Melbourne Veterinary School - Research Publications

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    Arylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus conforms in vitro
    Herath, HMPD ; Song, H ; Preston, S ; Jabbar, A ; Wang, T ; McGee, SL ; Hofmann, A ; Garcia-Bustos, J ; Chang, BCH ; Koehler, AV ; Liu, Y ; Ma, Q ; Zhang, P ; Zhao, Q ; Wang, Q ; Gasser, RB (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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    Selected alpha-pyrones from the plants Cryptocarya novoguineensis (Lauraceae) and Piper methysticum (Piperaceae) with activity against Haemonchus contortus in vitro
    Herath, HMPD ; Preston, S ; Jabbar, A ; Garcia-Bustos, J ; Addison, RS ; Hayes, S ; Rali, T ; Wang, T ; Koehler, A ; Chang, BCH ; Hofmann, A ; Davis, RA ; Gasser, RB (ELSEVIER SCI LTD, 2019-04-01)
    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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    Phenotypic screening of the "Kurz-box' of chemicals identifies two compounds (BLK127 and HBK4) with anthelmintic activity in vitro against parasitic larval stages of Haemonchus contortus
    Linh, TN ; Kurz, T ; Preston, S ; Brueckmann, H ; Lungerich, B ; Herath, HMPD ; Koehler, AV ; Wang, T ; Skalova, L ; Jabbar, A ; Gasser, RB (BMC, 2019-04-30)
    BACKGROUND: Due to anthelmintic resistance problems, there is a need to discover and develop new drugs for the treatment and control of economically important and pathogenic nematodes of livestock animals. With this focus in mind, we screened 236 compounds from a library (called the 'Kurz-box') representing chemically diverse classes such as heterocyclic compounds (e.g. thiazoles, pyrroles, quinolines, pyrimidines, benzo[1,4]diazepines), hydoxamic acid-based metalloenzyme inhibitors, peptidomimetics (bis- and tris-pyrimidoneamides, alkoxyamides) and various intermediates on Haemonchus contortus, one of the most important parasitic nematodes of ruminants. METHODS: In the present study, we tested these compounds, and measured the inhibition of larval motility and development of exsheathed third-stage (xL3) and fourth-stage (L4) larvae of H. contortus using an optimised, whole-organism phenotypic screening assay. RESULTS: Of the 236 compounds, we identified two active compounds (called BLK127 and HBK4) that induced marked phenotypic changes in the worm in vitro. Compound BLK127 induced an 'eviscerated' phenotype in the xL3 stage and also inhibited L4 development. Compound HBK4 exerted a 'curved' phenotype in both xL3s and L4s. CONCLUSIONS: The findings from this study provide a basis for future work on the chemical optimisation of these compounds, on assessing the activity of optimised compounds on adult stages of H. contortus both in vitro and in vivo (in the host animal) and against other parasitic worms of veterinary and medical importance.
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    First cross-sectional, molecular epidemiological survey of Cryptosporidium, Giardia and Enterocytozoon in alpaca (Vicugna pacos) in Australia
    Koehler, AV ; Rashid, MH ; Zhang, Y ; Vaughan, JL ; Gasser, RB ; Jabbar, A (BMC, 2018-09-05)
    BACKGROUND: Eukaryotic pathogens, including Cryptosporidium, Giardia and Enterocytozoon, have been implicated in neonatal diarrhoea, leading to marked morbidity and mortality in the alpaca (Vicugna pacos) and llama (Lama glama) around the world. Australia has the largest population of alpacas outside of South America, but very little is known about these pathogens in alpaca populations in this country. Here, we undertook the first molecular epidemiological survey of Cryptosporidium, Giardia and Enterocytozoon in V. pacos in Australia. METHODS: A cross-sectional survey of 81 herds, comprising alpacas of 6 weeks to 26 years of age, were sampled from the six Australian states (Queensland, New South Wales, Victoria, South Australia, Tasmania and Western Australia) across the four seasons. PCR-based sequencing was employed, utilising genetic markers in the small subunit of the nuclear ribosomal RNA (SSU) and 60-kilodalton glycoprotein (gp60) genes for Cryptosporidium, triose-phosphate isomerase (tpi) gene for Giardia duodenalis and the internal transcribed spacer region (ITS) for Enterocytozoon bieneusi. RESULTS: PCR-based analyses of 81 faecal DNA samples representing 1421 alpaca individuals detected Cryptosporidium, Giardia and/or Enterocytozoon on 15 farms in New South Wales, Victoria and South Australia, equating to 18.5% of all samples/herds tested. Cryptosporidium was detected on three (3.7%) farms, G. duodenalis on six (7.4%) and E. bieneusi on eight (9.9%) in two or all of these three states, but not in Queensland, Tasmania or Western Australia. Molecular analyses of selected faecal DNA samples from individual alpacas for Cryptosporidium, Giardia and/or Enterocytozoon consistently showed that alpacas of ≤ 6 months of age harboured these pathogens. CONCLUSIONS: This first molecular investigation of Cryptosporidium, Giardia and Enterocytozoon in alpaca subpopulations in Australia has identified species and genotypes that are of likely importance as primary pathogens of alpacas, particularly young crias, and some genotypes with zoonotic potential. Although the prevalence established here in the alpaca subpopulations studied is low, the present findings suggest that crias are likely reservoirs of infections to susceptible alpacas and/or humans. Future studies should focus on investigating pre-weaned and post-weaned crias, and on exploring transmission patterns to establish what role particular genotypes play in neonatal or perinatal diarrhoea in alpacas and in zoonotic diseases in different states of Australia.
