR]

• Month of treatment impacted resistance development in colder climates. • Increased treatment intensity increased resistance development in all climates. • Decreasing from six to four treatments a year had minor reduction of resistance development. • The impact of selective therapy on resistance development was climate- and season dependent. • Selective therapy delayed resistance development, when carried out in the spring. Anthelmintic resistance is widespread in equine cyathostomin populations across the world, and with no new anthelmintic drug classes in the pharmaceutical pipeline, the equine industry is forced to abandon traditional parasite control regimens. Current recommendations aim at reducing treatment intensity and identifying high strongylid egg shedders in a targeted treatment approach. But, virtually nothing is known about the effectiveness of these recommendations, nor their applicability to different climatic regions, making it challenging to tailor sustainable recommendations for equine parasite control. This study made use of a computer model of the entire cyathostomin life-cycle to evaluate the influence of climate and seasonality on the development of anthelmintic resistance in cyathostomin parasites. Furthermore, the study evaluated the impact of recommended programs involving selective anthelmintic therapy on delaying anthelmintic resistance development. All simulations evaluated the use of a single anthelmintic (i.e., ivermectin) over the course of 40 model years. The study made use of weather station data representing four different climatic zones: a cold humid continental climate, a temperate oceanic climate, a cold semi-arid climate, and a humid subtropical climate. Initially, the impact of time of the year was evaluated when a single anthelmintic treatment was administered once a year in any of the twelve months. The next simulations evaluated the impact of treatment intensities varying between 2 and 6 treatments per year. And finally, we evaluated treatment schedules consisting of a combination of strategic treatments administered to all horses and additional treatments administered to horses exceeding a predetermined fecal egg count threshold. Month of treatment had a large effect on resistance development in colder climates, but little or no impact in subtropical and tropical climates. Resistance development was affected by treatment intensity, but was also strongly affected by climate. Selective therapy delayed resistance development in all modelled scenarios, but, again, this effect was climate dependent with the largest delays observed in the colder climates. This study is the first to demonstrate the value of cyathostomin parasite refugia in managing anthelmintic resistance, and also that climate and seasonality are important. This modelling exercise has allowed an illustration of concepts believed to play important roles in anthelmintic resistance in equine cyathostomins, but has also identified knowledge gaps and new questions to address in future studies. [ABSTRACT FROM AUTHOR]

• A model for the complete cyathostomin lifecycle was constructed. • Multiple mechanisms for the genetics of anthelmintic resistance were incorporated. • Treatment strategies were compared for development of anthelmintic resistance. • The genetics of resistance had a large effect on the rate at which resistance developed. • Different strategies had similar relative ranking regardless of genetic mechanism assumed. Previously described models for the free-living and parasitic phases of the cyathostomin life-cycle were combined into a single model for the complete life-cycle. The model simulates a single free-living population on pasture utilising parasite egg output from the horses and localised temperature and rainfall data to estimate infective larval density on herbage. Multiple horses of different ages are possible, each with an individualised anthelmintic treatment programme. Genotypes for anthelmintic resistance are included allowing for up to three resistance genes with 2 alleles each. Because little is known of the genetics of resistance to anthelmintics in cyathostomins, the first use of this model was to compare the effect of different assumptions regarding the inheritance of resistance on model outputs. Comparisons were made between single and two-gene inheritance, where the heterozygote survival was dominant, intermediate or recessive under treatment, and with or without a fitness disadvantage associated with the resistance mechanism. Resistance developed fastest when the heterozygotes survived anthelmintic treatment (i.e. , were dominant) and slowest when they did not (i.e. , were recessive). Resistance was slower to develop when inheritance was poly-genic compared to a single gene, and when there was a fitness cost associated with the resistance mechanism, although the latter variable was the least influential. Importantly, while these genetic factors sometimes had a large influence on the rate at which resistant genotypes built up in the model populations, their order of ranking was always the same, when different anthelmintic use strategies were compared. Therefore, the described model is a useful tool for evaluating different treatment and management strategies on their potential to select for resistance. [ABSTRACT FROM AUTHOR]

