Identification of third stage larvae of strongyles and molecular diagnosis of Strongylus vulgaris in the feces of Thoroughbred horses kept in training centers in Rio de Janeiro, Brazil.

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals.

Diagnosing Strongylus vulgaris in pooled fecal samples.

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.

A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses.

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

Diagnostic performance of McMaster, Wisconsin, and automated egg counting techniques for enumeration of equine strongyle eggs in fecal samples.

Fecal egg counts are the cornerstone of equine parasite control programs. Previous work led to the development of an automated, image-analysis-based parasite egg counting system. The system has been further developed to include an automated reagent dispenser unit and a custom camera (CC) unit that generates higher resolution images, as well as a particle shape analysis (PSA) algorithm and machine learning (ML) algorithm. The first aim of this study was to conduct a comprehensive comparison of method precision between the original smartphone (SP) unit with the PSA algorithm, CC/PSA, CC/ML, and the traditional McMaster (MM) and Wisconsin (MW) manual techniques. Additionally, a Bayesian analysis was performed to estimate and compare sensitivity and specificity of all five methods. Feces were collected from horses, screened with triplicate Mini-FLOTAC counts, and placed into five categories: negative (no eggs seen), > 0 - ≤ 200 eggs per gram (EPG), > 200 - ≤ 500 EPG, > 500 - ≤ 1000 EPG, and > 1000 EPG. Ten replicates per horse were analyzed for each technique. Technical variability for samples > 200 EPG was significantly higher for MM than CC/PSA and CC/ML (p <  0.0001). Biological variability for samples> 0 was numerically highest for CC/PSA, but with samples > 200 EPG, MM had a significantly lower CV than MW (p =  0.001), MW had a significantly lower CV than CC/PSA (p <  0.0001), CC/ML had a significantly lower CV than both MW and SP/PSA (p <  0.0001, p =  0.0003), and CC/PSA had a significantly lower CV than CC/SP (p =  0.0115). Sensitivity was> 98 % for all five methods with no significant differences. Specificity, however, was significantly the highest for CC/PSA, followed numerically by SP/PSA, MM, CC/ML, and finally MW. Overall, the automated counting system is a promising new development in equine parasitology. Continued refinement to the counting algorithms will help improve precision and specificity, while additional research in areas such as egg loss, analyst variability at the counting step, and accuracy will help create a complete picture of its impact as a new fecal egg count method.

Characterisation of serum IgG(T) responses to potential diagnostic antigens for equine cyathostominosis.

Cyathostomins are ubiquitous parasitic nematodes of horses. These worms spend substantial periods as intestinal wall stage encysted larvae, which can comprise up to 90% of the total burden. Several million larvae have been reported in individuals. Emergence of these larvae from the gut wall can lead to life-threatening colitis. Faecal egg count tests, increasingly used by horse owners to inform anthelmintic treatments, do not correlate with the intra-host burden of cyathostomins; this represents a key gap in the diagnostic toolbox. Previously, a cyathostomin Gut Associated Larval Antigen was identified as a promising marker for the intra-host stages of infection. Here, cyathostomin Gut Associated Larval Antigen and an additional protein, Cyathostomin Immuno-diagnostic antigen, were investigated to examine their value in providing information on cyathostomin burden. ELISA analyses examined serum IgG(T) responses to recombinant proteins derived from individual cyathostomin species. Receiver Operator Characteristic curve analysis was performed on the ELISA data; proteins with the highest Area Under the Curve values were selected to test protein combinations to investigate which were the most informative in identifying the infection status of individuals. Three cocktail combinations were tested, comprising: (a) Cy-GALA proteins from two species and a Cy-CID protein from a third species (CT3), (b) Cy-GALA proteins from five species (CT5), and (c) all CT5 components, plus a Cy-CID protein from an additional species (CT6). The best predictive values for infection were obtained using CT3 and CT6, with similar values achieved for both. Proteins in CT3 are derived from the most commonly reported species, Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus longibursatus. This combination was selected for future development since it represents a more commercially viable format for a diagnostic test.

Reliability of three common fecal egg counting techniques for detecting strongylid and ascarid infections in horses.

