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

Mitchell, Mairi C.; Tzelos, Thomas; Handel, Ian; McWilliam, Hamish E G; Hodgkinson, Jane E.; Nisbet, Alasdair J.; Kharchenko, V. A.; Burgess, Stewart T. G.; Matthews, J. B.

doi:10.1017/s0031182016000627

Cited by 15 publications

(21 citation statements)

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“…An ELISA was developed based on recombinant Cy-GALA proteins, which allows the detection of the immune response to cyatostomin larvae. Cross-reactivity to other parasites was not observed and is unlikely, because of the diversity of orthologous GALA sequences of non-cyathostomin species (Mitchell et al, 2016). In the absence of experimental single species infections, cross-reactivity between cyathostomin species is hard to evaluate and diagnostic tests should therefore include a panel of different Cy-GALA proteins to detect most larval cyathostomin infections.…”

Section: Serological Methodsmentioning

confidence: 99%

Nematode Species Identification—Current Status, Challenges and Future Perspectives for Cyathostomins

Bredtmann

Krücken

Murugaiyan

et al. 2017

Front. Cell. Infect. Microbiol.

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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.

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Section: Serological Methodsmentioning

confidence: 99%

Nematode Species Identification—Current Status, Challenges and Future Perspectives for Cyathostomins

Bredtmann

Krücken

Murugaiyan

et al. 2017

Front. Cell. Infect. Microbiol.

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“…For this reason, it is currently recommended that FEC-directed protocols be augmented with at least one treatment aimed to be effective against these parasitic species and stages – for example, moxidectin administration in the late autumn/winter in temperate areas in the northern hemisphere (Hertzberg and others 2014). An ELISA diagnostic test is being developed to detect infection with encysted cyathostomins (Mitchell and others 2016) and a real-time quantitative PCR test has been developed to detect Strongylus vulgaris infection (Nielsen and others 2012a). …”

Section: Sustainable Control Strategiesmentioning

confidence: 99%

Anthelmintic resistance in equine helminths and mitigating its effects

Tzelos¹,

Matthews²

2016

In Practice

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Helminth control in horses has been based on the administration of broad-spectrum anthelmintics for over 50 years, but although this has reduced clinical disease, anthelmintic-resistant populations of nematodes (for example, cyathostomins and Parascaris equorum) have developed due to frequent ‘blanket’ treatments. Resistance to benzimidazole, pyrantel and, more recently, macrocyclic lactones is not uncommon so strategies that balance the use of anthelmintics for parasite control with the need to limit selection pressure for resistance should be adopted. An inability to control burdens of these nematodes could cause serious health problems, particularly in younger horses, as well as in the small number of individuals that have a higher susceptibility to high levels of infection throughout life. This article reviews anthelmintic resistance in equine nematodes and the measures that can be taken in practice to try to mitigate its spread.

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“…As no new anthelmintic classes are under development for use in horses in the short to medium term, it is important that the efficacy of currently effective compounds is preserved. Recommendations for control now place a strong emphasis on reducing anthelmintic treatment frequency (Matthews, 2014;Tzelos and Matthews, 2016). Because cyathostomin infections exhibit a negative binomial distribution amongst hosts (Lester et al, 2013;Relf et al, 2013;Wood et al, 2013), substantial reductions in treatment levels can be achieved by targeting anthelmintics based on worm egg shedding levels.…”

Section: Introductionmentioning

confidence: 99%

“…Because cyathostomin infections exhibit a negative binomial distribution amongst hosts (Lester et al, 2013;Relf et al, 2013;Wood et al, 2013), substantial reductions in treatment levels can be achieved by targeting anthelmintics based on worm egg shedding levels. Faecal egg count (FEC)-directed treatments are thus recommended (Sangster, 2003;Kaplan and Nielsen, 2010;Nielsen et al, 2014;Tzelos and Matthews, 2016), with good uptake in some regions (Easton et al, 2016, Tzelos et al, 2019. A main disadvantage of FEC analysis is the lack of information on total intra-host burden; in terms of targeting larvae to avoid disease, FEC tests therefore do not provide the relevant information.…”

Section: Introductionmentioning

confidence: 99%

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

Tzelos

Geyer

Mitchell

et al. 2020

International Journal for Parasitology

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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 an key gap in the diagnostic toolbox. Previously, a cyathostomin Gut Associated Larval Antigen (Cy-GALA) was identified as a promising marker for the intra-host stages of infection. Here, Cy-GALA and an additional protein, Cyathostomin Immuno-diagnostic (Cy-CID) 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 (ROC) curve analysis was performed on the ELISA data; proteins with the highest Area Under the Curve (AUC) values were selected to test protein combinations to investigate which were the most informative in identifying the infection status of individuals. Three cocktail (CT) 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.

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

Abstract: accession numbers: JN596964 (Cy-gala-ash), JN596966 (Cy-gala-cat), JN596967 (Cy-

Cited by 15 publications

References 54 publications

Nematode Species Identification—Current Status, Challenges and Future Perspectives for Cyathostomins

Nematode Species Identification—Current Status, Challenges and Future Perspectives for Cyathostomins

Anthelmintic resistance in equine helminths and mitigating its effects

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

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