Veterinary Biosciences - Research Publications
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ItemSpeciation in the genus Cloacina (Nematoda: Strongylida): species flocks and intra-host speciation(CAMBRIDGE UNIV PRESS, 2017-11)Sequences of the first and second internal transcribed spacers (ITS1 + ITS2) of nuclear ribosomal DNA were employed to determine whether the congeneric assemblages of species of the strongyloid nematode genus Cloacina, found in the forestomachs of individual species of kangaroos and wallabies (Marsupialia: Macropodidae), considered to represent species flocks, were monophyletic. Nematode assemblages examined in the black-striped wallaby, Macropus (Notamacropus) dorsalis, the wallaroos, Macropus (Osphranter) antilopinus/robustus, rock wallabies, Petrogale spp., the quokka, Setonix brachyurus, and the swamp wallaby, Wallabia bicolor, were not monophyletic and appeared to have arisen by host colonization. However, a number of instances of within-host speciation were detected, suggesting that a variety of methods of speciation have contributed to the evolution of the complex assemblages of species present in this genus.
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ItemScreening of a small, well-curated natural product-based library identifies two rotenoids with potent nematocidal activity against Haemonchus contortus(ELSEVIER SCIENCE BV, 2017-09-15)The control of parasitic roundworms (nematodes) is heavily reliant on the use of a limited number of anthelmintic drugs. However, drug resistance is now very widespread and no vaccines are available, such that the discovery of new chemical entities is crucial. Within this context, we screened a library of pure natural products (n=400) against exsheathed third-stage (xL3) larvae of the parasitic nematode Haemonchus contortus using a whole-organism screening method. We identified two plant-derived rotenoids, deguelin and rotenone, with inhibitory activity on xL3 motility. Rotenone was not investigated further, because of its toxicity to some vertebrates. The dose response and cytotoxicity studies showed potent and selective inhibitory activity of deguelin on motility of xL3 larvae of H. contortus. Detailed future work needs to be conducted to explore the mode of action of this compound on H. contortus and related nematodes, and to assess its potential as an anthelmintic candidate.
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ItemGenetic characterisation of Taenia multiceps cysts from ruminants in Greece(ELSEVIER, 2016-03)This study was designed to genetically characterise the larval stage (coenurus) of Taenia multiceps from ruminants in Greece, utilising DNA regions within the cytochrome c oxidase subunit 1 (partial cox1) and NADH dehydrogenase 1 (pnad1) mitochondrial (mt) genes, respectively. A molecular-phylogenetic approach was used to analyse the pcox1 and pnad1 amplicons derived from genomic DNA samples from individual cysts (n=105) from cattle (n=3), goats (n=5) and sheep (n=97). Results revealed five and six distinct electrophoretic profiles for pcox1 and pnad1, respectively, using single-strand conformation polymorphism. Direct sequencing of selected amplicons representing each of these profiles defined five haplotypes each for pcox1 and pnad1, among all 105 isolates. Phylogenetic analysis of individual sequence data for each locus, including a range of well-defined reference sequences, inferred that all isolates of T. multiceps cysts from ruminants in Greece clustered with previously published sequences from different continents. The present study provides a foundation for future large-scale studies on the epidemiology of T. multiceps in ruminants as well as dogs in Greece.
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ItemArylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus contortus in vitro.(Elsevier BV, 2018-12)Due to widespread drug resistance in parasitic nematodes, there is a need to develop new anthelmintics. Given the cost and time involved in developing a new drug, the repurposing of known chemicals can be a promising, alternative approach. In this context, we tested a library (n = 600) of natural product-inspired pesticide analogues against exsheathed third stage-larvae (xL3s) of Haemonchus contortus (barber's pole worm) using a whole-organism, phenotypic screening technique that measures the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from chemical scaffolds of arylpyrrole or fipronil. The seven most promising compounds, selected based on their anthelmintic activity and/or limited cytotoxicity, are arylpyrroles that reduced the motility of fourth-stage larvae (L4s) with significant potency (IC50 values ranged from 0.04 ± 0.01 μM to 4.25 ± 0.82 μM, and selectivity indices ranged from 10.6 to 412.5). Since the parent structures of the active compounds are uncouplers of oxidative phosphorylation, we tested the effect of selected analogues on oxygen consumption in xL3s using the Seahorse XF24 flux analyser. Larvae treated with the test compounds showed a significant increase in oxygen consumption compared with the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study have identified natural product-derived molecules that are worth considering for chemical optimisation as anthelmintic drug leads.
