Molecular and phylogenetic characterization of cytochromes c from Haemonchus contortus and Trichostrongylus vitrinus (Nematoda: Trichostrongylida)

Campbell, Bronwyn E.; Nisbet, Alasdair J.; Mulvenna, Jason; Loukas, Alex; Gasser, Robin B.

doi:10.1016/j.gene.2008.07.028

Cited by 9 publications

(3 citation statements)

References 33 publications

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“…The full-length cDNA isolated above was cloned into pGEM®-T Easy, employing T4 DNA ligase (3 U), and transformed into Escherichia coli (MACH-1 strain, Life Technologies, California, USA) using an established method [47]. Colonies were picked, resuspended in 20 μl of water, with 2 μl used as a PCR template for insert screening and the remaining grown overnight in Luria Bertani/ampicillin (100 g/ml) broth.…”

Section: Methodsmentioning

confidence: 99%

“…Colonies were picked, resuspended in 20 μl of water, with 2 μl used as a PCR template for insert screening and the remaining grown overnight in Luria Bertani/ampicillin (100 g/ml) broth. Plasmid DNA from positive colonies was isolated using the PureYield™ Plasmid Miniprep System (Promega, Wisconsin, USA) and inserts were sequenced in both directions using the T7 and SP6 primers of pGEM®-T Easy [47]. …”

Section: Methodsmentioning

confidence: 99%

“…All cDNAs were ligated into the SmaI restriction enzyme site of the pGEX3X glutathione S-transferase fusion protein expression vector (GE Healthcare Bio-Sciences, Pennsylvania, USA) using T4 DNA ligase (3 U) and transformed into E. coli (BL21 strain). Clones were induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 h and then analyzed via SDS PAGE (10 % gels) using an established protocol [47]. Expressed peptides were purified using EZview™ Red Glutathione Affinity Gel (Sigma Aldrich, Missouri, USA) according to the manufacturer’s instructions, without elution of the peptide from the resin.…”

Section: Methodsmentioning

confidence: 99%

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Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease

et al. 2016

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BackgroundOf increasing importance to the medical and veterinary communities is the zoonotic filarioid nematode Onchocerca lupi. Onchocercosis, thus far found in wolves, dogs, cats and humans, is diagnosed via skin snips to detect microfilariae and surgical removal of adults from the eye of the host. These methods are time-consuming, laborious and invasive, highlighting the need for new tools for the diagnosis of O. lupi in susceptible hosts. Symptoms related to the presence of the adults in the eye can range from none apparent to severe, including blindness. No reliable chemotherapeutic protocols are available, as yet, to eliminate the infection. Paramyosin, an invertebrate-specific protein, has been well-studied as an allergen, diagnostic marker and vaccine candidate. The aim of this study, therefore, was to isolate and characterise paramyosin from O. lupi to assess its suitability for the development of a serological diagnostic assay.MethodsThe adult and microfilarial stages of O. lupi were isolated from the eyes and skin of a 3-year-old male dog. Total RNA was extracted and reverse transcribed into single stranded cDNA. Reverse-transcription PCR was used to isolate a full-length paramyosin cDNA from adult worms and to investigate the temporal expression patterns of this gene. All amplicons were sequenced using dideoxy chain termination sequencing. Bioinformatics was used to predict the amino acid sequence of the gene, to compare the DNA and protein sequences with those available in public databases and to investigate the phylogenetic relationship of all molecules. Antibody binding sites were predicted using bioinformatics and mapped along with published antigenic epitopes against the O. lupi paramyosin protein. The native protein, and three smaller recombinantly expressed peptides, were subjected to western blot using serum from dogs both positive and negative for O. lupi.ResultsParamyosin of O. lupi was herein molecularly characterized, encoded by a transcript of 2,643 bp and producing a protein of 881 amino acids (101.24 kDa). The paramyosin transcript was detected, by reverse transcription PCR, in adults and microfilariae, but not in eggs. Phylogenetic analysis indicates that this molecule clusters with paramyosins from other filarioids to the exclusion of those from other taxa. A total of 621 unique antibody binding epitopes were predicted for this protein and another 28 were conserved in other organisms. This information was used to design three peptides, for recombinant expression, to identify the antibody binding epitope(s) and reduce potential cross-reactivity with serum from dogs infected with other filarioid nematodes. Native paramyosin, purified from microfilariae and adults, was detected by antibodies present in serum from dogs with known O. lupi infections.ConclusionsData provided herein may assist in the development of a serological diagnostic test, based on antibodies to O. lupi paramyosin, for the diagnosis of this infection, in order to gain more information on the real distribution of this l...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease

et al. 2016

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Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals

Rinaldi

Krücken

Martínez‐Valladares

et al. 2022

Advances in Parasitology

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Serine/threonine phosphatases in socioeconomically important parasitic nematodes—Prospects as novel drug targets?

Campbell

Hofmann

McCluskey

et al. 2011

Biotechnology Advances

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Little is known about the fundamental biology of parasitic nematodes (=roundworms) that cause serious diseases, affecting literally billions of animals and humans worldwide. Unlocking the biology of these neglected pathogens using modern technologies will yield crucial and profound knowledge of their molecular biology, and could lead to new treatment and control strategies. Supported by studies in the free-living nematode, Caenorhabditis elegans, some recent investigations have provided improved insights into selected protein phosphatases (PPs) of economically important parasitic nematodes (Strongylida). In the present article, we review this progress and assess the potential of serine/threonine phosphatase (STP) genes and/or their products as targets for new nematocidal drugs. Current information indicates that some small molecules, known to specifically inhibit PPs, might be developed as nematocides. For instance, some cantharidin analogues are known to display exquisite PP-inhibitor activity, which indicates that some of them could be designed and tailored to specifically inhibit selected STPs of nematodes. This information provides prospects for the discovery of an entirely novel class of nematocides, which is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock, and has the potential to lead to significant biotechnological outcomes.

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Molecular and phylogenetic characterization of cytochromes c from Haemonchus contortus and Trichostrongylus vitrinus (Nematoda: Trichostrongylida)

Cited by 9 publications

References 33 publications

Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease

Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease

Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals

Serine/threonine phosphatases in socioeconomically important parasitic nematodes—Prospects as novel drug targets?

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