Veterinary Science - Research Publications

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    Screening of the 'Stasis Box' identifies two kinase inhibitors under pharmaceutical development with activity against Haemonchus contortus
    Jiao, Y ; Preston, S ; Koehler, AV ; Stroehlein, AJ ; Chang, BCH ; Simpson, KJ ; Cowley, KJ ; Palmer, MJ ; Laleu, B ; Wells, TNC ; Jabbar, A ; Gasser, RB (BMC, 2017-07-05)
    BACKGROUND: In partnership with the Medicines for Malaria Venture (MMV), we screened a collection ('Stasis Box') of 400 compounds (which have been in clinical development but have not been approved for illnesses other than neglected infectious diseases) for inhibitory activity against Haemonchus contortus, in order to attempt to repurpose some of the compounds to parasitic nematodes. METHODS: We assessed the inhibition of compounds on the motility and/or development of exsheathed third-stage (xL3s) and fourth-stage (L4) larvae of H. contortus using a whole-organism screening assay. RESULTS: In the primary screen, we identified compound MMV690767 (also known as SNS-032) that inhibited xL3 motility by ~70% at a concentration of 20 μM after 72 h as well as compound MMV079840 (also known as AG-1295), which induced a coiled xL3 phenotype, with ~50% inhibition on xL3 motility. Subsequently, we showed that SNS-032 (IC50 = 12.4 μM) and AG-1295 (IC50 = 9.92 ± 1.86 μM) had a similar potency to inhibit xL3 motility. Although neither SNS-032 nor AG-1295 had a detectable inhibitory activity on L4 motility, both compounds inhibited L4 development (IC50 values = 41.24 μM and 7.75 ± 0.94 μM for SNS-032 and AG-1295, respectively). The assessment of the two compounds for toxic effects on normal human breast epithelial (MCF10A) cells revealed that AG-1295 had limited cytotoxicity (IC50 > 100 μM), whereas SNS-032 was quite toxic to the epithelial cells (IC50 = 1.27 μM). CONCLUSIONS: Although the two kinase inhibitors, SNS-032 and AG-1295, had moderate inhibitory activity on the motility or development of xL3s or L4s of H. contortus in vitro, further work needs to be undertaken to chemically alter these entities to achieve the potency and selectivity required for them to become nematocidal or nematostatic candidates.
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    Assessing the performance of multiplexed tandem PCR for the diagnosis of pathogenic genotypes of Theileria orientalis using pooled blood samples from cattle
    Gebrekidan, H ; Gasser, RB ; Stevenson, MA ; McGrath, S ; Jabbar, A (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2017-02-01)
    Oriental theileriosis caused by multiple genotypes of Theileria orientalis is an important tick-borne disease of bovines. Here, we assessed the performance of an established multiplexed tandem PCR (MT-PCR) for the diagnosis of the two recognized, pathogenic genotypes (chitose and ikeda) of T. orientalis in cattle using pooled blood samples. We used a total of 265 cattle blood samples, which were divided into two groups according to previous MT-PCR results for individual samples. Samples in group 1 (n = 155) were from a herd with a relatively high prevalence of T. orientalis infection; and those in group 2 (n = 110) were from four herds with a low prevalence. For group 1, 31 and 15 batches of five- and ten-pooled samples (selected at random), respectively, were formed. For group 2, 22 and 11 batches of five- and ten-pooled samples (selected at random), respectively, were formed. DNAs from individual pooled samples in each batch and group were then tested by MT-PCR. For group 1, the apparent prevalences estimated using the 31 batches of five-pooled samples (97%) and 15 batches of ten-pooled samples (100%) were significantly higher compared with individual samples (75%). For group 2, higher apparent prevalences (9% and 36%) were also recorded for the 22 and 11 batches of pooled samples, respectively, compared with individual samples (7%). Overall, the average infection intensity recorded for the genotypes of chitose and ikeda were considerably lower in pooled compared with individual samples. The diagnostic specificities of MT-PCR were estimated at 95% and 94%, respectively, when batches of five- and ten-pooled samples were tested, and 94% for individual samples. The diagnostic sensitivity of this assay was estimated at 98% same for all individual, five- and ten-pooled samples. This study shows that screening batches of five- and ten-pooled blood samples from cattle herds are similar to those obtained for individual samples, and, importantly, that the reduced cost for the testing of pooled samples represents a considerable saving to herd managers.
