[Show abstract][Hide abstract] ABSTRACT: Limited prior evidence suggests that 5'-nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti-tick vaccine. To assess this, recombinant 5'-nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti-nucleotidase antibodies showed significant protection against tick infestation, although protection was less than that found with the previously characterized antigen, Bm86. Cattle were vaccinated, in separate groups, with 5'-nucleotidase, Bm86 and both antigens combined. Cattle, as the natural host, were challenged with larval ticks. Although Bm86 showed typical efficacy, no significant protection was seen in cattle vaccinated with 5'-nucleotidase. Cattle receiving a dual antigen formulation were no better protected than those receiving Bm86 alone. One possible reason for the difference between host species, namely antibody titre, was examined and shown to be an unlikely explanation. This demonstrates a limitation of using a model host like sheep in vaccine studies.
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal for parasitology 11/2009; 40(4):499-507. DOI:10.1016/j.ijpara.2009.10.013 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The continued development of effective anti-tick vaccines remains the most promising prospect for the control of the cattle tick, Rhipicephalus (Boophilus) microplus. A vaccine based on midgut proteins could interfere with successful tick feeding and additionally interfere with midgut developmental stages of Babesia parasites, providing opportunities for the control of both the tick and the pathogens it transmits. Midgut proteins from partially fed adult female cattle ticks were analysed using a combination of 2-DE and gel-free LC-MS/MS. Analysis of the urea-soluble protein fraction resulted in the confident identification of 105 gut proteins, while the PBS-soluble fraction yielded an additional 37 R. microplus proteins. The results show an abundance of proteins involved in mitochondrial ATP synthesis, electron transport chain, protein synthesis, chaperone, antioxidant and protein folding and transport activities in midgut tissues of adult female ticks. Among the novel products identified were clathrin-adaptor protein, which is involved in the assembly of clathrin-coated vesicles, and membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4. The observations allow the formulation of hypotheses regarding midgut physiology and will serve as a basis for future vaccine development and tick-host interaction research.
[Show abstract][Hide abstract] ABSTRACT: It is widely acknowledged that changes in intracellular calcium ion (Ca(2+)) 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 Ca(2+) 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 Ca(2+) signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca(2+) and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.
[Show abstract][Hide abstract] ABSTRACT: A quick and reproducible microgel plate assay was adapted to screen bacteria from cattle gastrointestinal tracts for production of compounds inhibitory to the growth of three enterohemorrhagic Escherichia coli (EHEC) serotypes: O157:H7, O111:H-, and O26:H11. The inhibitory activity of 309 bacteria, isolated on several agar media, was assessed by a microgel assay performed in 96-well microtiter plates. Fifty-three isolates secreted inhibitory compounds with a molecular weight of less than 1,000. In 12 isolates, the inhibitory activity was attributable to compounds other than lactic or acetic acid. These compounds were highly heat tolerant, with varying sensitivity to digestion by proteolytic enzymes. The inhibitory isolates were identified as lactic acid-producing bacteria on the basis of a combination of analyses, including 16S-rDNA restriction fragment length polymorphisms, 16S-rDNA gene sequences, and fermentation end products. The lactic acid bacteria of ruminants may contain antibacterial compounds not yet described. Naturally occurring populations of lactic acid bacteria may have potential as probiotics, to reduce the carriage of EHEC in the gastrointestinal tract of ruminants.
Journal of food protection 01/2007; 69(12):2843-50. · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sixteen isolates of Bacillus thuringiensis, derived from various soil samples collected in Australia, are highly toxic to larvae of the sheep blowfly (Lucilia cuprina). The toxin gene from one of the strains (CAA890) was cloned by genome walking, and sequencing of the cloned fragments revealed a new cry gene, encoding a protein of 1134 amino acid residues, with a theoretical molecular mass of 139,209Da. Based on the amino acid sequence comparison with known Cry delta-endotoxins, the gene was designated cry47Aa. Homology modelling based on known crystal structures of the Cry toxins reveals the differences to be located in the loops of domain II in the putative toxin-receptor binding surfaces between Cry47Aa and the dipteran active Cry2Aa. We also showed that the cry47Aa gene is present in the other isolates that are highly toxic to the sheep blowfly.