Strongylus vulgaris has been found endemic in equine populations subject to parasite control by targeted selective anthelmintic therapy. This study investigated hemostasis in foals naturally infected with S. vulgaris and monitored this response over the course of progressing infection stages. The hemostatic indices D-dimer, antithrombin III (ATIII), fibrinogen, prothrombin time (PT), and activated partial thromboplastin time were evaluated in weekly blood samples for up to 50 weeks in 12 foals born into a herd with high prevalence of S. vulgaris . Results were compared with weekly S. vulgaris antibody enzyme-linked immunosorbent assay values in all foals using a linear mixed effects model with repeated measures and to total numbers of S. vulgaris larvae in nine foals at necropsy with Pearson linear correlation. In the first week of life, all evaluated indices of hemostasis were significantly different from those observed in the rest of the study weeks, corresponding to previously demonstrated aberrancies in neonates. Significant changes were seen for D-dimer in weeks 11–24, 26–27, 30, and 39 compared with week 2, for PT in weeks 12–13 compared with week 6, and for ATIII in week 15 compared with week 4. Strongylus vulgaris antibody levels were statistically associated with D-dimer ( P = .0076) and fibrinogen ( P = .0004) concentrations. Naturally acquired infection with S. vulgaris was associated with changes suggestive of mild activation of coagulation, fibrinolysis, and inflammation. The results of this study may help elucidate the pathogenesis of the endarteritis, thromboembolism, and nonstrangulating intestinal ischemia that is observed in horses with S. vulgaris infection. [ABSTRACT FROM AUTHOR]

Cyathostomins (small strongyles) are ubiquitous in grazing horses and are known pathogens as cause of larval cyathostominosis. As part of their life cycle, cyathostomin larvae invade the mucosal walls of the large intestines and undergo encystment. Newly ingested third stage larvae are known to undergo arrested development and this can lead to an accumulation of encysted burdens over the course of a grazing season. It is believed that the host immune system plays a significant role in triggering this arrestment. Little is known about the development and progression of larval stages in foals that are not expected to mount a pronounced immune response to ingested cyathostomin larvae. This study evaluated counts of encysted larvae measured in 37 foals. The foals were born in 2013, 2014, and 2015 into a parasitology research herd kept without anthelmintic intervention and were humanely euthanatized between 50 and 293 days of age as part of an ongoing parasite transmission study. A mucosal digestion technique was performed to enumerate encysted early third stage (EL3) and late third stage/fourth stage (LL3/L4) larvae in the cecum, ventral colon and dorsal colon. Counts were analyzed statistically to evaluate the influence of foal age, sex, and seasonality on the counts. Total counts as well as LL3/L4 counts were significantly higher during the grazing season (March–November). Three defined age groups (>100, 100–200, >200 days) did not have statistically different counts. Male foals had significantly higher total counts compared to females, and this has not been reported before. The study found that 41% of the recovered larvae were EL3s, but no indication of arrested development of these was observed. This indicates that cyathostomin infection in foals progresses in a manner substantially different from mature horses. [ABSTRACT FROM AUTHOR]

Cyathostomins can cause a severe inflammation of equine large intestine characterized by substantial ventral edema and pronounced protein loss. Anthelmintic treatment of horses can result in a localized inflammatory response in the colonic mucosa of clinically normal horses. The aim of this study was to evaluate the systemic inflammatory response of ponies naturally infected with cyathostomins to single dose representatives of three anthelmintic drug classes, namely, oxibendazole, pyrantel pamoate, and moxidectin. Thirty ponies aged between 1 and 18 years of age were allocated to one of three anthelmintic treatments groups. Anthelmintic efficacy was evaluated using the fecal egg count reduction test per-formed weekly between 2 and 8 weeks post-treatment. Inflammatory responses were evaluated on days 0,1,3, 5, and 14 after treatment using hematology, measurement of the acute phase inflammatory mark-ers serum amyloid A, fibrinogen, haptoglobin, and iron, and real-time PCR measurement of expression of the genes for interleukins 1-ß and -10, tumor necrosis factor-a, and interferon-γ. There were subtle inflammatory responses to treatment, but cytokine expression was significantly associated with the interaction term between treatment group and anthelmintic efficacy (P< 0.05). Of the acute phase markers, only fibrinogen associated with treatment group. The findings suggest that sys-temic inflammatory responses subsequent to anthelmintic treatment of cyathostomin infection are min-imal. It is possible that this response is 'buffered' by anti-inflammatory products of the parasites and/or the anti-inflammatory effects of the macrocyclic lactones. [ABSTRACT FROM AUTHOR]

