Biomedical subjects

I J East

Publications and source records attributed to I J East.

At least 19 recordsLinked to original sources

PubMed1998

Amelioration of virulent Babesia bovis infection in calves by administration of the nitric oxide synthase inhibitor aminoguanidine.

Calves undergoing initial infection with a virulent strain of the haemoprotozoan parasite Babesia bovis were treated with aminoguanidine (AG), an inhibitor of the inducible form of nitric oxide synthase (iNOS). The mean maximum parasitaemia of the AG treated calves was significantly lower than that of the control cattle. In addition, the febrile response and decrease in packed cell volume (PCV) observed during acute infection were significantly ameliorated in the AG treated cattle relative to the controls. However, AG had no effect on the multiplication of B. bovis in the microaerophilous stationary-phase (MASP) in-vitro culture system. These results provide evidence of a role for nitric oxide (NO) produced in response to acute infection in the pathology of bovine babesiosis.

Anemia↗

PubMed1998

Mapping the T cell epitopes of the Babesia bovis antigen 12D3: implications for vaccine design.

The Babesia bovis antigen 12D3 was analysed to identify potential T-cell epitopes. Two predictive algorithms identified 13 possible sites but there was minimal agreement between the different predictive methods. Experimental determination of the T-cell epitopes recognized by nine cattle was achieved using a panel of overlapping peptides which identified seven different epitopes, five of which were clustered together around residues 210-320 of the molecule. No T cell epitopes were located within the tightly disulphide bonded core of 12D3. Using a series of truncated peptides, the location of two of the epitopes was mapped to residues 35-43 and 266-275. The sequences of these two epitopes was compared with a database of previously described binding motifs for MHC II alleles and each epitope was found to contain three sequence motifs recognized by HLA-DR alleles. The BoLA-DRB3 alleles occurring in these cattle were determined by a sequence specific oligonucleotide hybridization assay. Within those cattle whose T cells proliferated in response to 12D3, there was a consistent pattern of epitope recognition and presence of particular DRB3 alleles. The implications for effective vaccine design are discussed.

Alleles↗

PubMed1997

Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles.

Vaccination of cattle against the haemoprotozoan parasite, Babesia bovis, with the recombinant antigen 11C5 resulted in 9 of 15 cattle being protected against challenge infection. The cellular immune responses of protected and unprotected cattle were compared in order to identify differences in response. No differences were observed in the pattern of change in various blood leukocyte populations throughout challenge infection. FACScan analysis revealed an increase in the proportion of cells bearing the CD2 marker in both protected and unprotected cattle over the course of infection. There were no observable differences in the frequency of various cell-surface markers between the unprotected and protected cattle. During the period of patent parasitaemia, in vitro cultures of peripheral blood mononuclear cells (PBMC) from protected cattle produced significantly more TNF-alpha (P < 0.05) than cultures from unprotected cattle. TNF-alpha concentrations remained at pre-challenge levels until day 10, when levels in the unvaccinated control and vaccinated/unprotected animals dropped. By peak parasitaemia, TNF-alpha production in vitro was significantly greater (P < 0.05) in cultures of PBMCs from protected cattle. Interferon production showed an initial peak at day 5 in all cattle, followed by a decrease and a second peak at days 10-13 in protected cattle only, which coincided with resolution of the infection.

Animals↗

PubMed1996

Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina. cDNA and deduced amino acid sequences.

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Amino Acid Sequence↗

PubMed1996

Babesia bovis: biosynthesis and localisation of 12D3 antigen in bovine erythrocytes.

The 12D3 antigen of Babesia bovis was found to be synthesised rapidly in cultured parasites, and localised to both the apical complex of the merozoite and the cytoplasm of the parasitised erythrocyte. Amino-terminal sequencing suggested that the nascent protein had been processed and differences between the predicted and measured molecular weights suggested post-translational modification. The major proportion of 12D3 appeared in the soluble compartment of the parasitised erythrocytes with a molecular weight consistent with no further processing. A significant proportion of the protein required extraction by sodium carbonate, suggesting association with membranous components. The timing of release of soluble 12D3 was coincident with haemoglobin release and this probably reflects a non-specific lysis of the erythrocyte. Synthesis of recombinant BV12D3 was achieved in baculovirus-infected SF9 insect epithelial cells. The product was of the same molecular weight as the native 12D3 and polyclonal antibodies raised against the recombinant protein reacted with both the recombinant and native forms of the antigen.