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    Using PCR-Based Sequencing to Diagnose Haycocknema perplexum Infection in Human Myositis Case, Australia
    Koehler, AV ; Leung, P ; McEwan, B ; Gasser, RB (CENTERS DISEASE CONTROL, 2018-12-01)
    We report a case of myositis in a male patient in Australia who had progressive weakness and wasting in his left lower limb. Although clinical, pathologic, and laboratory assessments were inconclusive, a new, nested PCR-coupled sequencing method enabled the unequivocal diagnosis of myositis caused by the enigmatic nematode Haycocknema perplexum.
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    Description of Cloacina atthis sp. nov. from the stomach of the euro (Macropus robustus) (Marsupialia: Macropodidae) from Western Australia based on morphological and molecular criteria
    Beveridge, I ; Hanh, N ; Nyein, S ; Cheng, C ; Koehler, A ; Shuttleworth, ME ; Gasser, RB ; Jabbar, A (SPRINGER, 2014-09-01)
    A new species of strongyloid nematode from the genus Cloacina (Chabertiidae: Cloacininae) is described from the stomach of the hill kangaroo or euro (Macropus robustus) (Marsupialia: Macropodidae) from Western Australia. Cloacina atthis sp. nov. was found only in euros from the Pilbara region in the northwest of Western Australia, in spite of extensive collecting of the same host species from around the Australian continent. C. atthis is most closely related to Cloacina clymene, a species found in the same host species but only in the eastern half of the continent; the two species differ in minor morphological features (the shape of the wall of the buccal capsule, spicule lengths, the degree of sclerotisation of the gubernaculum and the shape of the vagina) as well as in differences in the internal transcribed spacers of ribosomal DNA. This study highlights the importance of using molecular methods when investigating the apparently disjunct distributions of strongyloid nematodes.
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    Molecular investigation of Cryptosporidium and Giardia in pre- and post-weaned calves in Hubei Province, China
    Fan, Y ; Wang, T ; Koehler, AV ; Hu, M ; Gasser, RB (BIOMED CENTRAL LTD, 2017-10-25)
    BACKGROUND: The protistan pathogens Cryptosporidium and Giardia can cause significant intestinal diseases in animals and humans. Cattle, particularly calves, carrying these protists can be significant reservoirs for human infections and disease. However, little is known about the genetic make-up of Cryptosporidium and Giardia populations in cattle and other ruminants in some regions of China. RESULTS: In the present study, PCR-based tools were used to genetically characterise these protists in faecal samples from a total of 339 pre- and post-weaned calves from four distinct locations in Hubei Province using markers in the large (LSU) or small (SSU) subunits of nuclear ribosomal RNA genes. Cryptosporidium andersoni, C. bovis, C. ryanae and Giardia duodenalis assemblage E were detected in 0.6%, 10.9%, 4.1% and 22.6% of calves, respectively. CONCLUSIONS: This study is the first to report the prevalence of Cryptosporidium and Giardia in pre- and post-weaned calves in Hubei Province, and encourages large-scale molecular studies of animals and humans, in an effort to better understand the epidemiology of these enteric pathogens in China.
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    Use of a bioinformatic-assisted primer design strategy to establish a new nested PCR-based method for Cryptosporidium
    Koehler, AV ; Korhonen, PK ; Hall, RS ; Young, ND ; Wang, T ; Haydon, SR ; Gasser, RB (BMC, 2017-10-23)
    BACKGROUND: The accurate tracking of Cryptosporidium in faecal, water and/or soil samples in water catchment areas is central to developing strategies to manage the potential risk of cryptosporidiosis transmission to humans. Various PCR assays are used for this purpose. Although some assays achieve specific amplification from Cryptosporidium DNA in animal faecal samples, some do not. Indeed, we have observed non-specificity of some oligonucleotide primers in the small subunit of nuclear ribosomal RNA gene (SSU), which has presented an obstacle to the identification and classification of Cryptosporidium species and genotypes (taxa) from faecal samples. RESULTS: Using a novel bioinformatic approach, we explored all available Cryptosporidium genome sequences for new and diagnostically-informative, multi-copy regions to specifically design oligonucleotide primers in the large subunit of nuclear ribosomal RNA gene (LSU) as a basis for an effective nested PCR-based sequencing method for the identification and/or classification of Cryptosporidium taxa. CONCLUSION: This newly established PCR, which has high analytical specificity and sensitivity, is now in routine use in our laboratory, together with other assays developed by various colleagues. Although the present bioinformatic workflow used here was for the specific design of primers in nuclear DNA of Cryptosporidium, this approach should be broadly applicable to many other microorganisms.