Abstract: Strongyle parasites are ubiquitous in grazing horses and constitute a potential threat to equine health. Feces were collected from six horses four times daily over a period of 5 days. Fecal egg counts (FECs) were performed to identify any diurnal rhythms in strongyle egg shedding and to quantify variability at the different levels: individual horses, repeated counts, repeated subsamples, different time points, and different days. No significant differences in FECs were found between the different time points (P = .11). The variables—horse, day, subsample, and egg count—accounted for a variance of 104.83, 0.10, 7.24, and 5.61, respectively. The apparent lack of additional variability between the four different time points suggests that time of the day chosen for collecting fecal samples does not constitute a source of error in field studies. The majority of variability exists between different subsamples and repeated egg counts on the same subsamples, whereas the variability of FECs between following days can be considered negligible. The findings of this study have implication for designing and performing field surveillance of strongyle FEC levels and applying the FEC reduction test for evaluating anthelmintic efficacy. [Copyright &y& Elsevier]

Abstract: Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan® probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (C t) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (κ-value=0.78), but with a significantly higher performance of the PCR assay. An association between C t values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples. [Copyright &y& Elsevier]

Development of resistance to anthelmintic drugs by horse strongyles constitutes a growing threat to equine health because it is unknown when new drug classes can be expected on the market. Consequently, parasite control strategies should attempt to maintain drug efficacy for as long as possible. The proportion of a parasite population that is not exposed to anthelmintic treatment is described as being "in refugia" and although many factors affect the rate at which resistance develops, levels of refugia are considered the most important as these parasites are not selected by treatment and so provide a pool of sensitive genes in the population. Accordingly, treatment should be avoided when pasture refugia are small because such treatments will place significant selection pressure for resistance on worm populations. Given this new paradigm for parasite control, it has become important to identify seasons and circumstances wherein refugia are diminished. Free-living stages of equine strongyles are highly dependent on climatic influences, and this review summarises studies of strongyle development and survival under laboratory and field conditions in Northern (cool) temperate, Southern (warm) temperate and subtropical] tropical climates. In Northern temperate climates, refugia are smallest during the winter. In contrast, refugia are lowest during the summer in warm temperate and subtropical/tropical climates. Although adverse seasonal changes clearly have significant effects on the ability of free living stages of strongyle nematode parasites to survive and develop, available data suggest that climatic influences cannot effectively "clean" pastures from one grazing season to the next. [ABSTRACT FROM AUTHOR]

• Mixing neither influenced accuracy nor precision of equine strongylid egg counts. • Shaking the bottle positively affected accuracy, but not precision. • Standardized methodologies for fecal sample processing are warranted. Fecal egg counts are essential monitoring tools in veterinary parasite control. In recent years, several groups have developed automated egg counting systems based on image analysis and deep learning algorithms. Work in our laboratory demonstrated that an automated system performed with significantly better precision than traditional egg counting techniques. However, while the counting process is no longer operator dependent, the pre-analytical homogenization steps still are. This study aimed at evaluating the influence of sample homogenization on diagnostic performance on an automated equine strongylid egg counting system. Samples were collected from 12 horses and assigned to three egg count categories (four samples per category): Low (0–500 eggs per gram (EPG)), Moderate (501–1000 EPG), and High (1001–2000 EPG). Within each category, all samples were divided into four portions and each was analyzed with the automated system using the following four homogenizing procedures using a homogenizing device supplied with the system: 1) pressing the plunger five times and pouring directly into the counting chamber, 2) pressing the plunger five times and shaking the bottle prior to pouring, 3) pressing the plunger ten times with direct pouring, and 4) pressing the plunger ten times with shaking the bottle before pouring. There were no differences in precision expressed as coefficient of variation between these four procedures but shaking of the bottle prior to pouring was significantly associated with higher counts (p = 0.0068). These results demonstrate that the homogenization process can affect the diagnostic performance of an automated egg counting system and suggest that more efforts should be invested in standardizing and optimizing homogenization procedures. [ABSTRACT FROM AUTHOR]

Formulating and disseminating recommendations for parasite control is a dynamic process that requires constant interaction between parasitology researchers and the end-users in the field. Any set of guidelines requires continuous evaluation of efficacy and possible adverse effects, and adjustments are invariably needed along the way. Experience with formulating guidelines for equine parasite control illustrates that end-users struggle with striking the balance between treating too little and too much. [ABSTRACT FROM AUTHOR]