Animals↗

PubMed1996

Detection of a common BoLA-DRB3 deletion by sequence-specific oligonucleotide typing.

The bovine MHC clas II allele BoLA-DRB3*2A has an amino acid deletion of unknown function at codon 65 in the second exon, which codes for the antigen-binding site. Sequence-specific oligonucleotides were designed based on published nucleotide sequences on BoLA-DRRB3 alleles, and used to detect this deletion in 51 Hereford cattle. Probes 65+ and 65- detect the presence or absence of codon 65 respectively. Oligonucleotide probes were labelled with Digoxigenin (DIG), hybridized to dot blots of BoLA-DRB3 exon 2 polymerase chain reaction (PCR) product, and detected by chemiluminescence. Of the 51 animals screened, two were homozygous and 11 were heterozygous for the deletion at codon 65. The methodology described here provides the necessary tools to screen rapidly for this deletion in a large number of animals in order to study its effect on antigen binding and immune response.

Alleles↗

PubMed1995

Variation in immune responsiveness of sheep to the antigens of intestinal nematodes and blowfly larvae.

The total and IgG1 antibody responses to the intestinal nematode parasites Haemonchus contortus and Trichostrongylus colubriformis were measured in the serum of 160 lambs, 4 months of age. These antibodies had developed as the result of natural exposure to the parasites on pasture. Three sires were examined and strong sire effects on half-sib progeny were found. Plotting of ELISA antibody results in two dimensions revealed clustering of responses within sire groups. Bimodal antibody distributions were also observed within sire groups and the whole population for T. colubriformis. A bimodal distribution of antibodies to H. contortus was found for one sire group but not for the whole population. The injection of blowfly larvae (Lucilia cuprina) extract into 42/160 lambs at a later age (12 months) was followed by increased antibodies to L. cuprina and an apparent increase in antibodies to T. colubriformis. A bimodal distribution for antibodies to L. cuprina was found in one sire group and in the whole population. These bimodal distributions of antibodies to L. cuprina did not coincide with the distribution of antibodies to T. colubriformis or H. contortus, measured on the same serum samples. It was concluded that high and low responder sire groups could be differentiated in lamb populations for all three parasites. These effects persisted during lamb maturation and appeared to be genetic effects. Finally, cross-reacting antibodies between L. cuprina and T. colubriformis appear to be stimulated by injection of L. cuprina antigens.

Animals↗

PubMed1995

Anti-idiotypic antibodies as surrogate antigens for vaccinating against the cattle tick, Boophilus microplus.

QU13, a monoclonal antibody (mAb) raised against midgut (GM) antigens from Boophilus microplus and shown to recognise antigens which protect cattle from tick challenge was used to immunise cattle and rabbits to produce anti-idiotypic antibodies (AIA). Polyclonal antisera against mAb QU13 were produced in rabbits and cattle. AIA were purified from these antisera by affinity chromatography procedures. These purified AIA were found to block mAb QU13 binding to GM in enzyme-linked immunosorbent assay (ELISA). AIA purified from bovine antiserum elicited an immune response in cattle to antigens extracted by detergent from the midgut of B. microplus (TXGM) after the fourth vaccination (P = 0.06) compared with the bovine immunoglobulin (Ig) control. The mean antibody level in the group of experimental cattle vaccinated with AIA purified from rabbit antisera was significantly higher (P < 0.03) than that of bovine Ig control cattle after the fourth vaccination and an anamnestic response (P < 0.11) occurred in the rabbit AIA vaccinated group of cattle when a single booster dose of 300 micrograms of TXGM was given after the first tick challenge. The positive control group of cattle vaccinated with TXGM were significantly protected (P < 0.05) against tick infestation after the booster dose of 300 micrograms of TXGM. The AIA vaccinated groups of cattle were not protected against challenge with 20,000 larval ticks either before or after the booster injection of 300 micrograms of TXGM.

Animals↗

PubMed1995

Identification and characterization of new BoLA-DRB3 alleles by heteroduplex analysis and direct sequencing.