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ItemPhylogenetic analysis of the Australasian paralysis ticks and their relatives (Ixodidae: Ixodes: Sternalixodes)(BMC, 2017-03-02)BACKGROUND: The Australasian paralysis ticks and their relatives, Ixodes Latrielle, subgenus Sternalixodes Schulze, are some of the most important ticks in the region. However, very little is known about their phylogenetic relationships. The aim of this study was to elucidate the evolutionary relationships of members of the subgenus Sternalixodes by undertaking phylogenetic analyses of morphological and molecular datasets. METHODS: Adult females (n = 64) of Sternalixodes, including Ixodes anatis Chilton, 1904, Ixodes confusus Roberts, 1960, Ixodes cornuatus Roberts, 1960, Ixodes cordifer Neumann, 1908, Ixodes dendrolagi Wilson, 1967, Ixodes hirsti Hassall, 1931, Ixodes holocyclus Neumann, 1899, Ixodes myrmecobii Roberts, 1962 and Ixodes trichosuri Roberts, 1960, were examined morphologically. Subsequently, these Ixodes spp. were genetically characterised using cytochrome c oxidase subunit 1 (cox1) gene and the internal transcribed spacer 2 (ITS-2) of the rRNA. Both morphological and molecular datasets were analysed using various phylogenetic methods to assess the evolutionary relationship of various members of the subgenus Sternalixodes. RESULTS: Phylogenetic analyses of the cox1 sequences and morphological characters datasets revealed that the Australian and Papuan Sternalixodes formed a distinct clade with the New Zealand member of the group I. anatis positioned basally, in a separate clade. Ixodes holocyclus, I. cornuatus and I. myrmecobii formed a distinctive clade in both the cox1 and morphological phylogenies. However, based on phylogenetic analysis of the ITS-2 data, I. holocyclus formed a separate clade whereas I. cornuatus and I. myrmecobii grouped in a different clade. CONCLUSIONS: The cox1 and morphological data suggest that the subgenus Sternalixodes is paraphyletic, and I. anatis is not a sternalixodid tick; hence, it should not be included in the subgenus. Based on the phylogenetic analyses of cox1 and ITS-2 sequences, it appears that I. myrmecobii and I. cornuatus are not subspecies of I. holocyclus. Although this study provided better insights into the taxonomic status of the subgenus Sternalixodes, a complete morphological and molecular (using multiple markers) phylogenetic analysis including all members of the subgenus would be required to more accurately elucidate the evolutionary relationships within the subgenus.
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ItemScreening of the 'Open Scaffolds' collection from Compounds Australia identifies a new chemical entity with anthelmintic activities against different developmental stages of the barber's pole worm and other parasitic nematodes(ELSEVIER SCI LTD, 2017-12)The discovery and development of novel anthelmintic classes is essential to sustain the control of socioeconomically important parasitic worms of humans and animals. With the aim of offering novel, lead-like scaffolds for drug discovery, Compounds Australia released the 'Open Scaffolds' collection containing 33,999 compounds, with extensive information available on the physicochemical properties of these chemicals. In the present study, we screened 14,464 prioritised compounds from the 'Open Scaffolds' collection against the exsheathed third-stage larvae (xL3s) of Haemonchus contortus using recently developed whole-organism screening assays. We identified a hit compound, called SN00797439, which was shown to reproducibly reduce xL3 motility by ≥ 70%; this compound induced a characteristic, "coiled" xL3 phenotype (IC50 = 3.46-5.93 μM), inhibited motility of fourth-stage larvae (L4s; IC50 = 0.31-12.5 μM) and caused considerable cuticular damage to L4s in vitro. When tested on other parasitic nematodes in vitro, SN00797439 was shown to inhibit (IC50 = 3-50 μM) adults of Ancylostoma ceylanicum (hookworm) and first-stage larvae of Trichuris muris (whipworm) and eventually kill (>90%) these stages. Furthermore, this compound completely inhibited the motility of female and male adults of Brugia malayi (50-100 μM) as well as microfilariae of both B. malayi and Dirofilaria immitis (heartworm). Overall, these results show that SN00797439 acts against genetically (evolutionarily) distant parasitic nematodes i.e. H. contortus and A. ceylanicum [strongyloids] vs. B. malayi and D. immitis [filarioids] vs. T. muris [enoplid], and, thus, might offer a novel, lead-like scaffold for the development of a relatively broad-spectrum anthelmintic. Our future work will focus on assessing the activity of SN00797439 against other pathogens that cause neglected tropical diseases, optimising analogs with improved biological activities and characterising their targets.
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ItemTetrahydroquinoxalines induce a lethal evisceration phenotype in Haemonchus contortus in vitro(ELSEVIER SCI LTD, 2019-04)In the present study, the anthelmintic activity of a human tyrosine kinase inhibitor, AG-1295, and 14 related tetrahydroquinoxaline analogues against Haemonchus contortus was explored. These compounds were screened against parasitic larvae - exsheathed third-stage (xL3) and fourth-stage (L4) - using a whole-organism screening assay. All compounds were shown to have inhibitory effects on larval motility, development and growth, and induced evisceration through the excretory pore in xL3s. The estimated IC50 values ranged from 3.5 to 52.0 μM for inhibition of larval motility or development. Cytotoxicity IC50 against human MCF10A cells was generally higher than 50 μM. Microscopic studies revealed that this eviscerated (Evi) phenotype occurs rapidly (<20 min) and relates to a protrusion of internal tissues and organs (evisceration) through the excretory pore in xL3s; severe pathological damage in L4s as well as a suppression of larval growth in both stages were also observed. Using a relatively low concentration (12.5 μM) of compound m10, it was established that the inhibitor has to be present for a relatively short time (between 30 h and 42 h) during in vitro development from xL3 to L4, to induce the Evi phenotype. Increasing external osmotic pressure prevented evisceration and moulting, and xL3s remained unaffected by the test compound. These results point to a mode of action involving a dysregulation of morphogenetic processes during a critical time-frame, in agreement with the expected behaviour of a tyrosine kinase inhibitor, and suggest potential for development of this compound class as nematocidal drugs.
