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Cardiopulmonary nematodes cause health and fitness disorders in wild and domestic carnivores. The red fox (Vulpes vulpes) participates in the spread of these shared parasites at the domestic-wildlife interface. This study aimed to evaluate the sensitivity of post-mortem diagnostic method for detecting lungworms in carnivores, and its exactness to estimate the parasite intensity of each nematode species. Cardiorespiratory system of fifty-one foxes were examined through three consecutively methodological steps: first, the tracheobronchial tree, pulmonary arteries and their branches were opened (OT); next, lung parenchyma was immersed in water and squeezed (WS); finally, the parenchyma was artificially digested in a pepsin and chlorhydric acid solution (AD). Eucoleus aerophilus, Angiostrongylus vasorum, Crenosoma vulpis and Metathelazia capsulata were identified. The number of recovered nematodes in each step were 454 (OT), 285 (WS) and 141 (AD). The use of OT and WS helped to improve parasite intensity results and decreased false negative cases. Accordingly, when OT and WS were used together, the sensitivity in the detection of parasitized foxes was 96.1%, while the exactness of parasite intensity was 84%. When AD was performed, although sensitivity does not rise, results were more exact, increasing the total number of detected parasites by 16%. Moreover, AD improved the sensitivity in the detection of A. vasorum and M. capsulata, as well as quantifying more exactly the parasite intensity (92.5% and 92.3% of exactness without AD, respectively). Our study provides valuable information that should be taken into account when planning epidemiological studies based on cardiopulmonary nematode detection in carnivores., (© 2023. The Author(s).)

Helminth infections of cattle place significant burdens on livestock production and farm economic efficiency. Heavy infections are relatively easy to detect and treat with anthelmintics. However, subclinical infections have major but often hidden impacts on animals, necessitating more refined diagnostics to detect them and ideally inform farmers about the likely impact of anthelmintic treatment on animal and herd performance. Here, we review recent advances in diagnosing three major cattle helminth infections - gastrointestinal nematodes (GINs), liver flukes, and lungworms - and the search for subclinical infection thresholds to guide treatment decisions. Combining refined diagnostic thresholds with farm-specific information on grazing systems and animal history enables farmers to tailor helminth treatments to specific epidemiological circumstances, thereby limiting anthelmintic resistance (AR) and boosting agricultural efficiency and food security., Competing Interests: Declaration of interests J.C. and E.C. are involved in a license agreement between Ghent University and INDICAL Bioscience GmbH for commercialization of SVANOVIR® O. ostertagi-Ab and F. hepatica-Ab ELISA. The authors declare no other known conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Parasites cause losses in animal production. Parasite infection in ruminants has been estimated to be a major problem causing more than 3 billion USD per year, from which 60% corresponds to the sheep industry. Treatment is based on the use of synthetic anthelmintics; however, repeated application or under dosage have resulted in the selection of nematodes resistant to anthelmintics. The objective of the present work was to perform a diagnosis of gastrointestinal parasites in sheep kept under different zootechnical management. Ninety female sheep were used, most of them pregnant. Sampling was performed monthly from December 2015 to June 2016 (flock 5 until April). Fecal samples were collected from the rectum; the McMaster technique was performed, morphological characteristics were observed, oocysts and eggs were counted per gram of feces (opg and epg), frequency and intensity were obtained. Faecal culture was performed for feces that had a positive result, infective larvae were obtained and taxonomically identified. At the end of the study, a dewormer (fenbendazole) was administered and its effect was measured. The frequency of gastrointestinal parasites was 100%. The highest opg was 3,600 (flock 3, March, 2016), the epg for cestodes was 2800 (flock 1, January, 2016) and for gastrointestinal nematodes (GIN) was 25,000 (flock 1, May, 2016); the intensity was variable and it was increased by peripartum. Protists (Eimeria spp), cestodes (Moniezia) and nematodes (Haemonchus, Trichostrongylus, Cooperia, Chabertia ovina. Teladorsagia, Oesophagostomum, Nematodirus and Trichuris ovis) were identified. No previous diagnosis is performed in flocks, and sometimes dewormers are administered, even though resistance to ivermectin and benzimidazole is suspected. Flock management, its feeding system and its conditions were determinant for the observed results; therefore, it is necessary to count with a diagnosis that provides information about the parasitic population and its dynamic, in order to carry out a selective and comprehensive control that has an impact on the animal, human and environmental health., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Effective gastrointestinal nematode management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major gastrointestinal nematodes. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed gastrointestinal nematode management decisions. The traditional 'gold-standard' approach, larval culture followed by morphological differentiation, is laborious and potentially inaccurate. We developed a new diagnostic approach to identify gastrointestinal nematodes that integrates a remote-location digital faecal egg count platform, FECPAK G2 , with internal transcribed spacer 2 (ITS2) nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAK G2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing. The gastrointestinal nematode compositions and alpha diversity generated by our FECPAK G2 egg nemabiome metabarcoding approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAK G2 harvested eggs in either DNA isolation lysis buffer or 80% ethanol (v/v) had no impact on gastrointestinal nematode identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility for nemabiome metabarcoding, in the absence of a cold chain. We discovered that sustained gastrointestinal nematode egg embryonation in the lysis buffer storage solution lead to higher yields of DNA compared with ethanol-stored gastrointestinal nematode eggs or faeces with gastrointestinal nematode eggs. Taking advantage of an already well-established platform such as FECPAK G2 , and providing the livestock producers that use it with the option to identify gastrointestinal nematodesin their samples and contribute to large-scale gastrointestinal nematode distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAK G2 egg nemabiome metabarcoding approach., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Classic approaches for antemortem identification of gastrointestinal nematodes (GIN) require coproculture of eggs and morphological examination. While adequate for diagnosis, many PCR techniques cannot easily quantify mixed infections without controls and/or standard curves. Herein, we developed a simple and rapid test for differentiating and quantifying mixed infections of GIN using PCR products separated by capillary electrophoresis. Among the cattle GIN, the ITS2 region is sufficiently distinct in length to delineate among the most common infecting genera, Ostertagia ostertagi = 373 bases (b), Haemonchus contortus (placei) = 366b, Cooperia punctata (oncophora) = 376b, Trichostrongylus axei = 372b, and Oesophagostomum radiatum = 357b. Conserved primers were synthesized that span the ITS2 where one primer was fluorescently labeled with 6-FAM. DNAs from infective L3 were PCR amplified then loaded onto an ABI 3130 sequencer adapted for size fragment analysis. Resulting peak amplitudes were both diagnostic and quantitative on a relative basis. As proof of principle, quantification was performed on PCR fragments from mixed species pairs of Ostertagia ostertagi, Cooperia punctata, and Haemonchus contortus and analyzed using Gene Marker V1.85 software. In all cases, linear responses were observed where R 2  > 0.97 and line slopes ranged between 0.90 and 1.1. When tested on eggs from naturally infected animals, the assay showed superior results on two farms when compared to coproculture and morphological identification. Using wildlife-derived samples, results coincided well with deep amplicon sequencing. The assay is adaptable to large-scale studies, does not require comparative PCR controls, and should be compliant with GIN from small ruminant livestock., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies., Methods: Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC., Results: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp., Conclusions: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment., (© 2021. The Author(s).)