• Samples spiked with parasite ova can be used, but have several issues. • Sensitivity and specificity have limited implications for fecal egg counts. • Detection limit of a technique is not a performance parameter. • Low egg count samples negatively affect sensitivity and precision estimates. • Precision is the most important parameter for fecal egg count techniques. The first parasite fecal egg counting techniques were described over 100 years ago, and fecal egg counting remains essential in parasitology research as well as in clinical practice today. Several novel techniques have been introduced and validated in recent years, but this work has also highlighted several current issues in this research field. There is a lack of consensus on which diagnostic parameters to evaluate and how to properly design studies doing so. Furthermore, there is a confusing and sometimes incorrect use of terminology describing performance of fecal egg counting techniques, and it would be helpful to address these. This manuscript reviews qualitative and quantitative diagnostic performance parameters, discusses their relevance for fecal egg counting techniques, and highlights some of the challenges with determining them. Qualitative parameters such as diagnostic sensitivity and specificity may be considered classic diagnostic performance metrics, but they generally only have implications at low egg count levels. The detection limit of a given technique is often referred to as the "analytical sensitivity", but this is misleading as the detection limit is a theoretically derived number, whereas analytical sensitivity is determined experimentally. Thus, the detection limit is not a diagnostic performance parameter and does not inform on the diagnostic sensitivity of a technique. Quantitative performance parameters such as accuracy and precision are highly relevant for describing the performance of fecal egg counting techniques, and precision is arguably the more important of the two. An absolute determination of accuracy can only be achieved by use of samples spiked with known quantities of parasite ova, but spiking does not necessarily mimic the true distribution of eggs within a sample, and accuracy estimates are difficult to reproduce between laboratories. Instead, analysis of samples from naturally infected animals can be used to achieve a relative ranking of techniques according to egg count magnitude. Precision can be estimated in a number of different approaches, but it is important to ensure a relevant representation of egg count levels in the study sample set, as low egg counts tend to associate with lower precision estimates. Coefficients of variation generally provide meaningful measures of precision that are independent of the multiplication factor of the techniques evaluated. Taken together, there is a need for clear guidelines for studies validating fecal egg counting techniques in veterinary parasitology with emphasis on what should be evaluated, how studies could be designed, and how to appropriately analyze the data. Furthermore, there is a clear need for better consensus regarding use of terminology describing the diagnostic performance of fecal egg count techniques. [ABSTRACT FROM AUTHOR]

[Display omitted] • Coproculture and PCR had similar performance at or above 50 % Strongylus vulgaris -positive feces. • For pools with less than 50 % S. vulgaris -positive feces, the PCR was more sensitive. • Both methods detected significantly higher levels in pools at the 100 % infected level. • The qPCR results did not provide quantitative information across pool concentrations. Strongylus vulgaris is the most pathogenic intestinal helminth parasite infecting horses. The migrating larvae in the mesenteric blood vessels can cause non-strangulating intestinal infarctions, which have a guarded prognosis for survival. Infections are typically diagnosed by coproculture, but a PCR test is available in some countries. While it is ideal to test horses individually, many veterinarians and clients wish to pool samples to reduce workload and cost of the diagnostic method. The purpose of this study was to determine if pooling of fecal samples would negatively impact diagnostic performance of the coproculture and the PCR for determination of S. vulgaris infection. Ten horses with strongylid eggs per gram (EPG) >500 and confirmed as either S. vulgaris positive or negative were selected as fecal donors. Eight pools with feces from five horses were created with 0%, 10 %, 20 %, 30 %, 40 %, 50 %, 80 %, and 100 % S. vulgaris positive feces. From each pool, 20 subsamples of 10 g each were collected and analyzed. Half of these samples were set up for coproculture and the other half for PCR. All pools containing 50 % or greater S. vulgaris positive feces were detected positive by both PCR and coproculture. In the pools with less than 50 % S. vulgaris positive feces, the PCR detected 33 positive samples compared to 24 with the coproculture. Three samples from the 0% pool were detected as low-level PCR positives, but this could be due to contamination. These results indicate that diagnosing S. vulgaris on pooled samples is reliable, when at least 50 % of the feces in a pool are from S. vulgaris positive animals. Since S. vulgaris remains relatively rare in managed horses, however, some diagnostic sensitivity is expected to be lost with a pooled sample screening approach. Nonetheless, pooled sample screening on farms could still be considered useful under some circumstances, and the PCR generally performed better at the lower proportions of S. vulgaris positive feces. [ABSTRACT FROM AUTHOR]

