Neil H. Bagnall

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.

Lucilia cuprina genome unlocks parasitic fly biology to underpin future interventions

Lucilia cuprina is a parasitic fly of major economic importance worldwide. Larvae of this fly invade their animal host, feed on tissues and excretions and progressively cause severe skin disease (myiasis). Here we report the sequence and annotation of the 458-megabase draft genome of Lucilia cuprina. Analyses of this genome and the 14,544 predicted protein-encoding genes provide unique insights into the flys molecular biology, interactions with the host animal and insecticide resistance. These insights have broad implications for designing new methods for the prevention and control of myiasis.

Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus.

While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype.

Activation of several key components of the epidermal differentiation pathway in cattle following infestation with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick, Rhipicephalus (Boophilus) microplus, and the diseases it transmits pose a persistent threat to tropical beef production. Genetic selection of host resistance has become the method of choice for non-chemical control of cattle tick. Previous studies have suggested that larval stages are most susceptible to host resistance mechanisms. To gain insights into the molecular basis of host resistance that occurs during R. microplus attachment, we assessed the abundance of proteins (by isobaric tag for relative and absolute quantitation (iTRAQ) and Western blot analyses) and mRNAs (by quantitative reverse transcription PCR (qRT-PCR)) in skin adjacent to tick bite sites from high tick-resistant (HR) and low tick-resistant (LR) Belmont Red cattle following challenge with cattle tick. We showed substantially higher expression of the basal epidermal keratins KRT5 and KRT14, the lipid processing protein, lipocalin 9 (LCN9), the epidermal barrier catalysing enzyme transglutaminase 1 (TGM1), and the transcriptional regulator B lymphocyte-induced maturation protein 1 (Blimp1) in HR skin. Our data reveals the essential role of the epidermal permeability barrier in conferring greater resistance of cattle to tick infestation, and suggest that the physical structure of the epidermal layers of the skin may represent the first line of defence against ectoparasite invasion.

Expression of intracellular calcium signalling genes in cattle skin during tick infestation.

It is widely acknowledged that changes in intracellular calcium ion (Ca2+) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real‐time PCR was used to analyse gene expression of 14 Ca2+ signalling proteins in skin obtained from high tick‐resistant (HR) and low tick‐resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up‐regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca2+ signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca2+ and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.

The complexity of the secreted NPA and FAR lipid-binding protein families of Haemonchus contortus revealed by an iterative proteomics-bioinformatics approach.

Two different classes of small nematode specific lipid-binding proteins, the nematode polyprotein allergens/antigens (NPAs) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in Haemonchus contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to reveal the expression and complexity of these proteins in the excretory/secretory products (ESP) of adult H. contortus at the protein and gene levels. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to Ostertagia ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and Caenorhabditis elegans homologues retrieved diverse sequences encoding H. contortus NPA and FAR proteins. H. contortus sequences were then integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the diversity in structures of the encoding genes was revealed by assembling the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least one Hc-NPA gene and six Hc-FAR genes in H. contortus, and life stage gene expression of Hc-FAR-1 to -6 revealed unique transcription patterns for each of these genes.

Identification of Genes Involved with Tick Infestation in Bos taurus and Bos indicus

Tick resistant cattle could provide a potentially sustainable and environmentally sound method of controlling cattle ticks. Advances in genomics and the availability of the bovine genome sequence open up opportunities to identify useful and selectable genes controlling cattle tick resistance. Using quantitative real-time PCR and theAffymetrix bovine array platform, differences in gene expression of skin biopsies from tick resistant Bos indicus (Brahman) and tick susceptible Bos taurus (Holstein-Friesian) cattle following tick challenge were examined. We identified 138 significant differentially-expressed genes, including several immunologicallhost defence genes, extracellularmatrix proteins, and transcription factors as well as genes involved in lipid metabolism. Three key pathways, represented by genes differentially expressed in resistant Brahmans, were identified; the development of the cell-mediated immune response, structural integrity of the dermis and intracellular Ca2+ levels. Ca2+, which is implicated in host responses to microbial stimuli, may be required for the enhancement or fine-tuning of transcriptional activation of Ca2+ -dependant host defence signalling pathways.

Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae.

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

Histone deacetylase enzymes as drug targets for the control of the sheep blowfly, Lucilia cuprina

The Australian sheep blowfly, Lucilia cuprina, is an ecto-parasite that causes significant economic losses in the sheep industry. Emerging resistance to insecticides used to protect sheep from this parasite is driving the search for new drugs that act via different mechanisms. Inhibitors of histone deacetylases (HDACs), enzymes essential for regulating eukaryotic gene transcription, are prospective new insecticides based on their capacity to kill human parasites. The blowfly genome was found here to contain five HDAC genes corresponding to human HDACs 1, 3, 4, 6 and 11. The catalytic domains of blowfly HDACs 1 and 3 have high sequence identity with corresponding human and other Dipteran insect HDACs (Musca domestica and Drosophila melanogaster). On the other hand, HDACs 4, 6 and 11 from the blowfly and the other Dipteran species showed up to 53% difference in catalytic domain amino acids from corresponding human sequences, suggesting the possibility of developing HDAC inhibitors specific for insects as desired for a commercial insecticide. Differences in transcription patterns for different blowfly HDACs through the life cycle, and between the sexes of adult flies, suggest different functions in regulating gene transcription within this organism and possibly different vulnerabilities. Data that supports HDACs as possible new insecticide targets is the finding that trichostatin A and suberoylanilide hydroxamic acid retarded growth of early instar blowfly larvae in vitro, and reduced the pupation rate. Trichostatin A was 8-fold less potent than the commercial insecticide cyromazine in inhibiting larval growth. Our results support further development of inhibitors of blowfly HDACs with selectivity over human and other mammalian HDACs as a new class of prospective insecticides for sheep blowfly.

Cloning, recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly, Lucilia cuprina.

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non‐specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC50 values at µg levels. Trimethoprim and pyrimethamine were 5–30‐fold less active. Relative IC50 values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active‐site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.