Since the EU ban on battery cages, many studies have listed Ascaridia galli and Heterakis gallinarum as the most common roundworms in the European laying hen population. A complicating factor is that the eggs of these parasites are almost identical. Thus, lack of molecular diagnostic approaches has driven epidemiological studies to take on necropsy for species discrimination, which is labor and cost intensive. Here, we describe a novel diagnostic tool based on droplet digital PCR for simultaneous identification and absolute quantification of the eggs of both of these ascarids in chickens' droppings using two different genus-specific primer-probe sets targeting the second internal transcribed spacer region (ITS-2) in the nuclear ribosomal (rRNA) gene array. No cross-reaction was observed when different combinations of DNA and species-specific primers and probes were tested. The lowest obtained frequency threshold for the detection of H. gallinarum in the presence of a constant A. galli DNA concentration was determined to be 0.8 %. After validation, we used the assay to analyze field samples collected from several Swedish laying hen farms. Out of 134 samples, 86 (64 %) were positive for A. galli while 11 (8.3 %) samples were positive for H. gallinarum. These samples were initially analyzed with flotation technique for detection of ascarid eggs. The results of the Cohen's kappa indicated substantial agreement (85.8 %) between the two tests. In conclusion, we have validated a novel molecular-based diagnostic tool for quantification and differentiation between intestinal parasites of major importance in chickens with high precision. Although this study focuses on identification of parasites of laying hens, the findings may well have a bearing on all types of chicken production systems. The present study lays the groundwork for future research into epidemiology of these two important chicken parasite species., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Heterakis gallinarum, H. beramporia, and H. indica are common nematodes in gallinaceous poultry in Asian countries, and the infections occasionally lead to declining health of the hosts. These three Heterakis spp. can be identified by the morphological characteristics of the male worms; however, the female worms and eggs cannot be identified because they have no reliable morphological characteristics for discrimination. In addition, H. gallinarum is a well-known vector of fetal protozoan Histomonas meleagridis, making the discrimination between these three Heterakis species important in basic and clinical veterinary parasitology. We analyzed nuclear ribosomal 18S-ITS1-5.8S-ITS2-28S DNA sequences of these three Heterakis species. The 18S, 5.8S, and 28S DNA sequences had very high homology between the species; however, the ITS1 and ITS2 sequence similarity was 68.5 %-93.2 %. H. gallinarum, H. beramporia, and H. indica were divided into separate clades in the ITS1 and ITS2-concatenated phylogenetic tree. Therefore, to develop a multiplex PCR method for discriminating between the three Heterakis species, we designed species-specific reverse primers within the ITS2 region as follows: H. gallinarum-specific HgI2-R, H. beramporia-specific HbI2-R5, and H. indica-specific HiI2-R. The multiplex PCR amplified 396-bp, 272-bp, and 482-bp fragments specific to H. gallinarum, H. beramporia, and H. indica DNA, respectively, and did not amplify the fragments using other chicken nematode DNAs such as Ascaridia galli, Oxyspirura mansoni, Dispharynx nasuta, and Cheilospirura hamulosa. These results suggest that the multiplex PCR would serve as a useful tool for identifying and diagnosing infections of H. gallinarum, H. beramporia, and H. indica in poultry., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Diffuse unilateral subacute neuroretinitis (DUSN) is a rare form of chorioretinitis caused by retinal infestation by nematode larvae. Definitive diagnosis requires visualization of a motile nematode, but this can be challenging because the nematode is small, translucent, and usually motionless with overlying media opacity from cellular inflammation. The authors describe a case of DUSN in which widefield swept-source optical coherence tomography angiography was used to localize a midperipheral intraretinal nematode facilitating subsequent clinical observation of movement. Inflammation resolved after focal laser photocoagulation of the nematode with recovery of visual acuity to 20/20. [ Ophthalmic Surg Lasers Imaging Retina . 2021;52:345-349.] .