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    Molecular characterisation of Theileria orientalis in imported and native bovines from Pakistan
    Gebrekidan, H ; Abbas, T ; Wajid, M ; Ali, A ; Gasser, RB ; Jabbar, A (ELSEVIER, 2017-01-01)
    The epidemiological aspects of Theileria orientalis in Pakistan are unknown; therefore, investigations using sensitive and precise molecular techniques are required. This study reports the first molecular characterisation of T. orientalis detected from imported (Bos taurus) and native cattle (Bos indicus×Bos taurus) and buffaloes (Bubalus bubalis) selected from four districts of Punjab, Pakistan. DNA samples from blood (n=246) were extracted and tested using conventional PCR utilising the major piroplasm surface protein (MPSP) gene and multiplexed tandem PCR (MT-PCR). Theileria orientalis DNA was detected (15%; 22/147) only in imported cattle by conventional PCR, whereas 24.5% (36/147), 6% (3/50) and 6.1% (3/49) of the imported cattle and native Pakistani cattle and buffaloes, respectively were test-positive for T. orientalis using MT-PCR. Using MT-PCR, the prevalence of T. orientalis was significantly higher (P<0.0001) in imported cattle compared to that of detected in native Pakistani bovines. The prevalence of T. orientalis and DNA copies of chitose and ikeda were significantly higher (P<0.05) in imported cattle than those detected in native Pakistani bovines. DNA sequencing of amplicons of the conventional PCR revealed the presence of buffeli, chitose and ikeda genotypes of T. orientalis. Phylogenetic analysis revealed that the MPSP sequences of buffeli, chitose and ikeda from imported cattle were closely related to those sequences reported previously from Australia and other regions. This study provides the first survey of T. orientalis infection in imported and native bovines in Pakistan, and highlights the need for future studies to understand the spread of transboundary animal diseases.
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    Assessing the anthelmintic activity of pyrazole-5-carboxamide derivatives against Haemonchus contortus
    Jiao, Y ; Preston, S ; Song, H ; Jabbar, A ; Liu, Y ; Baell, J ; Hofmann, A ; Hutchinson, D ; Wang, T ; Koehler, AV ; Fisher, GM ; Andrews, KT ; Laleu, B ; Palmer, MJ ; Burrows, JN ; Wells, TNC ; Wang, Q ; Gasser, RB (BIOMED CENTRAL LTD, 2017-05-31)
    BACKGROUND: In this study, we tested five series of pyrazole-5-carboxamide compounds (n = 55) for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm), one of the most pathogenic parasites of ruminants. METHODS: In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility and development of H. contortus. RESULTS: Amongst the 55 compounds, we identified two compounds (designated a-15 and a-17) that reproducibly inhibit xL3 motility as well as L4 motility and development, with IC50 values ranging between ~3.4 and 55.6 μM. We studied the effect of these two 'hit' compounds on mitochondrial function by measuring oxygen consumption. This assessment showed that xL3s exposed to each of these compounds consumed significantly less oxygen and had less mitochondrial activity than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. CONCLUSIONS: The present findings provide a sound basis for future work, aimed at identifying the targets of compounds a-15 and a-17 and establishing the modes of action of these chemicals in H. contortus.
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    Molecular characterization of species of Cloacina (Strongyloidea: Cloacininae) from the common wallaroo, Macropus robustus (Marsupialia: Macropodidae) in Australia
    Shuttleworth, MA ; Beveridge, I ; Koehler, AV ; Gasser, RB ; Jabbar, A (ELSEVIER, 2016-10-01)
    This study examined genetic variation within and among species of Cloacina found in the common wallaroo (Macropus robustus) collected at different localities from mainland Australia, and evaluated geographical distance as a potential driver for genetic variation. The first and second internal transcribed spacers (ITS-1 and ITS-2=ITS) of nuclear ribosomal DNA were used to characterize individuals of 17 morphospecies of Cloacina that parasitize Macropus robustus and its sub-species. Results revealed intraspecific variation in ITS within some morphospecies of Cloacina. Phylogenetic analyses showed little correlation between host speciation patterns and geographical location for the majority of the nematode species, although it did suggest geographical distance was a driver for speciation within Cloacina communis, C. phaethon and C. parva. Our results suggest that nucleotide variation within Cloacina species is complex, and is likely to be propagated by factors such as geographical distance and host sub-species. Further studies determining factors involved in speciation, such as host-parasite relationships, are needed to improve our understanding of the diversity of populations of species of Cloacina.