A sample of 52 mixed-breed dairy cattle (Holstein Friesian and Jersey) and 51 beef cattle (Hereford) from south-east Queensland was studied. The second exon of BoLA-DRB3 was amplified by polymerase chain reaction (PCR), and polymorphisms were detected by heteroduplex analysis. A large number of different heteroduplex patterns indicated extensive sequence polymorphism. Direct sequencing of PCR products from 17 homozygotes and cloning and sequencing of PCR product from two heterozygotes resulted in the identification and characterization of four novel alleles. The previously described allele BoLA-DRB3*2A is characterized by an amino acid deletion at position 65. We have identified three animals that are homozygous for this amino acid deletion, indicating that the deletion is unlikely to result in loss of function. Two of these animals had allele BoLA-DRB3*2A, and one had a novel allele with codon 65 deleted but differing from BoLA-DRB3*2A at a number of other amino acid positions. In conclusion, heteroduplex analysis allows rapid discrimination between homozygotes and heterozygotes, and enables rapid identification of new BoLA-DRB3 alleles.

Alleles↗

PubMed1995

Isolation and characterization of a trypsin-like enzyme from the buffalo fly, Haematobia irritans exigua.

The incorporation of soybean trypsin inhibitor (SBTI) into the diet of the buffalo fly, Haematobia irritans exigua (De Meijere), results in increased mortality and reduced fecundity. A trypsin-like enzyme which binds to SBTI was isolated by affinity chromatography on a Sepharose-SBTI column followed by ion-exchange chromatography. The enzyme was inhibited by benzamidine, phenylmethylsulfonyl fluoride, ovomucoid, leupeptin and alpha-2 macroglobulin. The enzyme was not inhibited by EDTA or p-chloromecuribenzoic acid and had a broad pH optimum of pH 7-9. Vaccination of sheep produced antibodies specific for the trypsin-like enzyme which inhibited enzyme activity in vitro but did not affect the survival of flies maintained in in vitro culture.

Animals↗

PubMed1994

An association between successful vaccination against the bovine nodular worm, Oesophagostomum radiatum and induction of eosinophilia.

Immunization of 48 cattle with somatic antigen extracts of the nodular worm, Oesophagostomum radiatum, reduced faecal egg counts (FEC) by 51% and worm burden by 47%. The antibody titre of each animal was correlated to both worm burden (r = -0.40, P < 0.005) and FEC (r = -0.37, P < 0.01). The vaccinated cattle showed wide variation in their response to vaccination with worm burdens after challenge infection varying from 0 to 4060. Of these 48 vaccinated animals, eight developed sterile immunity (worm burden = 0, FEC = 0) whereas a further 10 had no protective response to immunization (worm burden > 1,000, FEC > 100). A comparison of these two sub-groups showed that the group with sterile immunity had a higher antibody titre (P < 0.05). In addition, the group with sterile immunity developed a blood eosinophilia between 21 and 29 days after challenge infection. Vaccinated, unprotected calves and control, unvaccinated calves did not develop eosinophilia.

Animals↗

PubMed1993

Lucilia cuprina: inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane.

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

Animals↗

PubMed1993

Oesophagostomum radiatum: the effect of different adjuvants on vaccination with a partially purified protective antigen.

The components of a soluble extract of adult Oesophagostomum radiatum were separated into four fractions by gel filtration chromatography on a Superose-12 column. The second fraction had a molecular weight range of approximately 60,000-65,000 as judged by SDS gel electrophoresis. Vaccination of calves with this fraction significantly (P < 0.05) reduced faecal egg output after challenge infection. In addition, the packed cell volume of vaccinated calves was higher than that of control calves (P < 0.05). The effect of three adjuvants on the vaccine activity was compared. Fraction 2 presented in dextran sulfate reduced the faecal egg count by 93.6% (P < 0.05), Fraction 2 in Freund's complete adjuvant reduced the faecal egg count by 27% (P = N.S.). Quil A was ineffective as an adjuvant.

Adjuvants, Immunologic↗

PubMed1993

Vaccination against Lucilia cuprina: the causative agent of sheep blowfly strike.

The sheep blowfly, Lucilia cuprina, is responsible for over 80% of cases of blowfly strike in Australia and the losses in production and sheep deaths due to flystrike exceed $200 million per annum. Traditional methods of control are becoming less effective because of the blowfly's resistance to insecticides and thus other methods of control are necessary. In general, sheep develop very little immunity to flystrike even after repeated infestation, however vaccination against L. cuprina has shown considerable potential for controlling flystrike. The most successful sources of antigens have been the larvae's secreted proteases and several extracts from gut or peritrophic membrane. Immunization with these antigens results in retardation of larval growth and in some cases larval mortality. On sheep immunized with peritrophic membrane extracts, the growth retardation appears to be caused by a blocking of the peritrophic membrane which results in the larvae being starved of nutrients. The prospects for vaccine development and the remaining barriers to be overcome are discussed.

Animals↗

PubMed1992

Vaccines against blowfly strike: the effect of adjuvant type on vaccine effectiveness.

Vaccination of sheep with a partially purified extract of Lucilia cuprina larvae in some cases resulted in marked reduction of growth in larvae which fed on the sheep. Twelve adjuvants were assessed, in vitro and in vivo, to determine which induced the largest inhibitory effect on larval growth. The Freund's complete adjuvant and Quil A groups produced ELISA antibody levels significantly higher (P less than 0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P less than 0.05). When the sheep were assessed by in vivo larval culture, only larvae feeding on sheep vaccinated with the antigen presented in Freund's complete adjuvant or dextran sulphate or a dextran sulphate/Freund's incomplete adjuvant mixture weighed significantly less (P less than 0.05) than larvae feeding on control sheep. The effect on larvae was monitored in vitro for 70 days after vaccination, by which time significant reduction in larval weight was no longer observed. The loss of larval growth inhibition was not associated with a corresponding reduction in overall antibody levels.

Adjuvants, Immunologic↗

PubMed1992

Potent immunosuppression by secretory/excretory products of larvae from the sheep blowfly Lucilia cuprina.

Secretory/excretory products (sec/ex) of parasitic larvae of the sheep blowfly Lucilia cuprina potently inhibited proliferation of peripheral blood leucocytes stimulated by mitogens in vitro. Suppression of proliferation was not due to irreversible damage because cells cultured for 24 h in high concentrations of sec/ex appeared viable (assessed by Trypan blue exclusion) and did not show impaired proliferation after washing. Furthermore, suppression induced by sec/ex could be overcome by increasing concentrations of mitogen. The inhibitory activity could be demonstrated in cultures where sec/ex was added at different times during the culture period. Inhibitory activities in sec/ex were heat-labile and sensitive to treatment with trypsin. In addition to effects in vitro, sec/ex was strongly immunosuppressive in vivo. Sheep given combined injections of myoglobin and sec/ex had markedly lower anti-myoglobin antibody levels in sera than sheep that received injections of myoglobin alone. There was no significant antibody response to sec/ex itself. Immunosuppressive moieties in sec/ex produced by blowfly larvae may promote parasite survival by inhibiting the immune response of host sheep.

Animals↗

PubMed1992

The survival and fecundity of buffalo flies after treatment of cattle with three anthelmintics.

Two anthelmintics with known insecticidal action (ivermectin and closantel) and one with no recorded effect on insects (levamisole) were tested to evaluate their effects on buffalo fly (Haematobia irritans exigua). Blood from animals given closantel or levamisole had no significant effect on mortality of buffalo flies in an in-vitro assay. In contrast, blood from animals given ivermectin showed a dose-dependent effect on the mortality of buffalo flies. At 24 h after one injection of the recommended dose of ivermectin, 98% of the flies applied to cattle in an in-vivo assay are killed. Blood from cattle injected with ivermectin killed 95% of flies 8 d after injection and still killed 15% of flies at 18 days after injection. Surviving flies laid almost no eggs and this effect on flies was significant up to 33 d after injection. The results indicate that ivermectin may be useful to control buffalo fly populations in the field.

Animals↗

PubMed1992

Reduced growth of Lucilia cuprina larvae fed serum from sheep treated with anthelmintics.

The effect of three commonly used anthelmintics, levamisole hydrochloride, ivermectin and closantel, on the development of the sheep blowfly, Lucilia cuprina, was determined. Sheep were treated with each anthelmintic using the manufacturers' recommended dose for helminth control. Both ivermectin and closantel significantly (p < 0.05) reduced the growth rate of larvae of L cuprina cultured in vitro on serum from these sheep. Levamisole hydrochloride had no effect. Ivermectin was effective for less than 6 days after treatment, whereas closantel significantly reduced larval growth 21 days after treatment. Dose-response experiments showed that lower concentrations of both ivermectin and closantel were not as effective in reducing larval growth.

Animals↗