Two adult African pygmy geese ( Nettapus auritus ) were found dead with minimal clinical disease signs. Necropsy revealed aberrant migration of the nematode parasite Echinuria uncinata , as confirmed morphologically and through DNA sequencing. This common waterfowl parasite typically lives in the proventriculus, burying headfirst into the mucosa and laying eggs into the gastrointestinal lumen. In these geese, the parasites tunneled through the gastrointestinal tract wall to invade the coelomic cavity; from which, a substantial quantity of eggs found their way into the coelomic space and into the air sacs and lungs. This potential parasite migration should be monitored for in Anseriformes species that present with similar disease conditions, and the use of Daphnia species, the intermediate host, as a waterfowl feed source is not recommended.

Although zebrafish continue to increase in popularity as a vertebrate animal model for biomedical research, chronic infectious diseases in laboratory populations remain prevalent. The presence of pathogens such as Pseudocapillaria tomentosa, a parasitic nematode found in the intestine of infected zebrafish, can significantly influence experimental endpoints and negatively impact reproducibility of research findings. Thus, there is a need for screening tests for zebrafish with the sensitivity to detect even low levels of pathogens present in tissues. Assays based on the detection of DNA are commonly used for such screening tests. Newer technologies such as digital PCR provide an opportunity to improve the sensitivity and precision of these assays, so they can be reliably used to detect pathogen DNA in water, reducing the need for lethal testing. We have designed a qPCR-based assay with the sensitivity to detect less than 5 copies of the P. tomentosa SSU-rDNA gene in tissues of infected zebrafish and environmental DNA from aquarium water housing infected fish. In addition, we adapted this test to a dPCR platform to provide a precise quantification of P. tomentosa DNA and demonstrate the resistance of this assay to inhibitors commonly found in freshwater aquaria., (© 2020 John Wiley & Sons Ltd.)

Background: Faecal egg count (FEC) techniques are commonly used to detect gastrointestinal nematodes (GINs) in cattle and to determine anthelmintic efficacy/resistance through the faecal egg count reduction test (FECRT). Mini-FLOTAC is one of the techniques recommended for a standardised FEC/FECRT of helminth eggs in cattle. However, only one paper evaluated the recovery rate of GIN eggs by Mini-FLOTAC (compared to McMaster and modified-Wisconsin method) in cattle, using only a level of contamination of 200 eggs per gram (EPG) of faeces and using GIN eggs collected from goat faeces to spike faecal samples from cattle. To further study the recovery rate of GIN eggs from cattle faeces, this study was conducted in two laboratories, one in Belgium and one in Italy to evaluate the sensitivity, accuracy, precision and reproducibility of the Mini-FLOTAC and McMaster techniques (at two reading levels: grids and chambers) for the detection of GIN eggs in spiked bovine faecal samples., Methods: In both countries, spiked cattle faecal samples with five different levels of egg contamination (10, 50, 100, 200 and 500 EPG) of GINs were used. The study was performed in both laboratories by the same expert operator and using the same standard operating procedures (SOPs) for the Mini-FLOTAC and McMaster techniques. Sensitivity, accuracy and precision were calculated for each technique and for each level of contamination. Statistical analyses were performed to evaluate differences in performance between the two techniques., Results: Mini-FLOTAC had a higher sensitivity (100% at all EPG levels for Mini-FLOTAC vs 0-66.6% for McMaster chambers and grids at levels< 100 EPG), a higher accuracy (98.1% mean value for Mini-FLOTAC vs 83.2% for McMaster grids and 63.8% for McMaster chambers) and a lower coefficient of variation (10.0% for Mini-FLOTAC vs 47.5% for McMaster grids and 69.4% for McMaster chambers) than McMaster. There was no significant difference in the recovery of GIN eggs between the two studies performed in Belgium and in Italy., Conclusions: The high GIN egg recovery rate detected by Mini-FLOTAC and the similar results obtained in Belgium and in Italy indicated that the diagnostic performance of a FEC technique was not dependent on the laboratory environment.

Parasitic myelopathy caused by Gurltia paralysans in domestic cats is a disease commonly reported in several South American countries. The adult parasite is lodged in the meningeal veins and spinal cord, often causing clinical manifestations of vascular proliferation, thrombophlebitis, and medullary compression. Wild felines are believed to be the definitive hosts of this parasite. The infection occurs through the ingestion of paratenic hosts, but the life cycle of G. paralysans is not yet clearly understood. In this paper, we discuss a case of parasitic myelopathy in a margay (Leopardus wiedii) that died during post-surgical care. Necropsy revealed focal hemorrhages in the thoracolumbar spinal cord. A microscopic examination revealed adult nematodes and eggs inside the veins of subarachnoid space in spinal cord, suggesting G. paralysans infection. This is first description of parasitic myelopathy in a margay in Brazil.