[Show abstract][Hide abstract] ABSTRACT: Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.
[Show abstract][Hide abstract] ABSTRACT: A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pH<or=3) showed greatly reduced toxicity in subsequent bioassays with nematode larvae, highlighting the need to protect the toxin from the acidic conditions of the sheep abomasum if it were to be administered per os as an anthelmintic. This study indicates that both the parasitic adult stages and the free-living larval stages of economically significant nematode parasites are susceptible to the effects of Bt, thus identifying this group of toxins as potential biocontrol agents.
International Journal for Parasitology 09/2005; 35(9):1013-22. DOI:10.1016/j.ijpara.2005.03.010 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The leguminous tropical multipurpose tree Acacia angustissima is a potential source of protein for ruminants fed roughage diets. Proximate analysis and in vitro techniques were used to evaluate the nutritive value of 15 accessions of A. angustissima in comparison with the tanniniferous legumes (Calliandra calothyrsus and Leucaena leucocephala) and lucerne (Medicago sativa). Freeze- and oven-dried (50°C) leaf samples were used and polyethylene glycol (PEG) was added to some in vitro fermentations (5mg PEG/50mg plant substrate) to assess the effects of tannins on digestion of dry matter (DM) and nitrogen (N). The acacia accessions were also screened for secondary compounds that could effect the nutritive value of the plant.Average NDF and ADF content did not differ markedly between A. angustissima accessions (417 and 189g/kg), L. leucocephala (447 and 178g/kg), C. calothyrsus (416 and 205g/kg) and lucerne (399 and 168g/kg). Average N content of A. angustissima (38g/kg) and C. calothyrsus (36g/kg) were similar but tended to be lower than L. leucocephala (43g/kg) and lucerne (50g/kg). Oven drying at 50°C compared with freeze drying did not significantly effect fermentation characteristics of NH3, branched chain (BCVFA) and short chain volatile fatty acid (SCVFA) production in the plants. There was a two-fold difference in SCVFA production amongst acacia accessions without PEG while variation in NH3 and BCVFA production was small due to negligible production or a net consumption of these metabolites during fermentation. Inclusion of PEG in fermentations of A. angustissima resulted in a marked and significant increase in DM digestibility and production of SCVFA (2–4.4-fold increase), NH3 (>4-fold increase) and BCVFA (>10-fold increase).All acacia accessions had a high free condensed tannin content which ranged from 98 to 180g/kg and only minor amounts of tannin were in a bound form (oxalylalbizziin>α-acetyldiaminobutyric acid>albizziin>oxalyldiaminobutyric acid and oxalyldiaminopropionic acid. The main non-protein amino acid was γ-acetyldiaminobutyric acid which ranged in concentration from 181 to 293g/kg DM.In conclusion, in vitro digestibility was low for all acacia accession due to the presence of tannins. These accessions all contained significant amounts of non-protein amino acids that need to be evaluated further for potential toxicity.
Fuel and Energy Abstracts 06/2005; 121(1):175-190. DOI:10.1016/j.anifeedsci.2005.02.017
[Show abstract][Hide abstract] ABSTRACT: To determine the effect of different carbohydrate-based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces.
The size of faecal E. coli populations and fermentation characteristics were ascertained in three experiments where cattle were maintained on a range of finishing diets including high grain, roughage, and roughage + molasses (50%) diets. Increased E. coli numbers, decreased pH and enhanced butyrate and lactate fermentation pathways were associated with grain diets, whereas roughage and roughage + molasses diets resulted in decreased concentrations of ehxA, eaeA and stx(1) genes, this trend remaining at lairage. In one experiment, faecal E. coli numbers were significantly lower in animals fed roughage and roughage + molasses, than animals fed grain (4.5, 5.2 and 6.3 mean log10 g(-1) digesta respectively). In a second experiment, faecal E. coli numbers were 2 log lower in the roughage and roughage + molasses diets compared with grain-fed animals prior to lairage (5.6, 5.5 and 7.9 mean log10 g(-1) digesta respectively) this difference increasing to 2.5 log at lairage.