• Estimating cyathostomin larval counts by mucosal digestion had low precision. • No spatial trends were observed within intestinal segments. • The dorsal colon had significantly lower counts of developing larvae. • Increasing sample replicates improved confidence intervals. Cyathostomins are pervasive parasites of equids across the world. Larval stages encyst in the mucosa of the cecum, ventral and dorsal colon and can induce an inflammatory response leading to larval cyathostominosis, a life-threatening generalized typhlocolitis. Mucosal digestion is the only gold standard procedure for identifying and quantifying all larval stages. There is a lack of standardization of this technique and several aspects are ambiguous, such as precision of the method and the possibility of spatial variation of mucosal larval counts. The aim of this study was to estimate precision for enumeration of early third stage larvae (EL3) and late third stage/fourth stage (LL3/L4) larvae and investigate spatial variation of encysted counts within large intestinal organs. Six naturally infected and untreated horses aged 2–5 years were euthanized as part of an anthelmintic efficacy study, and the cecum (Cec), ventral colon (VC) and dorsal colon (DC) were collected. Each organ was rinsed, weighed, and visually separated into 3 equally sized sections. Two 5% tissue samples were collected from each section, a total of six replicates per organ. The mucosae were digested, and 2% examined under the microscope for presence of EL3 and LL3/L4 stage larvae. Overall, 59 % of the harvested larvae were EL3s, and 41 % were LL3/L4s. The ventral colons represented 45 % of the total organ weight, and contributed 37 and 41 % of the EL3s and LL3/L4s harvested, respectively. The Cec, representing only 27 % of the weight contributed 23 % of EL3s and 47 % of LL3/L4s. The DC represented 28 % of the total organ weight, and 28 % and 12 % of the total EL3s and LL3/L4s, respectively. Coefficients of variation varied from 33 to 183 % for EL3 counts and 38–245% for LL3/L4 counts. There were no statistically significant associations between EL3 counts and either organ or location. For LL3/L4 counts there were no statistically significant differences between the three locations within organs (p = 0.1166), but the DC had significantly lower counts than the other two organs (p < 0.0001). Increasing the number of mucosal replicates from each organ improved estimation, but required a considerably increased workload. In conclusion, mucosal larval cyathostomin counts are highly variable, complicating their use for treatment efficacy estimation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Commercial translation of an ELISA for mucosal/luminal cyathostomins is described. • The test demonstrates high (>0.9) ROC-AUC values for various cyathostomin burden thresholds. • The test has high sensitivity (>90%)/specificity (>70%) at selected cyathostomin burden ELISA cut-off values. • The percentage of test-positives associates with egg shedding levels in populations. • Logistic regression modelling shows promise as a method for predicting burden. 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. [ABSTRACT FROM AUTHOR]

The use of fecal egg count techniques to indirectly assess intestinal parasite burdens and determine anthelmintic efficacy is common in parasitological research and veterinary practice. The McMaster method is one of the most widely used techniques in veterinary practice, but recently, the Mini-FLOTAC technique has been introduced as a possible alternative. Studies comparing the two methods in precision and accuracy are needed. This study aimed at evaluating the Mini-FLOTAC technique for determining equine strongyle egg counts through a two-part procedure. First, a set of fecal counts was executed using both methods. Next, blind counts were performed on spiked fecal samples with true egg counts at 0, 5, 50, 500, and 1,000 eggs per gram. All counts were performed in triplicates, and each sample was counted using both methods. Mini-FLOTAC and McMaster had 83.2% and 53.7% precision, respectively. The accuracy was found to be 42.6% and 23.5% for Mini-FLOTAC and McMaster, respectively. In conclusion, this study found that Mini-FLOTAC exhibited both higher precision and accuracy than the McMaster technique and appears to be a more reliable alternative. Using a more precise egg-counting method can help assure that changes in egg counts before and after treatment reflect a genuine reduction and are not due to chance variability. [ABSTRACT FROM AUTHOR]