The detection and quantification of nematode eggs using fecal egg count techniques have an irreplaceable role in equine parasitic control. The reliability, particularly precision and accuracy, of individual techniques have been described only for strongylid infections. The aim of this study was to compare three fecal egg count techniques used for the detection of the two most common equine nematode infections: strongylid and ascarid. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques were tested on spiked fecal samples with various levels of egg concentration (50, 100, 200, 500, 1000 and 3000 eggs per gram) and naturally infected mixed strongylid-ascarid samples with 30 replicates. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques had precision coefficients of variation of 44.33, 35.64 and 18.25% for the strongylid infection and 62.95, 35.71 and 18.95% for the ascarid infection, and percent accuracies (mean count/number of eggs spiked) of 97.53, 88.39 and 74.18% for the strongylid infection and 65.53, 83.18 and 90.28% for the ascarid infection, respectively. Accuracy depended greatly on the type of nematode, but precision did not. The Mini-FLOTAC technique was more precise than the Simple and Concentration McMaster techniques regardless of nematode type. Simple McMaster was the most accurate technique for detecting strongylid eggs, and Mini-FLOTAC was the most accurate technique for detecting ascarid eggs. Our results indicated that none of the current techniques were universally and sufficiently reliable for the simultaneous quantification of both of these common equine nematodes.

Identification of third stage larval types of cyathostomins of equids: An improved perspective.

Cyathostomins comprise around 50 parasite species of equids, offering a great challenge regarding their individual identification. The objective of our work was to improve identification of infective third stage larvae (L3) with a morphological key supplemented with detailed scientific illustrations based on our research and available literature. The highlighted features were; the number, arrangement, and shape of intestinal cells (IC), general features and the total body length of the eight different Cyathostomin sensu latum types (Type A, B, C, D, E, F, G, H), Gyalocephalus capitatus, and Posteriostomum spp. Due to variability, we were unable to define final body length measurements to differentiate L3 of cyathostomins. However, IC characteristics displayed a higher difference between L3 types than total body length. Through the number and arrangement of IC, 14 species were classified within three larval types. The classification of L3 into distinct larval types sensu latum gives us the advantage of reducing the number of probable species presented in equine faecal samples using a low-cost technique when monitoring the parasite fauna present in individual horses or on the farm level. The present improved identification key shall increase the diagnostic capabilities of classical equine parasitology techniques, using general L3 morphology thereby pragmatically improving regional and transnational epidemiological and biodiversity studies. The present key may also assist in defining the cyathostomin community in cyathostominosis clinical cases and within drug resistant populations across different management systems and geographical locations.

Are small strongyles (Cyathostominae) involved in horse colic occurrence?

Strongyle infections have been traditionally regarded as a possible cause of colic in horses. Aim of the study was the comparison of parasitological status between subjects with or without colic syndrome, with particular attention to small strongyle infections. Coprological analyses were performed on 86 horses: 43 with colic and 43 controls. Strongyle eggs were found in 34/86 horses (prevalence 39.5%), the mean number of strongyles eggs per gram of faeces (EPG)2 was 145.34 (standard deviation 398.28). All those 34 positive animals had small strongyles infections. Negative binomial multiple regression highlighted no influence of horse sex on strongyle EPG, while there was a negative relationship between age and EPG (p <0.05); the same analysis revealed a significant difference of EPG (p <0.05) between control horses (mean EPG=178.1; standard deviation: 411.4) and horses with surgical colic (mean EPG=68.6 standard deviation: 259.8) when controlling for S. vulgaris presence including it in the model. On the contrary, the intensity of infection in horses with non-surgical colic (mean EPG=154.5; standard deviation: 480.4) did not significantly differed from controls. Similar results were obtained having estimated cyathostomine EPGs as dependent variable. Multinomial logistic regression confirmed the negative relationship between cyathostonine presence and surgical colic occurrence. It is possible that (1) the presence of adult luminal parasites, would have a protective effect against the pathogenic action exerted by the development and emergence of small strongyles larvae from intestinal mucosa; (2) the management practices able to reduce the risk of colics are the same that cause higher exposure to strongyle infective larvae.

Detection of Strongylus vulgaris in equine faecal samples by real-time PCR and larval culture - method comparison and occurrence assessment.