The type of dietary carbohydrate has a significant effect on E. coli numbers and concentration of EHEC virulence genes in faeces of cattle.
The study provides a better understanding of the impact finishing diet and commercial lairage management practices may have on the shedding of E. coli and EHEC virulence factors, thus reducing the risk of carcass contamination by EHEC.
[Show abstract][Hide abstract] ABSTRACT: In this manuscript, the authors have sought to gain a better understanding of the interactions between Escherichia coli and lactic acid bacteria (LAB) isolated from Rogossa MRS agar along the digestive tract of grain- and forage-fed cattle. E. coli from cattle receiving a high-grain diet were more numerous (P<0.05) than from the high-forage diet and the highest numbers were in the faeces. Isolates on Rogossa MRS agar were always greater in the high-grain diet (P<0.05) and contained a significant number of LAB. A random set of Rogossa MRS agar colonies was selected and artificial neural networks were used to develop a relationship between colony description and species which was validated using sequence analysis (16S rDNA). The neural networks correctly predicted species in more than 80 % of cases and was composed, primarily, of Lactobacillus vitulinus, Lactobacillus ruminis, Selenomonas ruminantium, Streptococcus bovis, Acidaminococcus fermentans and Megasphaera elsdenii. In conjunction with statistical diversity indices, it was demonstrated that diversity in the high-fibre diet was always lower and was a consequence of the dominance of Str. bovis. In contrast, the diversity in the high-grain diet was greater (P<0.05) and was a consequence of the decline in Str. bovis. These data demonstrate that there is a positive relationship between coliform and LAB isolates throughout the digestive tract of cattle, and diet is the major factor regulating bacterial composition.
[Show abstract][Hide abstract] ABSTRACT: Buffalo fly (Haematobia irritans exigua), sheep blowfly (Lucilia cuprina), and the sheep body louse (Bovicola ovis) are important pests of the Australian livestock industry. Current control measures rely heavily on chemical insecticides. Because all three pests have developed resistance and insecticide residues in animal products threaten trade, alternative control strategies need to be investigated. The bacterium, Bacillus thuringiensis (Bt), produces protein (δ-endotoxin) crystals that are specifically toxic to some insects. In this study, feeding assays were used to screen Bt isolates for toxicity to adult and larval buffalo fly, larval sheep blowfly, and adults and nymphs of the sheep louse. Some Bt isolates were found to have high toxic activity against larvae of the two fly species and moderate activity against the sheep louse. This is the first report of insecticidal activity of Bt toxin against buffalo fly. Bacillus thuringiensis isolates with toxic activity due to δ-endotoxin were characterized further by polymerase chain reaction to identify toxin genes. In four isolates, no cry genes were identified with the primer sets used. These isolates were analyzed further by amino-terminal sequencing of the protein bands. Two amino acid sequences, unrelated to any known Cry proteins, have been identified by database searches.