• Equine strongyle type, ascarid, and anoplocephalid eggs move at different speeds through flotation solution. • Strongyle type eggs moved the fastest, followed by ascarid and then anoplocephalid egg types. • The difference in mean flotation speed was statistically significant between egg types. The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotation protocols exist, little is known about the characteristics of these eggs with respect to their movement through a flotation solution. In this study, we have demonstrated a novel method for the observation and quantification of microscopic (65–100 µm) objects as they experience unassisted flotation. This also represents, to our knowledge for the first time, that the flotation of parasite eggs has been observed and their movement characteristics quantified as they float through solution. Particle tracking and video analysis software were utilised to automatically detect and track the movement of individual eggs as they floated. Three 30 s videos and one 2 min video of each egg type were analysed. If the first 30 s of video were discounted, the differences in mean flotation speed among all videos was statistically significant between egg types (P = 0.0004). Strongyle type eggs (n = 201) moved the fastest with a mean 51.08 µm/s (95% confidence interval: 47.54–54.62). This was followed by Parascaris spp. (n = 131) and Anoplocephala perfoliata eggs (n = 322), with mean speeds of 44.43 µm/s (95% confidence interval: 39.47–49.4) and 31.11 µm/s (95% confidence interval: 29.6–32.61), respectively. This method for evaluating the mean speed of passive flotation may represent a first step towards further optimizing fecal egg flotation and be of interest to parasitologists and veterinary practitioners. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate whether aged horses demonstrate statistically higher fecal egg counts (FECs) compared with middle-aged horses and to investigate systemic expression of proinflammatory cytokines in old and middle-aged horses treated with moxidectin compared with horses treated with pyrantel pamoate. Old horses (n = 21) and middle-aged horses (n = 19) were blocked by levels of inflammation and randomly allocated to one of the following treatment groups: group 1 (n = 8) old treated with moxidectin gel; group 2 (n = 9) old treated with pyrantel pamoate paste; group 3 (n = 4) old receiving no treatment; group 4 (n = 8) middle-aged treated with moxidectin gel; group 5 (n = 7) middle-aged treated with pyrantel pamoate paste, and group 6 (n = 4) middle-aged receiving no treatment. Fecal samples were collected on day 0 and again 14 days after treatment to determine FECs and presence of Strongylus vulgaris by polymerase chain reaction. Peripheral blood was also collected on day 0 and again at days 3, 5, and 14 after deworming for inflammatory cytokine analysis, along with routine hematological analyses. Results indicated that old horses have significantly higher FEC than middle-aged adults. Fecal egg counts declined significantly after anthelmintic treatment in both age groups of horses. Inflammatory markers exhibited differences between age groups and by anthelmintic treatment. In summary, this study provided evidence of different inflammatory and immunologic reactions to anthelmintic treatment in old horses. Moreover, the higher FECs found in the old horse group may have practical implications for parasite management routines on farms with representation of this age group. [ABSTRACT FROM AUTHOR]

Parasitic nematodes, particularly cyathostomins, are ubiquitous in grazing horses world-wide. Considerable burdens of cyathostomin larvae can encyst in the large intestinal wall. The most recommended treatment against these pathogenic stages is moxidectin. Information is required on how effective moxidectin is against cyathostomin populations in different regions. The objectives here were to determine the efficacy of moxidectin treatment and estimate the strongyle egg reappearance period (ERP) after treatment in several equine populations, to confirm the type of strongyle nematodes present and to identify other (i.e. management) factors associated with shortened ERP. Eight yards were recruited and moxidectin in combination with praziquantel administered to all horses (n = 261). Faecal egg count (FEC) analysis was performed at weeks 0, 2, 6, 10 and 12 after treatment to determine efficacy and ERP. The ERP was estimated using two previously published methods. Morphological identification of cultured third stage larvae from the sample population was compared to a S trongylus vulgaris -specific end-point PCR to examine the presence of S. vulgaris in samples before and after treatment. Strongyle egg shedding patterns were also compared to worm management practices at each site. At 2 weeks post-treatment, moxidectin was highly effective (faecal egg count reduction range, 99.9–100%). The strongyle ERP ranged from 6 weeks to >12 weeks depending on the calculation method applied. Only cyathostomin larvae were detected by morphological identification. The results from the coprocultures and PCR showed that S. vulgaris was absent before and after treatment. Analysis revealed that regular faecal removal from pasture was associated with lower average FEC and lower prevalence of egg shedding. [ABSTRACT FROM AUTHOR]