BACKGROUND: Strongylus vulgaris has become a rare parasite in Germany during the past 50 years due to the practice of frequent prophylactic anthelmintic therapy. To date, the emerging development of resistance in Cyathostominae and Parascaris spp. to numerous equine anthelmintics has changed deworming management and the frequency of anthelmintic usage. In this regard, reliable detection of parasitic infections, especially of the highly pathogenic S. vulgaris is essential. In the current study, two diagnostic methods for the detection of infections with S. vulgaris were compared and information on the occurrence of this parasite in German horses was gained. For this purpose, faecal samples of 501 horses were screened for S. vulgaris with real-time PCR and an additional larval culture was performed in samples of 278 horses. A subset of 26 horses underwent multiple follow-up examinations with both methods in order to evaluate both the persistence of S. vulgaris infections and the reproducibility of each diagnostic method. RESULTS: The real-time PCR revealed S. vulgaris-DNA in ten of 501 investigated equine samples (1.9%). The larval culture demonstrated larvae of S. vulgaris in three of the 278 samples (1.1%). A direct comparison of the two methods was possible in 321 samples including 43 follow-up examinations with the result of 11 S. vulgaris-positive samples by real-time PCR and 4 S. vulgaris-positive samples by larval culture. The McNemar's test (p-value = 0.016) revealed a significant difference and the kappa values (0.525) showed a moderate agreement between real-time PCR and larval culture. CONCLUSIONS: The real-time PCR detected a significantly higher proportion of positives of S. vulgaris compared to larval culture and should thus be considered as a routine diagnostic method for the detection of S. vulgaris in equine samples.

Repeatability of strongyle egg counts in naturally infected horses.

The selective treatment of horses is used to decrease the number of anthelmintic treatments by only treating a proportion of animals in the population. One way to select animals for treatment is to identify low and high egg-shedders using faecal egg counts (FEC); then to treat only the high egg-shedders. The value of this method is enhanced if differences among individuals in the level of egg-shedding remain consistent over time. One way to assess the stability of the rankings of animals over time is to measure the repeatability which is defined as the variance between horses divided by the total variance. The repeatability varies between 0 (no consistency in the values) to 1 (perfect consistency). To determine the repeatability of egg-shedding in naturally infected horses over time, 2637 FEC and raw egg counts (REC; i.e. originally counted eggs without multiplication factor) from 303 horses were analysed. The distribution of FEC was more overdispersed than a Poisson distribution. Therefore, a negative-binomial model was used. The within-horse-repeatability of RECs was 0.52. In a second analysis, we excluded horses that were treated with anthelmintic drugs during the study by eliminating all REC within the egg-reappearance-period. Here, the within-horse-repeatability was very similar at 0.53. The results show that egg-shedding of individual horses stays fairly consistent over time. They also show that animals which shed relatively high numbers of nematode eggs can be identified and targeted for treatment.

Development of a recombinant protein-based ELISA for diagnosis of larval cyathostomin infection.

Cyathostomins are ubiquitous nematodes of horses. Once ingested, they can spend a substantial time as encysted larvae in the intestinal wall. The larvae can comprise up to 90% of the total burden, with up to several million worms reported in individuals. These stages can emerge in large numbers to cause life-threatening colitis. Direct methods for detection of encysted larval burdens in live horses do not exist. Previously, two antigen complexes were identified as promising markers for infection. A component of these, cyathostomin gut associated larval antigen-1 (Cy-GALA-1), was identified following immunoscreening of a complementary DNA library. Serum immunoglobulin G(T) (IgG(T)) responses to Cy-GALA-1 were shown to inform on larval infection. Sequence analysis of polymerase chain reaction products amplified from individual worms indicated that Cy-GALA-1 was derived from Cyathostomum pateratum. As cyathostomin infections always comprise multiple species, a diagnostic test must account for this. Here, segments of the Cy-gala gene were isolated from four common species, Cyathostomum catinatum, Cylicocyclus ashworthi, Cylicostephanus goldi and Cylicostephanus longibursatus, and the associated proteins expressed in recombinant form. The specificity and immunogenicity of each protein was confirmed. Each protein was assessed by enzyme linked immuno sorbent assay (ELISA) for its ability for informing on the presence of encysted larval infection and the level of burden.

Effect of vacuum packing and temperature on survival and hatching of strongyle eggs in faecal samples.