Biological Control 02/2002; 23(2):179-189. DOI:10.1006/bcon.2001.1006 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several peritrophins of larvae of Lucilia cuprina (sheep blowfly) have demonstrated potential as vaccine antigens, and some have been characterised and cloned. These proteins are tightly associated with the peritrophic matrix, a chitinous tube or sac lining the lumen of the gut of most insects. The peritrophins require strong denaturants for their removal from peritrophic matrix. We now report the preliminary characterisation of peritrophins of the adult stage of L. cuprina and Haematobia irritans exigua (buffalo fly). Similar SDS-PAGE profiles were obtained for proteins extracted in SDS or urea from isolated adult peritrophic matrices of both species. Radioiodination of urea-extracted peritrophins improved sensitivity, indicating numerous proteins of 15-75 kDa. Direct radioiodination of L. cuprina peritrophic matrix preferentially labelled high molecular weight complexes and proteins of 80-90 kDa. Two-dimensional gel analyses of a urea extract of adult L. cuprina peritrophic matrix revealed that most proteins were moderately acidic. Antibodies produced against SDS-extracted peritrophins, or against sonicated peritrophic matrices of these two flies were crossreactive. The sera also appeared to recognise SDS-extracted components of Triton X-100 treated and washed adult peritrophic matrix of the mosquito, Aedes vigilax (Skause). This profile altered as the peritrophic matrix matured. In concordance with extracts from the adult L. cuprina and H.i. exigua peritrophic matrices, proteins in the 50-75 kDa region were immunodominant. The vaccine potential of the peritrophins of these Diptera were examined following vaccination of cattle and rabbits with adult H.i. exigua or L. cuprina peritrophins. When the adult life stages of H.i. exigua or two mosquitoes, A. vigilax and A. aegypti (Linnaeus), were fed on the sera or blood of vaccinated hosts, there were no detrimental effects to any life cycle stages of these Diptera.
International Journal for Parasitology 10/1999; 29(9):1363-77. DOI:10.1016/S0020-7519(99)00070-3 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family. This PCR product was labelled with digoxigenin-dUTP and used as a probe to screen a lambdagt-11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da. Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (DRIP) of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full-length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap-sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full-length coding sequence of AqpBF1 was cloned into the (His)6-fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)6-fusion protein was localized predominantly in the membrane fraction of E. coli. The protein was solubilized from E. coli membranes with n-octyl beta-D-glucopyranoside and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.
[Show abstract][Hide abstract] ABSTRACT: HieACE, a soluble 70 kDa protein related to the angiotensin-converting enzyme (ACE) has recently been identified, characterized and cloned from the adult buffalo fly (Haematobia irritans exigua). HieACE is enzymatically similar to the mammalian ACEs and its predicted amino acid sequence has 42% identity with the mammalian testicular ACEs. In adult H.i. exigua, HieACE expression is restricted to the compound ganglion and posterior midgut, and the maturing male reproductive system. Western blot analysis was used to investigate the expression of HieACE and its homologues in the larvae of H.i. exigua, Drosophila melanogaster, the sheep blowfly (Lucilia cuprina), the Old World screwworm fly (Chrysomya bezziana) and a secondary strike fly, Chrysomya rufifacies. Dipteran ACE homologues of 65-70 kDa were detected in all the larval instars investigated. Most of the immunoreactive proteins were concentrated in the soluble fraction. The first and second larval instars of L. cuprina and C. bezziana appeared to express two ACE homologues. These larvae were also found to secrete (or excrete) the ACE homologue in larval cultures. The presence of ACE-like enzymes in these larvae was confirmed by the measurement of carboxydipeptidase activity that was inhibited by the specific ACE inhibitor, captopril. The tissue distributions of the ACE homologues in the third instar larvae of H.i. exigua and L. cuprina were examined. As in adult H.i. exigua, HieACE was detected in the larval ganglion, but in contrast to the restricted distribution in the adult stage midgut, HieACE was found throughout the digestive system, and in the salivary glands of H.i. exigua larvae. The expression pattern in the gut of L. cuprina larvae was similar despite the differences in diet and habitat. The most striking difference from the adult stage H.i. exigua was the expression of HieACE and its L. cuprina homologues in the hindgut and Malpighian tubules of these larvae. These results suggest that the role(s) played by the dipteran ACE-like enzymes differ between the adult and larval stages.
[Show abstract][Hide abstract] ABSTRACT: The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.
European Journal of Biochemistry 05/1996; 237(2):414-23. DOI:10.1111/j.1432-1033.1996.0414k.x · 3.58 Impact Factor