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    Organometallic Derivatization of the Nematocidal Drug Monepantel Leads to Promising Antiparasitic Drug Candidates
    Hess, J ; Patra, M ; Rangasamy, L ; Konatschnig, S ; Blacque, O ; Jabbar, A ; Mac, P ; Jorgensen, EM ; Gasser, RB ; Gasser, G (WILEY-V C H VERLAG GMBH, 2016-11-01)
    The discovery of novel drugs against animal parasites is in high demand due to drug-resistance problems encountered around the world. Herein, the synthesis and characterization of 27 organic and organometallic derivatives of the recently launched nematocidal drug monepantel (Zolvix® ) are described. The compounds were isolated as racemates and were characterized by 1 H, 13 C, and 19 F NMR spectroscopy, mass spectrometry, and IR spectroscopy, and their purity was verified by microanalysis. The molecular structures of nine compounds were confirmed by X-ray crystallography. The anthelmintic activity of the newly designed analogues was evaluated in vitro against the economically important parasites Haemonchus contortus and Trichostrongylus colubriformis. Moderate nematocidal activity was observed for nine of the 27 compounds. Three compounds were confirmed as potentiators of a known monepantel target, the ACR-23 ion channel. Production of reactive oxygen species may confer secondary activity to the organometallic analogues. Two compounds, namely, an organic precursor (3 a) and a cymantrene analogue (9 a), showed activities against microfilariae of Dirofilaria immitis in the low microgram per milliliter range.
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    The complement of family M1 aminopeptidases of Haemonchus contortus - Biotechnological implications
    Mohandas, N ; Young, ND ; Jabbar, A ; Korhonen, PK ; Koehler, AV ; Hall, RS ; Hu, M ; Hofmann, A ; Gasser, RB (PERGAMON-ELSEVIER SCIENCE LTD, 2016-03-01)
    Although substantial research has been focused on the 'hidden antigen' H11 of Haemonchus contortus as a vaccine against haemonchosis in small ruminants, little is know about this and related aminopeptidases. In the present article, we reviewed genomic and transcriptomic data sets to define, for the first time, the complement of aminopeptidases (designated Hc-AP-1 to Hc-AP-13) of the family M1 with homologues in Caenorhabditis elegans, characterised by zinc-binding (HEXXH) and exo-peptidase (GAMEN) motifs. The three previously published H11 isoforms (accession nos. X94187, FJ481146 and AJ249941) had most sequence similarity to Hc-AP-2 and Hc-AP-8, whereas unpublished isoforms (accession nos. AJ249942 and AJ311316) were both most similar to Hc-AP-3. The aminopeptidases characterised here had homologues in C. elegans. Hc-AP-1 to Hc-AP-8 were most similar in amino acid sequence (28-41%) to C. elegans T07F10.1; Hc-AP-9 and Hc-AP-10 to C. elegans PAM-1 (isoform b) (53-54% similar); Hc-AP-11 and Hc-AP-12 to C. elegans AC3.5 and Y67D8C.9 (26% and 50% similar, respectively); and Hc-AP-13 to C. elegans C42C1.11 and ZC416.6 (50-58% similar). Comparative analysis suggested that Hc-AP-1 to Hc-AP-8 play roles in digestion, metabolite excretion, neuropeptide processing and/or osmotic regulation, with Hc-AP-4 and Hc-AP-7 having male-specific functional roles. The analysis also indicated that Hc-AP-9 and Hc-AP-10 might be involved in the degradation of cyclin (B3) and required to complete meiosis. Hc-AP-11 represents a leucyl/cystinyl aminopeptidase, predicted to have metallopeptidase and zinc ion binding activity, whereas Hc-AP-12 likely encodes an aminopeptidase Q homologue also with these activities and a possible role in gonad function. Finally, Hc-AP-13 is predicted to encode an aminopeptidase AP-1 homologue of C. elegans with hydrolase activity, suggested to operate, possibly synergistically with a PEPT-1 ortholog, as an oligopeptide transporter in the gut for protein uptake and normal development and/or reproduction of the worm. An appraisal of structure-based amino acid sequence alignments revealed that all conceptually translated Hc-AP proteins, with the exception of Hc-AP-12, adopt a topology similar to those observed for the two subgroups of mammalian M1 aminopeptidases, which possess either three (I, II and IV) or four (I-IV) domains. In contrast, Hc-AP-12 lacks the N-terminal domain (I), but possesses a substantially expanded domain III. Although further work needs to be done to assess amino acid sequence conservation of the different aminopeptidases among individual worms within and among H. contortus populations, we hope that these insights will support future localisation, structural and functional studies of these molecules in H. contortus as well as facilitate future assessments of a recombinant subunit or cocktail vaccine against haemonchosis.