Strongyle eggs of helminths of livestock usually hatch within a few hours or days after deposition with faeces. This poses a problem when faecal sampling is performed in the field. As oxygen is needed for embryonic development, it is recommended to reduce air supply during transport and refrigerate. The present study therefore investigated the combined effect of vacuum packing and temperature on survival of strongyle eggs and their subsequent ability to hatch and develop into L3. Fresh faecal samples were collected from calves infected with Cooperia oncophora, pigs infected with Oesophagostomum dentatum, and horses infected with Strongylus vulgaris and cyathostomins. The samples were allocated into four treatments: vacuum packing and storage at 5 °C or 20 °C (5 V and 20 V); normal packing in plastic gloves closed with a loose knot and storage at 5 °C or 20 °C (5 N and 20 N). The number of eggs per gram faeces (EPG) was estimated every fourth day until day 28 post set up (p.s.) by a concentration McMaster-method. Larval cultures were prepared on day 0, 12 and 28 p.s. and the larval yield determined. For C. oncophora, the EPG was significantly higher in vacuum packed samples after 28 days as compared to normal storage, regardless of temperature. However, O. dentatum EPG was significantly higher in samples kept at 5 °C as compared to 20 °C, irrespective of packing. For the horse strongyles, vacuum packed samples at 5 °C had a significantly higher EPG compared to the other treatments after 28 days. The highest larval yield of O. dentatum and horse strongyles were obtained from fresh faecal samples, however, if storage is necessary prior to setting up larval cultures O. dentatum should be kept at room temperature (aerobic or anaerobic). However, horse strongyle coprocultures should ideally be set up on the day of collection to ensure maximum yield. Eggs of C. oncophora should be kept vacuum packed at room temperature for the highest larval yield.

[Helminth control in the adult horse: the need for a re-orientation]. / Helminthenmanagement beim adulten Pferd: Notwendigkeit einer Neuorientierung.

The epidemiological situation of strongyle infections in adult horses in Switzerland is characterized by a strong dominance of small strongyles (Cyathostominae) and an overall low level of egg shedding in the faeces. The prevailing attitude towards anthelmintic therapy considers neither husbandry conditions nor pasture hygiene measures. Instead, calendar-based routine medication, comprising usually 3 to 4 annual treatments, is the typical strategy. Such an approach, however, often results in an excessive administration of anthelmintics. With respect to the continuous spread of drug resistant cyathostomins a change of strategy seems inevitable. A consensus has been agreed on between equine parasitologists and clinicians of the Vetsuisse Faculty in Zurich and Berne to focus on the concept of a selective control approach, based on individual faecal egg counts as the central element. It is now recommended that clinically healthy horses (> 4 y) are treated only when their strongyle egg count is equal to or higher than 200 eggs per gram of faeces. A regular analysis of the strongyle population based on larval cultures, the control of drug efficacy, and quarantine measures for incoming horses are mandatory components of the concept. Recent experiences in several pilot farms have indicated that only 4 % of the McMaster analyses resulted in a deworming treatment. For horses that did not receive any nematicidal anthelmintic during the current season, a "safety" treatment is recommended at the end of the grazing period.

En Suisse, la situation épidémiologique des infestations des chevaux adultes par les strongylidés est caractérisée par une nette dominance des petits strongles (Cyathostominae) et par un faible niveau d'excrétion des œufs de parasites dans les selles. Cars les conditions de détention des chevaux et les mesures relatives à l'hygiène des pâtures ne sont que rarement prises en compte dans la planification des mesures de contrôle des parasitoses, il en résulte un schéma de traitement de routine basé sur 3 à 4 traitements par année, ce qui représente un usage d'anthelminthiques souvent supérieur à la nécessité. Vu le développement continu de populations de cyatostomes résistants aux anthelminthiques, un changement de stratégie dans le contrôle des helminthes est nécessaire. Le contrôle sélectif propagé par les parasitologues et les cliniciens des deux sites de la faculté Vetsuisse propose de seulement traiter les chevaux sains adultes (> de 4 ans) si l'excrétion des œufs de strongles dépasse 200 œufs par gramme de selles. Une différentiation régulière des populations de strongles, le contrôle de l'efficacité des anthelminthiques et des mesures de quarantaines chez les nouveaux venus sont des composants indispensables de ce concept. Les expériences faites jusqu'à présent avec cette stratégie dans plusieurs exploitations-pilotes montrent que seulement 4 % des analyses coprologiques sont suivies par une application d'anthelminthiques. Pour les chevaux qui n'ont pas été vermifugés pendant toute la saison, un traitement de sécurité à la fin de la saison de pâturage est recommandé.

Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis.

Strongyle and trichostrongyle parasites are ubiquitous nematodes of grazing livestock. Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well known that such eggs undergo development during storage, but it remains unknown to which extent developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralized water. A total of 25 wells containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were examined on an inverted microscope at 100× magnification, where the developmental stages of the eggs were noted, and then eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted using a commercial kit previously validated for strongyle type eggs. PCR reactions were performed with a primer set specific for the ribosomal DNA region for all strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis.

Recent advances in diagnosing pathogenic equine gastrointestinal helminths: the challenge of prepatent detection.