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ItemArylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus conforms in vitro(Elsevier Inc., 2018-12-01)Due to widespread drug resistance in parasitic nematodes, there is a need to develop new anthelmintics. Given the cost and time involved in developing a new drug, the repurposing of known chemicals can be a promising, alternative approach. In this context, we tested a library (n=600) of natural product-inspired pesticide analogues against exsheathed third stage-larvae (xL3s) of Haemonchus contortus (barber's pole worm) using a wholeorganism, phenotypic screening technique that measures the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from chemical scaffolds of arylpyrrole or fipronil. The seven most promising compounds, selected based on their anthelmintic activity and/or limited cytotoxicity, are arylpyrroles that reduced the motility of fourth-stage larvae (L4s) with significant potency (IC50 values ranged from 0.04 ± 0.01 μM to 4.25 ± 0.82 μM, and selectivity indices ranged from 10.6 to 412.5). Since the parent structures of the active compounds are uncouplers of oxidative phosphorylation, we tested the effect of selected analogues on oxygen consumption in xL3s using the Seahorse XF24 flux analyser. Larvae treated with the test compounds showed a significant increase in oxygen consumption compared with the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study have identified natural product-derived molecules that are worth considering for chemical optimisation as anthelmintic drug leads.
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ItemSelected α-pyrones from the plants Cryptocarya novoguineensis (Lauraceae) and Piper methysticum (Piperaceae) with activity against Haemonchus contortus in vitro(ELSEVIER SCI LTD, 2019-04)Due to the widespread occurrence and spread of anthelmintic resistance, there is a need to develop new drugs against resistant parasitic nematodes of livestock animals. The Nobel Prize-winning discovery and development of the anti-parasitic drugs avermectin and artemisinin has renewed the interest in exploring natural products as anthelmintics. In the present study, we screened 7500 plant extracts for in vitro-activity against the barber's pole worm, Haemonchus contortus, a highly significant pathogen of ruminants. The anthelmintic extracts from two plants, Cryptocarya novoguineensis and Piper methysticum, were fractionated by high-performance liquid chromatography (HPLC). Subsequently, compounds were purified from fractions with significant biological activity. Four α-pyrones, namely goniothalamin (GNT), dihydrokavain (DHK), desmethoxyyangonin (DMY) and yangonin (YGN), were purified from fractions from the two plants, GNT from C. novoguineensis, and DHK, DMY and YGN (= kavalactones) from P. methysticum. The three kavalactones induced a lethal, eviscerated (Evi) phenotype in treated exsheathed third-stage larvae (xL3s), and DMY and YGN had moderate potencies (IC50 values of 31.7 ± 0.23 μM and 23.7 ± 2.05 μM, respectively) at inhibiting the development of xL3s to fourth-stage larvae (L4s). Although GNT had limited potency (IC50 of 200-300 μM) at inhibiting L4 development, it was the only compound that reduced L4 motility (IC50 of 6.25-12.50 μM). The compounds purified from each plant affected H. contortus in an irreversible manner. These findings suggest that structure-activity relationship studies of α-pyrones should be pursued to assess their potential as anthelmintics.
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ItemNew species and new records of species of Cloacina von Linstow, 1898 (Nematoda: Strongylida) parasitic in the western scrub wallaby, Notamacropus irma (Jourdan) (Marsupialia: Macropodidae) from Western Australia(SPRINGER, 2019-07)The helminth parasites of the western scrub wallaby or black-glove wallaby, Notamacropus irma (Jourdan) which occurs in Western Australia are relatively poorly documented. Six new species of the strongyloid genus Cloacina von Linstow, 1898 (Strongylida: Chabertiidae) are described namely C. asymmetrica n. sp., C. brazellei n. sp., C. harriganae n. sp., C. hobbsi n. sp., C. middletoni n. sp. and C. woodi n. sp. A redescription of C. laius Beveridge, 1999 from the same host species is included. Molecular sequence data (ITS1 and ITS2 ribosomal DNA) were obtained for C. asymmetrica, C. brazellei, C. hobbsi, C. middletoni and from the previously described species C. themis Beveridge, 1998 occurring in the same host species. Phylogenetically, C. asymmetrica, C. hobbsi and C. middletoni formed a distinct clade, suggesting the possibility of within-host speciation. Cloacina themis clustered with a group of morphologically distinctive species in a separate clade and C. brazellei clustered in a third clade but with poor support. This pattern of congeners in a single host species occurring in multiple clades mirrors the situation in other kangaroos and wallabies. Species of Cloacina from N. irma reported thus far therefore consist of a series of species found only in this host, with two species (C. brazellei and C. laius) shared with the sympatric macropodid Setonix brachyurus (Quoy & Gaimard).