Recently, feline cardio-pulmonary nematodes have attracted high scientific interest, as they are increasingly reported from various areas. Most of these parasites have similar transmission patterns and/or host reservoirs, thus they may affect domestic and wild felids living in sympatry. In the present study, a case of multiple cardio-pulmonary parasitism in co-infection with other parasites in a European wildcat is presented. The animal, found exhausted, was hospitalised for recovery and parasitological, haematological, clinical and imaging examinations were performed. The parasitological examinations revealed 4 cardio-pulmonary nematodes, i.e. Aelurostrongylus abstrusus, Troglostrongylus brevior, Eucoleus aerophilus, Angiostrongylus chabaudi, 3 intestinal parasites, i.e. Toxocara cati, ancylostomatids, Cystoisospora felis, 2 haemoparasites, i.e. Hepatozoon felis and elements morphologically compatible with small Babesia/Cytauxzoon spp., and Ixodes ricinus and Haemaphysalis erinacei ticks. Treatment with a spot-on formulation containing imidacloprid 10% and moxidectin 1% (Advocate® spot-on solution for cats, Bayer) was decided and follow-up faecal examinations were performed until the release of the animal. By the end of the hospitalisation, all metazoan endoparasites were no longer detectable in faecal examinations, with the exception of a low number of A. abstrusus larvae. Thus, the animal was released after a second treatment with the same product. This is the first description of an apparently successful treatment of multiple cardio-respiratory parasitosis in a naturally infected wildcat showing compatible clinical signs. The evidence that Advocate® may be effective against A. chabaudi could be useful for treating infected, hospitalised, wildcats and it is promising in the case A. chabaudi infection will spread to domestic cats in a near future., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In October 2018, two Atelerix algirus hedgehogs were admitted to the Wildlife Rehabilitation Hospital in Mallorca (Balearic Islands, Spain) with signs of acute neurological disease. Necropsy detected immature, fully developed nematodes in the subarachnoid space of both hedgehogs, including a gravid female worm. DNA-based molecular tools confirmed the nematode as Angiostrongylus cantonensis , an important aetiological agent of eosinophilic meningitis in humans. So far this zoonotic parasite in has not been reported in western European wildlife.

Aims: Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay., Methods: We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode ( cox1 gene) and 33 nematode ( cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested., Results: The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples., Conclusions: The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Microscopic methods which employ active or passive flotation have been used to detect parasite diagnostic stages in the feces of companion animals for many years. More recently, coproantigen ELISAs for the detection of excretory/secretory products from intestinal nematodes have been introduced. These assays can identify the presence of parasites when eggs are not recovered by flotation (e.g. prepatent infection or intermittent egg shedding). The study was designed to assess the added benefit of these coproantigen tests in canine fecal diagnostics. The work was performed at 3 separate sites where canine fecal samples were each independently evaluated by both centrifugal flotation with an expert examiner (CFE) and passive flotation with a less experienced examiner. All samples were also tested using coproantigen ELISA to detect ascarid, hookworm, or whipworm antigen (IDEXX Laboratories, Inc, Westbrook, Maine). A total of 1202 samples were collected; 626 were from shelter dogs and 576 were from pet dogs. CFE recovered ascarid eggs in 58 samples, hookworm eggs in 229 samples, and whipworm eggs in 95 samples. Of the positive samples identified by CFE, the PFE and ELISA identified 40 and 51 ascarid samples, 188 and 203 hookworm samples, and 65 and 67 whipworm positive samples, respectively. The coproantigen ELISA identified 8 ascarid, 82 hookworm, and 22 whipworm positive samples that were not detected by CFE. The combined results of passive flotation and the coproantigen ELISA improved the percent agreement with centrifugal flotation, suggesting that greater sensitivity of detection may be achieved through the use of complementary diagnostic methods. However, errors of misidentification and poor recovery apparently introduced by less experienced examiners using an inferior flotation method remained. A diagnostic approach that combines coproantigen assays with centrifugal flotation and examination by an expert allows detection of more ascarid, hookworm, and whipworm infections., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Control of equine nematodes has relied on benzimidazoles (BZs), tetrahydropyrimidines and macrocyclic lactones. The intensive use of anthelmintics has led to the development of anthelmintic resistance (AR) in equine cyathostomins and Parascaris equorum. Field studies indicate that BZ and pyrantel resistance is widespread in cyathostomins and there are also increasing reports of resistance to macrocyclic lactones in cyathostomins and P. equorum. The unavailability of reliable laboratory-based techniques for detecting resistance further augments the problem of nematode control in horses. The only reliable test used in horses is the fecal egg count reduction test; therefore, more focus should be given to develop and validate improved methodologies for diagnosing AR at an early stage, as well as determining the mechanisms involved in resistance development. Therefore, equine industry and researchers should devise and implement new strategies for equine worm control, such as the use of bioactive pastures or novel feed additives, and control should increasingly incorporate alternative and evidence-based parasite control strategies to limit the development of AR. This review describes the history and prevalence of AR in equine nematodes, along with recent advances in developing resistance diagnostic tests and worm control strategies in horses, as well as giving some perspective on recent research into novel control strategies.

Aims: This case report describes a clinical case of patent and natural infection with trichuroid Eucoleus aerophilus in a 5-month-old, mixed-breed female dog in Bosnia and Herzegovina., Methods: A detailed history was taken from the owner. Physical exam and thoracic radiography were performed. Faecal sample was processed by the passive flotation tube test with saturated zinc sulphate solution. After the final diagnosis, a dose of 0.4 mg /kg ivermectin was applied subcutaneously to the diseased dog., Results: The history revealed intermittent, non-productive cough after physical exercise. During the physical exam, mild signs of general distress, moderate cachexia with bad body condition, increased heart and respiratory rates, mild dyspnoea and increased bronchovesicular respirations were observed. Thoracic radiography showed mixed bronchial-alveolar lung pattern compatible with chronic bronchopneumonia. Trichuroid eggs of Eucoleus aerophilus were detected in faecal samples from the affected dog. Ivermectin administration at a dose of 0.4 mg/kg was sufficient to eliminate parasitic infection with Eucoleus aerophilus., Conclusion: This is the first report of respiratory eucoleosis in a dog from Bosnia and Herzegovina.