Parasites infecting horses are ubiquitous and clinically important across the world. The major parasitic threats to equine health are cyathostomins, Parascaris equorum, Anoplocephala perfoliata, and Strongylus vulgaris. Increasing levels of anthelmintic resistance reported world wide in equine parasites have led to recommendations of constructing sustainable parasite control programmes based on systematic surveillance of parasite levels. Regulations at the European Union level now make anthelmintics available on prescription-only basis and disallow prophylactic treatment. This emphasizes the needs for reliable and practical diagnostic tools for detection of major parasites infecting equines. The current, widely used coprological techniques are important and useful, but they do have considerable limitations as they are incapable of diagnosing the pathogenic migrating stages. Species-specific molecular assays have been developed for diagnosing patent infections with 21 cyathostomin species, A. perfoliata, and S. vulgaris, but none of these have found use in practice. An antibody-directed enzyme-linked immunosorbent assay (ELISA) has been developed, validated and made commercially available for diagnosing A. perfoliata infection, but interpretation is complicated by the fact that horses not harbouring tapeworms can maintain elevated antibody titres. Recent work with a coproantigen ELISA has shown promise for reliable detection of current A. perfoliata infection. Perhaps most remarkable is the fact that the pathogenic larval stages of cyathostomins and large strongyles cannot be detected by any of the available diagnostics. With the lengthy prepatency periods characterizing these parasites, there is a huge need for developing such assays. The recent identification of a possible diagnostic marker for encysted cyathostomins holds great promise, and could become very useful in clinical practice. Several attempts have been made to construct assays for diagnosing the highly pathogenic migrating larvae of S. vulgaris, but none of these have performed sufficiently to make a useful test. The present review illustrates that classical coprological techniques remain the cornerstone of equine parasitology diagnosis and surveillance, and will remain so in a foreseeable future. However, promising progress has been made for developing assays capable of diagnosing prepatent stages of strongyle infection, and there is reason to hope for validated and useful assays in the relative near future.

[Effect of distribution of eggs of strongyles and Parascaris equorum in faecal samples of horses on detection with a combined sedimentation-flotation method]. / Einfluss der Verteilung von Strongyliden- und Ascarideneiern in Pferdekotproben auf den Nachweis mit einem kombinierten Sedimentations-Flotations-Verfahren.

OBJECTIVE: Results of parasitological examination of faecal aliquots may vary between diagnostic laboratories. To examine whether inhomogeneous distribution of worm eggs in faecal samples is responsible for this observation, horse faeces provided for routine diagnosis of helminth infection were examined. Distribution of worm eggs was assessed by examining aliquots taken from different locations of the faecal sample by a combined sedimentation-flotation method (KSFV). In addition, it was tested, whether the homogenization of a larger amount (minimum of 40 g) of faeces before performing KSFV improved reproducibility of the method. MATERIAL AND METHODS: 51 faecal samples of horses were examined three times in parallel by KSFV with ZnSO4 solution. 10 g aliquots were taken from the margin (R), from inside (I) and from both locations (G). The remaining amount of faeces was weighed, suspended with water 1:1 and homogenized. Subsequently, three subsamples, each consisting of 20 g of this suspension, were taken and examined by KSFV. RESULTS: The egg numbers of the nematodes (strongyles and Parascaris equorum ) found in samples that originated from different locations were similar and variation was low. The homogenization of a larger amount of faeces had no relevant impact on egg counts of these nematodes. CONCLUSION AND CLINICAL RELEVANCE: Nematode infections are relevant and frequently occurring in the horse, and reliable assessment of worm egg excretion is a critical aspect for rational planning of control measures. It could be shown that the distribution of nematode eggs (strongyles and Parascaris equorum ) in horse faeces is quite even and results are in principle reproducible if 10 g faeces are examined by KSFV. The homogenization of a larger amount of faeces does not improve the sensitivity or reproducibility of KSFV, and is thus dispensable. For diagnostic purposes, it is advisable to ship approximately 50g of horse faeces to the laboratory.

Evaluation of conventional PCR for detection of Strongylus vulgaris on horse farms.