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    Practical and low cost whole-organism motility assay: A step-by-step protocol
    Preston, S ; Jabbar, A ; Nowell, C ; Joachim, A ; Ruttkowski, B ; Cardno, T ; Hofmann, A ; Gasser, RB (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2016-02-01)
    Here, we provide a step-by-step protocol for a practical and low cost whole-organism assay for the screening of chemical compounds for activity against parasitic worms. This assay has considerable advantages over conventional methods, mainly in relation to ease of use, throughput, time and cost. It is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation, and should be applicable to many different parasites and other organisms commensurate with the size of wells in the microtiter plates used for phenotypic screening.
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    Molecular characterization of Theileria orientalis from cattle in Ethiopia
    Gebrekidan, H ; Gasser, RB ; Baneth, G ; Yasur-Landau, D ; Nachum-Biala, Y ; Hailu, A ; Jabbar, A (ELSEVIER GMBH, 2016-01-01)
    This study reports the first molecular characterization of Theileria orientalis in local breeds of cattle in Ethiopia. A conventional PCR utilizing major piroplasm surface protein (MPSP) gene and an established multiplexed tandem PCR (MT-PCR) were used to characterize T. orientalis and to assess the infection intensity, respectively. Of 232 blood samples tested, T. orientalis DNA was detected in only 2.2% of samples using conventional PCR; two genotypes buffeli (1.3%; 3/232) and type 5 (0.9%; 2/232) of T. orientalis were detected. Phylogenetic analysis revealed that the buffeli MPSP sequences from Ethiopia were closely related to those reported from Kenya, Sri Lanka and Myanmar, and type 5 sequences from Ethiopia grouped with those from Korea, Japan, Vietnam and Thailand. A higher number of samples (3.9%; 9/232) were test-positive by MT-PCR and four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected. The average intensity of infections with genotypes buffeli (DNA copy numbers 11,056) and type 5 (7508) were significantly higher (P<0.0001) than the pathogenic genotype ikeda (61 DNA copies). This first insight into T. orientalis from cattle in Ethiopia using MPSP gene provides a basis for future studies of T. orientalis in various agroclimatic zones and of the impact of oriental theilerosis on cattle in this and other countries of Africa.
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    Assessment of the nematocidal activity of metallocenyl analogues of monepantel
    Hess, J ; Patra, M ; Jabbar, A ; Pierroz, V ; Konatschnig, S ; Spingler, B ; Ferrari, S ; Gasser, RB ; Gasser, G (ROYAL SOC CHEMISTRY, 2016-01-01)
    In this study, we present the design, synthesis, characterization and biological evaluation of structurally new ferrocenyl and ruthenocenyl derivatives of the organic anthelmintic monepantel (Zolvix®). All seven metallocenyl derivatives prepared (4a/b, 5a/b, 6a/b and 7) were isolated as racemates and characterized by 1H, 13C and 19F NMR spectroscopies, mass spectrometry, IR spectroscopy and elemental microanalysis. The molecular structures of four compounds (4a/b, 6a and 7) were further confirmed by X-ray crystallography. The biological activities of the organometallic intermediates (4a/b) and organometallic derivatives of monepantel (5a/b, 6a/b and 7) were evaluated in vitro using parasitic nematodes of major importance in livestock, namely Haemonchus contortus and Trichostrongylus colubriformis. Two ferrocenyl compounds (4a and 6a) showed nematocidal activity, while the analogous ruthenocenyl compounds (4b and 6b) were not active at the highest concentration tested (10 μg mL-1). In order to obtain insight into the difference in activity between ferrocenyl and ruthenocenyl derivatives, the potential of the compounds for reactive oxidative species (ROS) production in live cells was assessed. Interestingly, neither the ferrocenyl nor the ruthenocenyl compounds (4a/b and 6a/b) produced significant ROS in HeLa cells when checked after 22 h, potentially indicating a redox-independent activity of 4a and 6a on the parasites. The selectivity of the compounds on parasites was confirmed by investigating their cytotoxicity profiles. None of these compounds was toxic either to HeLa or MRC-5 cells. Thus, 4a and 6a could be considered as interesting leads for further development of new classes of anti-parasitic agents.