Background: Gastrointestinal nematodes (GINs) frequently infect South American camelids (alpacas and llamas) and cause economic losses due to reduced production of fiber, meat and/or leather. Our knowledge about the epidemiology and diagnosis of GINs in llamas and alpacas is limited, and reliable keys for the identification of the third-stage larvae (L3s) of some common nematodes (such as Camelostrogylus mentulatus) that infect alpacas and llamas remain undescribed. In this study, we modified two existing semi-quantitative multiplexed-tandem (MT)-PCR assays, originally developed for the GINs of sheep and cattle, to reliably detect and differentiate the common genera/species of GINs in the faeces of alpacas., Results: Following the establishment of the MT-PCR assay using positive and negative control samples, alpaca faecal samples were tested to validate the assay to detect and differentiate nematode genera/species, including C. mentulatus, Cooperia spp., Haemonchus spp., Oesophagostomum spp., Ostertagia ostertagi, Teladorsagia circumcincta and Trichostrongylus spp. Sequencing of the MT-PCR products demonstrated specific (100%) amplification of the target nematode genera/species. Additionally, a comparison of results of the MT-PCR assay and the morphological identification of adult worms collected from the same 35 alpacas revealed that there was a good agreement (37-94%) between the two methods. However, some discrepancies were observed between the results of the MT-PCR assay and the morphological identification of adult worms., Conclusions: The MT-PCR platform is an accurate, sensitive and rapid method for the diagnosis of GINs in alpacas, and it can be used as a substitute to larval culture to identify common nematodes in the faeces of alpacas and llamas.

Although various studies have provided novel insights into how to best design, analyze and interpret a fecal egg count reduction test (FECRT), it is still not straightforward to provide guidance that allows improving both the standardization and the analytical performance of the FECRT across a variety of both animal and nematode species. For example, it has been suggested to recommend a minimum number of eggs to be counted under the microscope (not eggs per gram of feces), but we lack the evidence to recommend any number of eggs that would allow a reliable assessment of drug efficacy. Other aspects that need further research are the methodology of calculating uncertainty intervals (UIs; confidence intervals in case of frequentist methods and credible intervals in case of Bayesian methods) and the criteria of classifying drug efficacy into 'normal', 'suspected' and 'reduced'. The aim of this study is to provide complementary insights into the current knowledge, and to ultimately provide guidance in the development of new standardized guidelines for the FECRT. First, data were generated using a simulation in which the 'true' drug efficacy (TDE) was evaluated by the FECRT under varying scenarios of sample size, analytic sensitivity of the diagnostic technique, and level of both intensity and aggregation of egg excretion. Second, the obtained data were analyzed with the aim (i) to verify which classification criteria allow for reliable detection of reduced drug efficacy, (ii) to identify the UI methodology that yields the most reliable assessment of drug efficacy (coverage of TDE) and detection of reduced drug efficacy, and (iii) to determine the required sample size and number of eggs counted under the microscope that optimizes the detection of reduced efficacy. Our results confirm that the currently recommended criteria for classifying drug efficacy are the most appropriate. Additionally, the UI methodologies we tested varied in coverage and ability to detect reduced drug efficacy, thus a combination of UI methodologies is recommended to assess the uncertainty across all scenarios of drug efficacy estimates. Finally, based on our model estimates we were able to determine the required number of eggs to count for each sample size, enabling investigators to optimize the probability of correctly classifying a theoretical TDE while minimizing both financial and technical resources., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Anthropogenic disturbance via resource acquisition, habitat fragmentation and climate change, amongst other factors, has led to catastrophic global biodiversity losses and species extinctions at an accelerating rate. Amphibians are currently one of the worst affected classes with at least a third of species categorised as being threatened with extinction. At the same time, they are also critically important for many habitats and provide man with a powerful proxy for ecosystem health by acting as a bioindicator group. Whilst the causes of synchronised amphibian losses are varied recent research has begun to highlight a growing role that macroparasites are playing in amphibian declines. However, diagnosing parasite infection in the field can be problematic, principally relying on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. The current study developed a non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair, thus avoiding the difficulties of using species specific primers in situations where common parasite species are unknown. Used in conjunction this protocol and primer pair was tested on a wide range of faecal samples from captive and wild amphibians. The primers and protocol were validated and detected infections, including a Railletnema nematode infection in poison dart frogs from ZSL London Zoo and Mantella cowani frogs in the wild. Furthermore, we demonstrate the efficacy of our PCR-based protocol for detecting nematode infection in other hosts, such as the presence of pinworm (Aspiculuris) in two tortoise species and whipworm (Trichuris muris) in mice. Our environmental DNA approach mitigates problems associated with microscopic identification and can be applied to detect nematode parasitoses in wild and captive hosts for infection surveillance and maintenance of healthy populations.

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification.