Strongyle parasites are ubiquitous in grazing horses. Of these, the bloodworm Strongylus vulgaris is regarded as most pathogenic. Increasing levels of anthelmintic resistance in strongyle parasites has led to recommendations of decreased treatment intensities, and there is now a pronounced need for reliable tools for detection of parasite burdens in general and S. vulgaris in particular. The only method currently available for diagnosing S. vulgaris in practice is the larval culture, which is laborious and time-consuming, so veterinary practitioners most often pool samples from several horses together in one culture to save time. Recently, molecular tools have been developed to detect S. vulgaris in faecal samples. The aim of this study was to compare the performance of a conventional polymerase chain reaction (PCR) assay with the traditional larval culture and furthermore test the performance of pooled versus individual PCR for farm screening purposes. Faecal samples were obtained from 331 horses on 18 different farms. Farm size ranged from 6 to 56 horses, and horses aged between 2 months and 31 years. Larval cultures and PCR were performed individually on all horses. In addition, PCR was performed on 66 faecal pools consisting of 3-5 horses each. Species-specific PCR primers previously developed were used for the PCR. PCR and larval culture detected S. vulgaris in 12.1 and 4.5% of individual horses, respectively. On the farm level, eight farms tested positive with the larval culture, while 13 and 11 farms were positive with the individual and pooled PCRs, respectively. The individual PCR method was statistically superior to the larval culture, while no statistical difference could be detected between pooled and individual PCR for farm screening. In conclusion, pooled PCR appears to be a useful tool for farm screening for S. vulgaris.

Statistical and biological considerations in evaluating drug efficacy in equine strongyle parasites using fecal egg count data.

Anthelmintic resistance (AR) is a serious problem for the control of equine gastrointestinal nematodes, particularly in the cyathostomins. The fecal egg count reduction test (FECRT) is the most common method for diagnosing AR and serves as the practical gold standard. However, accurate quantification of resistance and especially accurate diagnosis of emerging resistance to avermectin/milbemycin (A/M) drugs, is hampered by a lack of accepted standards for study design, data analysis, and data interpretation. In order to develop rational evidence-based standards for diagnosis of resistance, one must first take into account the numerous sources of variability, both biological and technical, that affect the measurement of fecal egg counts (FECs). Though usually ignored, these issues can greatly impact the observed efficacy. Thus, to accurately diagnose resistance on the basis of FECRT data, it is important to reduce levels of variability through improved study design, and then deal with inherent variability that cannot be removed, by performing thorough and proper statistical analysis. In this paper we discuss these issues in detail, and provide an explanation of the statistical models and methods that are most appropriate for analyzing these types of data. We also provide several examples using data from laboratory, field, and simulation experiments illustrating the benefits of these approaches.

Control de parásitos gastrointestinales en caballos pura sangre de carrera (Equus Caballus) en el Hipódromo "La Rinconada" Caracas, Venezuela / Control of gastrointestinal parasites in thoroughbred horses (Eqqus Caballus) the Racetrack

Se realizo un estudio coprológico por la técnica de flotación Mc Master (Willis-Molloy), realizando contaje de Huevos por gramo de Heces (HPG), a 100 equinos Pura Sangre de Carreras, durante el periodo de cuarentena Hipodromo “La Rinconada” Caracas Venezuela. Se procedió a la desparasitacióncon una terapéutica a base de Febantel, dosis 6mg/kg, presentación en pasta, vía oral (CALOXBANTEL) Febantel 88.7 mg; Excipientes c.s.p. 1g. A los 7 días post-desparasitación se realizo un estudio coprológico por la técnica de flotación Mc Master (Willis-Molloy), realizando contaje de Huevos por gramo de Heces (HPG). El estudio coprológico evidencio un 60% de infestación (60/100) en los caballos estudiados. El 40% (40/100) fue negativo al examen coprológico. Los resultados post-tratamientos fueron 1% de infestación persistente (01/100) y un 99% (99/100) negativos al examen coprológico. En todos los casos la infestación parasitaria fue por Strongylus sp. La presencia de Strongylus sp. se mantuvo por equino infestado entre un rango de 400-1200 HPG

We study 100 Thoroughbred horses, in the Racetrack “La Rinconada” Caracas, Venezuela, by McMaster flotation technique (Willis-Molloy), making counting of eggs per gram of feces (EPG) before deworming, each of the copies, then proceeded to the parasite with a Febantel based therapy, dose 6mg/kg, pasta presentation, oral (CALOXBANTEL) Febantel 88.7 mg, Excipients 1g. At 7 days post-parasite stool study was conducted by the McMaster flotation technique (Willis- Molloy), by counting eggs per gram of faeces (EPG). The coprology study showed 60% infestation (60/100) in horses studied. The 40% (40/100) was negative. The post-treatment were 1% infestation (01/100) and 99% (99/100) was negative. In all cases, parasite infestation was by Strongylus sp. within the range of 400-1200 HPG

Isolation of potentially useful antigens from cyathostomin third-stage larvae by using a fast protein liquid chromatography one-step method.

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes.