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Anthelmintic resistance (AR) in parasitic nematodes poses a global health problem in livestock and domestic animals and is an emerging problem in humans. Consequently, we must understand the mechanisms of AR, including target-site resistance (TSR), in which mutations affect drug binding, and non-target site resistance (NTSR), which involves alterations in drug metabolism and detoxification processes. Because much of the focus has been on TSR, NTSR has received less attention. Here, we describe how metabolomics approaches using Caenorhabditis elegans offer the ability to disentangle nematode drug metabolism, identify metabolic changes associated with resistance, uncover novel biomarkers, and enhance diagnostic methods., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.)

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.)

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.)

Parasitic nematodes (roundworms) of livestock have major economic impact globally. In spite of the diseases caused by these nematodes and some advances in the design of new therapeutic agents (anthelmintics) and attempts to develop vaccines against some of them, there has been limited progress in the establishment of practical diagnostic techniques. The specific and sensitive diagnosis of gastrointestinal nematode infections of livestock underpins effective disease control, which is highly relevant now that anthelmintic resistance (AR) is a major problem. Traditional diagnostic techniques have major constraints, in terms of sensitivity and specificity. The purpose of this article is to provide a brief background on gastrointestinal nematodes (Strongylida) of livestock and their control; to summarize conventional methods used for the diagnosis and discuss their constraints; to review key molecular-diagnostic methods and recent progress in the development of advanced amplification-based and sequencing technologies, and their implications for epidemiological investigations and the control of parasitic diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The accurate diagnosis of strongylid nematode infections is central to investigating their epidemiology and for parasite control. To overcome major limitations in sensitivity or specificity of traditional methods, including faecal egg count (FEC) and/or larval culture (LC), we evaluated and established a semi-automated, high throughput multiplexed-tandem PCR (MT-PCR) platform for the diagnosis of gastrointestinal strongylid nematode infections in sheep, and established its diagnostic sensitivity (100%) and specificity (87.5%) based on the testing of 100 faecal DNA samples from helminth-free sheep and 30 samples from sheep with infections confirmed by necropsy. Subsequently, the platform was employed to test 219 faecal samples from sheep with naturally acquired infections from various geographical localities within Australia and the results compared with those from conventional LC using 139 of the 219 samples. The results obtained using both MT-PCR and LC correlated significantly for most nematodes examined, but revealed that Oesophagostomum venulosum and Chabertia ovina (parasites of the large intestine) were significantly under-represented in the LC results. The results showed that Trichostrongylus spp. (87%), Teladorsagia circumcincta (80%) and Haemonchus contortus (67%) had the highest prevalences, followed by O. venulosum (51%) and C. ovina (12%). The molecular-diagnostic platform established can be used for species- or genus-specific diagnosis of patent nematode infections within 24h (compared with 7-10 days for LC), and is a sensitive and cost effective tool for routine application in research and service laboratories., (Copyright © 2012. Published by Elsevier Ltd.)

Purpose: To describe the determinant clinical signs to diagnose diffuse unilateral subacute neuroretinitis (DUSN) and the main features related to identification of the live worm., Design: Retrospective study performed at the Federal University of Rio Grande do Norte, Rio Grande do Norte, Brazil, between 2003 and 2008., Methods: Patients with early- or late-stage diffuse unilateral subacute neuroretinitis were included. All patients underwent complete ophthalmologic examination and had a minimum of 6 months of follow-up., Results: A total of 121 patients were included. Most patients were younger than 20 years (69.42%). Visual acuity was 20/400 or worse in 86 patients (71.7%). Nine patients (7.43%) presented in the early stage and 112 (92.57%) patients presented in the late stage. Subretinal tracks (91.7%), focal alterations of the retinal pigment epithelium (89.3%), small white spots (80.2%), and optic nerve atrophy (76.9%) were the most frequent clinical features. The subretinal worm was identified in 48 patients (39.66%), and laser treatment to destroy it was performed in all cases. The most common location of the nematode was the posterior pole (21 patients). It was observed that the younger the age, the higher the indices of larvae identification (P=.022). Multifocal yellow-white lesions and vitritis were correlated with identification of the worm (P=.001). Mean logarithm of the minimal angle of resolution visual acuity was 1.466 (20/600) and 1.281 (20/400) before and after laser treatment, respectively (P<.005)., Conclusions: Identification of clinical signs and diagnosis of diffuse unilateral subacute neuroretinitis in its early stage, followed by prompt location and destruction of the worm by photocoagulation, may improve the vision of affected patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The binding of 10 different lectins to the surface of trichostrongylid nematode eggs of sheep were investigated. Osage orange seed agglutinin bound to eggs from Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis, jack bean agglutinin to H. contortus and O. circumcincta eggs and peanut agglutinin to H. contortus eggs only. Staining of egg mixtures with fluorescein-labelled peanut agglutinin showed that the lectin correctly identified the percentage of H. contortus eggs present. Identification of eggs by lectin staining correlated well with results from larval differentiation. The findings suggest that peanut agglutinin can be used in routine diagnostic parasitology for the rapid identification of H. contortus eggs.

The protein profile, determined by SDS-PAGE, from different geographical strains (Slovakia and Spain) of Dictyocaulus filaria and Protostrongylus rufescens parasitizing Capra hircus and Ovis aries has been assayed. This protein profile has also been comparatively determined in D. viviparus isolated from lungs of Bos taurus killed in Slovakia, by SDS-PAGE. Protein profiles of D. viviparus and both strains of D. filaria were very similar while in P. rufescens a quite different protein profile was found. Furthermore, the isoenzymatic pattern of Lactate Dehydrogenase (LDH) has been studied in two different geographical strains of D. filaria and P. rufescens and in D. viviparus by starch gel electrophoresis. From both strains of D. filaria and from both sexes, the isoenzymatic pattern of LDH was characterized by the presence of five isoenzymes, four anodical and one cathodical. In contrast, males and females of D. viviparus showed a different LDH isoenzymatic pattern; males presenting two isoenzymes with anodical and cathodical migration, respectively, and females showed only a single isoenzyme with anodical migration. Moreover, the electrophoretic mobility of the D. viviparus isoenzymes was different to that of D. filaria. Therefore, LDH has been designated as an important diagnostic tool to differentiate between species of genus Dictyocaulus. Finally, the LDH isoenzymatic pattern in P. rufescens (Slovakian and Spanish strain) was identical in both sexes appearing as a single band with cathodical migration.

An immunoenzymatic test (DIG-ELISA) was serologically evaluated for the serodiagnosis of onchocerciasis. Control and infected sera from the onchocerciasis endemic area of Mexico was collected and the donors assessed for onchocerciasis according to parasitological, clinical, and epidemiological data. The sera were submitted to the DIG-ELISA test using a crude extract prepared from O. volvulus adult worms which had been preserved in nodules in 67% glycerol. The test showed a 100% sensitivity with sera from 38 microfilariae carriers and 96% specificity with sera from 133 non-infected people living outside the endemic zone. In addition, seropositivity was 52.9% with samples from non-onchocercotic people living inside the endemic area, while 82.5 and 90.5% of sera from patients with clinical symptoms and subcutaneous nodules, respectively, were positive. A high rate (30%) of cross-reactivity with serum samples from people infected with Wuchereria bancrofti or Brugia timori was obtained, which is in contrast with the low seropositivity rates (7.4%) obtained with sera from patients infected with other parasites. These results suggest that DIG-ELISA test may be a useful serological test for antibody detection in onchocerciasis, especially for epidemiological surveillance of disease, but because of the high cross-reactivity observed, its use must be limited to areas where this parasitic infection does not coexist with other human filariasis. Finally, the arrangement of sera in groups according to the relative likelihood to have onchocerciasis seems to be a useful procedure for evaluation of serological tests.

Specific antigen of G. spinigerum which has been shown to be a protein with a relative mol. wt of 24,000 (24K) was prepared from the advanced third-stage larvae (L3) obtained from the livers of naturally infected eels. The L3 were ground and extracted with water. Purification procedures involved gel filtration, chromatofocussing and anion exchange column chromatographies, while characterization of the specific antigen was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining, Western blot analysis and isoelectric focussing. The specific antigen which has a pI of 8.5 was used as antigen in the indirect enzyme-linked immunosorbent assay (ELISA) to detect specific antibody in four groups of individuals, namely five parasitologically diagnosed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); 136 patients with other parasitic infections (group 3); and 25 normal healthy parasite-free controls. Sensitivity, specificity and predictive values (positive and negative) of the assay were 100%.

Sera from four patients with parasitologically confirmed gnathostomiasis, 15 patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite's antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis.

In the analysis of excretory-secretory (ES) antigens from infective third-stage larvae (L3) of Angiostrongylus cantonensis, one major component of mol.wt 91,000 was not precipitated by pooled sera of patients with eosinophilic meningoencephalitis. Monoclonal antibodies (Mc Ab) secreted from two hybridoma cell lines, established against somatic antigens of L3, recognized this molecule but with different epitope specificities indicated by an additivity index (A.I.) of 83%. The 2 Mc Ab (TD2 and 3A5) belonged to IgG2a and IgM classes, respectively. Combinations of TD2 and 3A5 were used in a sensitive enzyme-linked fluorescent assay (ELFA) for the immunodiagnosis of human angiostrongyliasis. The double-antibody sandwich ELFA method was applied firstly to sera from experimentally infected rats using either TD2 or 3A5 to coat the assay plates. Two fluorescence unit (F.U.) peaks appeared in sera from infected rats collected 18 and 44 days after infection. Specimens from 35 patients were tested, all cerebrospinal fluids (CSF) and most sera (88%) showed positive reactions and the average F.U. of CSF was greater than that of serum.

A 7-year-old girl had a white fundus mass and vitreous cells, compatible with an endophytic retinoblastoma. Aqueous aspiration revealed normal lactate dehydrogenase levels and large numbers of eosinophils, suggesting the diagnosis of nematode endophthalmitis. Treatment with corticosteroids brought about resolution of the inflammation and an excellent visual result.

A young Thai woman was afflicted with aural gnathostomiasis. The only symptom she had was sudden intermittent otalgia without apparent hearing loss, tinnitus, vertigo or otorrhea. Presumptive diagnosis was made from the recent history of ingesting raw fish with subsequent migratory swellings. Definite diagnosis rested on identification of the worms, pertinent eosinophilia and positive skin test. This case was different from those previously reported because it was a primary aural gnathostomiasis without neurological involvement.

We studied the clinical and pathologic features of 22 cases of granulomas of the conjunctiva or eyelids. All cases showed the histologic features of the Splendore-Hoeppli phenomenon, that is, a giant cell and eosinophil granulomatous reaction to an antigen-antibody precipitate originally described in relation to parasites or fungi. In four of seven typical cases selected for detailed description unidentified nematodes were found to be the cause of the condition. In light of these findings together with a review of similar "allergic granulomas" reported both in ocular tissues and elsewhere in the body, we considered the cause in the remaining cases. All 22 cases may have been caused by nematodes, as seems, probable in 14 of them, or the causative antigens may have been of widely different kinds. Although in our cases all ocular granulomas had an identical histology, this study did not resolve the problem of those cases where no causative agent was found. Thorough investigation of such cases in the future and the demonstration of their cause may elucidate the wider problem of nonocular allergic granulomas.