Resistance to fenbendazole, ivermectin, and moxidectin was explored by a fecal egg count reduction test in four meat sheep flocks in southwestern France where anthelmintic resistance was suspected. The FECR test results of the present study confirmed the presence of benzimidazole resistance in three out of the four farms and the presence of ivermectin resistance in one flock. In addition, a suspicion of moxidectin resistance was shown in this latter farm. Both conventional morphological and molecular identifications were performed on larval cultures before and after the treatment in the studied farms. A high positive correlation was found between the number of larvae counted under binocular microscope and the number of larvae estimated by the qPCR analysis (R 2  = 0.88) and a high Cohen's Kappa value (0.91) in the detection of strongylid larvae in larval cultures. According to qPCR results, Trichostrongylus species demonstrated high levels of BZ resistance and Teladorsagia circumcincta was involved in the IVM resistance in one farm. The molecular procedures used in this study have the potential to be beneficial for anthelmintic resistance surveillance in sheep industry.

Parasitic infections influence the health of captive forest musk deer (Moschus berezovskii) and affect population increases. Nevertheless, there are few quantitative studies regarding forest musk deer parasites, and there is no common preservation method or flotation solution used for detection of faecal parasites because of the biology of the worms and the host physiological state. The objective of this study was to evaluate preservation and flotation methods for the detection of nematode eggs and coccidian oocysts in faeces of the forest musk deer. The McMaster technique was used to count nematode eggs and coccidian oocysts in 33 samples of faeces. For the nematode eggs, the differences among flotation solutions were significant (P< 0.01), with sodium nitrate being the best flotation solution, and the combination of freezing and sodium nitrate resulted in the greatest number of eggs per gram (EPG = 209.4 ± 67.8). For the coccidian oocysts, the interaction between preservation method and flotation solution was significant (P< 0.01), and the combination of formalin and sodium chloride yielded the greatest number of oocysts per gram (OPG = 1010.7 ± 162.3). The forest musk deer had a high prevalence of parasitic infections, with the parasite load of coccidia (96.4%) significantly greater than that of nematodes (71.9%, P< 0.01). These results confirm that captive forest musk deer suffer from serious parasitic invasions and demonstrate that the novel method described here could be utilized for parasitological diagnosis, detection and prevention in species of Moschidae and Cervidae.

Within the framework of the Federal External Quality Assessment (EQA) System and in the context of postgraduate training improvement for health workers in 2010-2014, specialists from the laboratories of the therapeutic-prophylactic organizations and institutions of the Russian Federal Service for Supervision of Consumer Rights Protection and Human Welfare were examined for their professional competence in microscopically identifying the pathogens of parasitic diseases in feces. The virtual remote educational computer technology tools that included different combinations of 16 helminthic species, 5 intestinal protozoan species, and a number of artefacts, were used. The specialists from 984 laboratories of multidisciplinary therapeutic-prophylactic organizations and hygiene and epidemiology centers in all Federal Districts of the Russian Federation were covered. A total of 8245 replies were analyzed. The detection rate for helminths was 64.0%, including those by a taxonomic group (nematodes, 65.0%; cestodes, 72.0%; trematodes, 55.1%). There was a dynamic decrease in the above indicators. There were low detection rates for trematodes parasitizing the small intestine (Metagonimus, 10.2%; Nanophyetus, 26.2%) and hepatobiliary organs (Fasciola, 59.6%; Clonorchis, 34.9%). The similar trend was seen in the detection rates for the pathogens of geohelminthisms (ascariasis, trichocephaliasis, etc.) and contagious helminthisms (enterobiasis, hymenolepiasis). The level of competence in detecting and identifying intestinal protozoa was much lower than the similar rates for helminthism pathogens. EQA for the laboratory diagnosis of the pathogens of parasitic diseases, by using the virtual tools is a leading element of the postgraduate training system for laboratory specialists. The results of EQA for the laboratory diagnosis of the pathogens of parasitic diseases are a basic material for the development, and improvement of training modernization programs, by applying a modular approach.

The review chronologically considers the main classes of the currently available anthelminthic substances: early anthelmintic compounds, benzimidazoles, imidazolthiazoles, tetrahydropyrimidines, avermectins and milbemycins, and salicylanilides. Great attention is paid to novel substances (emodepside, monepantel, derquantel, tribendimidine) and promising developments. Some aspects of the molecular mechanisms of action of anthelmintics, their resistance, and alternative dehelmintization methods are discussed.

Nematodes infecting the cardiorespiratory system of cats have recently stimulated high scientific interest. Over the past few years, different aspects of these parasites have been clarified and various issues elucidated. This increased knowledge has improved awareness on this topic but, at the same time, some dilemmas have not been solved and new questions have been raised. In this review, we underline and discuss current knowledge of, and new doubts relating to, feline lungworms and heartworms, with the aim of stimulating new studies to fill gaps of basic (i.e., epidemiology and biology) and applied (i.e., clinical aspects) knowledge of the old and new parasites affecting the cardiorespiratory system of cats., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the 21st century, despite increased globalization through international travel for business, medical volunteerism, pleasure, and immigration/refugees into the United States, there is little published in the dermatology literature regarding the cutaneous manifestations of helminth infections. Approximately 17% of travelers seek medical care because of cutaneous disorders, many related to infectious etiologies. This review will focus on the cutaneous manifestations of helminth infections and is divided into 2 parts: part I focuses on nematode infections, and part II focuses on trematode and cestode infections. This review highlights the clinical manifestations, transmission, diagnosis, and treatment of helminth infections. Nematodes are roundworms that cause diseases with cutaneous manifestations, such as cutaneous larval migrans, onchocerciasis, filariasis, gnathostomiasis, loiasis, dracunculiasis, strongyloidiasis, ascariasis, streptocerciasis, dirofilariasis, and trichinosis. Tremadotes, also known as flukes, cause schistosomiasis, paragonimiasis, and fascioliasis. Cestodes (tapeworms) are flat, hermaphroditic parasites that cause diseases such as sparganosis, cysticercosis, and echinococcus., (Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

The clinical presentation of an intraocular nematode unusually affixed to the posterior lens capsule is described. A 64-year-old female patient presented with a 7-year history of gradually declining vision and enlarging central scotoma, but no inflammation. On follow-up 2 years later, vision had further declined and a non-motile, 8-mm nematode was seen affixed to the posterior lens capsule that remained unchanged through final follow-up. The patient disclosed having resided in Africa as a child. Systemic review revealed no evidence of extraocular involvement. Nematode carcasses may remain preserved in the human eye for extended periods without ongoing inflammation., (Copyright 2015, SLACK Incorporated.)

Lungworms are important parasites of wildlife and host infection status is often evaluated using coprologic techniques, most commonly the Baermann method. Recently, the FLOTAC® has emerged as a new tool for diagnosing lungworm infections, and methodologic comparison studies in domestic species suggest that this method outperforms many other established techniques. We compared a modified FLOTAC with the beaker-modified (bm)-Baermann to evaluate the relative performance of the two techniques for counting lungworm larvae in bighorn sheep (Ovis canadensis) feces. Both methods generated equivalent larval counts and both were highly repeatable. The major difference between the two methods was that the FLOTAC was poorer at detecting mixed infections. The ultimate choice between using the FLOTAC and bm-Baermann methods for quantifying lungworm larvae in wildlife studies may depend on the specific nature of the research questions being addressed, balanced by practical constraints.

Most hosts are concurrently or sequentially infected with multiple parasites; thus, fully understanding interactions between individual parasite species and their hosts depends on accurate characterization of the parasite community. For parasitic nematodes, noninvasive methods for obtaining quantitative, species-specific infection data in wildlife are often unreliable. Consequently, characterization of gastrointestinal nematode communities of wild hosts has largely relied on lethal sampling to isolate and enumerate adult worms directly from the tissues of dead hosts. The necessity of lethal sampling severely restricts the host species that can be studied, the adequacy of sample sizes to assess diversity, the geographic scope of collections and the research questions that can be addressed. Focusing on gastrointestinal nematodes of wild African buffalo, we evaluated whether accurate characterization of nematode communities could be made using a noninvasive technique that combined conventional parasitological approaches with molecular barcoding. To establish the reliability of this new method, we compared estimates of gastrointestinal nematode abundance, prevalence, richness and community composition derived from lethal sampling with estimates derived from our noninvasive approach. Our noninvasive technique accurately estimated total and species-specific worm abundances, as well as worm prevalence and community composition when compared to the lethal sampling method. Importantly, the rate of parasite species discovery was similar for both methods, and only a modest number of barcoded larvae (n = 10) were needed to capture key aspects of parasite community composition. Overall, this new noninvasive strategy offers numerous advantages over lethal sampling methods for studying nematode-host interactions in wildlife and can readily be applied to a range of study systems., (© 2015 John Wiley & Sons Ltd.)

Agricultural ruminants usually harbour mixed infections of gastrointestinal nematodes. A specific diagnosis is important because distinct species can differ significantly in their fecundity and pathogenicity. Haemonchus spp. and Cooperia spp. are the most important gastrointestinal nematodes infecting ruminants in subtropical/tropical environments. In Brazil, C. punctata is more adapted to cattle than sheep. Additionally, C. spatulata appears to be more adapted to cattle, whereas C. curticei is more adapted to sheep. However, infection of sheep with C. punctata is common when cattle and sheep share the same pasture. Although morphological analyses have been widely used to identify nematodes, molecular methods can overcome technical limitations and help improve species-specific diagnoses. Genetic markers in the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA (rDNA) have been used successfully to detect helminths. In the present study, the ITS-1 region was analysed and used to design a species-specific oligonucleotide primer pair to identify C. curticei. The polymerase chain reaction (PCR) product was sequenced and showed 97% similarity to C. oncophora partial ITS-1 clones and 99% similarity to the C. curticei sequence JF680982. The specificity of this primer pair was corroborated by the analysis of 17 species of helminths, including C. curticei, C. punctata and C. spatulata. Species-specific diagnosis, which has implications for rapid and reliable identification, can support studies on the biology, ecology and epidemiology of trichostrongylid nematodes in a particular geographical location.

The specific diagnosis of gastrointestinal nematode infections in ruminants is routinely based on larval culture technique and on the morphological identification of developed third-stage larvae. However, research on the ecology and developmental requirements of different species suggests that environmental conditions (e.g., temperature and humidity) for optimal development to occur vary between the different species. Thus, employing a common culture protocol for all species will favour the development of certain species over others and can cause a biased result in particular when species proportions in a mixed infection are to be determined. Furthermore, the morphological identification of L3 larvae is complicated by a lack of distinctive, obvious features that would allow the identification of all key species. In the present paper we review in detail the potential limitations of larval culture technique and morphological identification and provide account to some modern molecular alternatives to the specific diagnosis of gastrointestinal nematode infection in ruminants., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Three methods, FLOTAC, FECPAK and McMaster were compared for accuracy and sensitivity for counting numbers of nematode eggs in faeces of naturally infected cattle with high or low nematode egg counts. Only FLOTAC gave positive results for 12 replicates from pooled samples with low egg counts making it more sensitive than FECPAK (67%) and McMaster (41.7%). FLOTAC resulted in generally higher egg counts and lower coefficients of variation than the other two methods used. The reliability of FECPAK and McMaster is depended on the area under the slide counted. All three methods can be used for making decisions whether to treat but FLOTAC or Mini-FLOTAC should be used for faecal egg count reduction tests when lower egg counts are present.

The third part of the overview "Helminths and helminthoses in Central Europe" deals with the medically relevant nematodes (roundworms) and nematode-caused diseases occurring in Central Europe. The paper comprises data on the biology of the parasites and their ways of transmission, describes the symptomatology of the diseases, summarizes the possibilities of diagnosis and refers to the prophylactic means.

Zoonotic anatrichosomiasis in a mother and daughter is reported. Both presented with a 10-week history of multiple painful oral ulcers. Biopsy specimens revealed the presence of small, coiled trichuroid nematodes with distinctive morphological features, including stichocytes and paired bacillary bands. This represents an unusual infection by a zoonotic Anatrichosoma species., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Ashworthius sidemi, a blood-sucking nematode, is a primary parasite of Asiatic cervides, primarily sika deer (Cervus nippon). As A. sidemi infections are common in bison, red and roe deer, and gastrointestinal nematodes are often exchanged between animals, it is possible that other farm animals such as cows and sheep that may use the same pastures can be infected. Hence, histopathological changes observed in the walls of the abomasa and duodena of infected wildlife caused by a strong parasite presence may become an important health problem also for farm animals., Methods: In the present study, a simple PCR test for the detection of A. sidemi infection in European bison based on DNA from third stage infective larvae (L3) has been optimized., Results: The species-specific primers generated a 406 bp fragment, and A. sidemi DNA could be detected at concentrations of 0.1 pg/μl. The specificity of PCR was confirmed by the use of the genomic DNA of adult Ostertagia ostertagi, Haemonchus contortus, Cooperia oncophora as negative controls., Conclusion: It is possible to detect A. sidemi infection in European bison using DNA from L3. If this nematode infection is transmitted to cows this method may be effective to diagnose invasion in breeding animals in vivo.

Gastrointestinal nematode (GIN) parasites pose a significant economic burden particularly in small ruminant production systems. Anthelmintic resistance is a serious concern to the effective control of GIN parasites and has fuelled the focus to design and promote sustainable control of practices of parasite control. Many facets of sustainable GIN parasite control programs rely on the ability to diagnose infection both qualitatively and quantitatively. Diagnostics are required to determine anthelmintic efficacies, for targeted treatment programs and selection of animals for parasite resistant breeding. This review describes much of the research investigated to date to improve the current diagnostic for the above practices which is based on counting the number of parasite eggs in faeces.

The presence of gastrointestinal nematode eggs in faecal samples is diagnostic of infection by these parasites. However, this technique cannot be used to distinguish between species of importance. The faecal culture technique and subsequent microscopic analysis of developed larvae is currently used to determine which parasite species are present in the samples, but these techniques take a week to perform and have inherent limitations. To overcome these parasite detection and identification problems, we have developed a DNA extraction method for sheep faeces, and a quantitative multiplex PCR (qPCR) test which can both enumerate and identify Haemonchus, Trichostrongylus and Teladorsagia. We demonstrate that the technique is sensitive to 10 eggs per gram and that dilution of DNA to 0.1 fold can overcome PCR inhibition issues for samples obtained from the field, while maintaining assay sensitivity. Further development of these tests for commercial use is warranted, given their potential to provide consistently faster and more accurate diagnoses of these parasites using simple sample collection and laboratory methods which can be easily adapted for the detection of a variety of pathogens from the same faecal sample., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Many hundreds of millions of people throughout the world are infected by nematodes found in the intestine or tissues with a high prevalence in developing countries. Despite their frequency and morbidity, these infections, which may affect migrants and travelers, remain difficult to diagnosis even in developed countries. This is primarily due to the variety of clinical signs often associated with a lack of highly sensitive and specific diagnostic tools. Parasitological diagnosis is often difficult to achieve and can neither be applied during the pre-patent period nor for parasitic impasses. Serological diagnosis is frequently hampered by a lack of specificity due to cross-reaction with others helminthes. Molecular biology methods still require optimization. The diagnostic approach applied by a clinician of a suspected nematode infection is based on a vast set of data including patient history and way of life, clinical examination, non-specific biological tests and, when available, specific diagnostic tests.

A 65-year-old man presented with decreased visual acuity in his left eye of 10 days' duration. Ocular examination revealed visual acuity of 20/200 in the left eye caused by a visible retinal nematode (roundworm) located close to the fovea. Spectral-domain optical coherence tomography imaging showed the nematode in the retinal nerve fiber layer. The patient was followed up without treatment, and the nematode disappeared spontaneously after 5 weeks. Visual acuity in the affected eye improved to 20/25., (Copyright 2013, SLACK Incorporated.)

A 65-year-old man presented with decreased visual acuity in his left eye of 10 days' duration. Ocular examination revealed visual acuity of 20/200 in the left eye caused by a visible retinal nematode (roundworm) located close to the fovea. Spectral-domain optical coherence tomography imaging showed the nematode in the retinal nerve fiber layer. The patient was followed up without treatment, and the nematode disappeared spontaneously after 5 weeks. Visual acuity in the affected eye improved to 20/25., (Copyright 2013, SLACK Incorporated.)