Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis.

A monoclonal antibody that had been raised against a protease-containing fraction of Babesia bovis, and shown to bind to a protein located in the rhoptries, was used to screen a B. bovis cDNA expression library. The sequence of the protein encoded by a positive clone was almost identical to the equivalent region of a previously described B. bovis 60-kDa rhoptry protein (Bv60). A tandem repeat of the gene encoding Bv60 was identified in all Australian isolates of B. bovis examined. Genes encoding homologous of Bv60 were cloned from Babesia ovis and Babesia canis. In B. ovis, 5 closely linked genes were identified. Four of these genes appeared to encode very similar proteins (Bo60.1-4). The protein (Bo60.5) encoded by the fifth B. ovis gene had 72% amino acid identity to Bo60.1-4 in the amino-terminal 306 amino acids, but no significant similarities in the carboxy-terminal region. In B. canis one gene (Bc60.2) was sequenced and a second closely linked gene was identified. A further member of the family, p58, has also been described previously from Babesia bigemina. Tandemly repeated genes subject to extensive gene conversion appear to be a feature of this family of babesial rhoptry protein homologous. No proteins significantly related to any members of the gene family were identified in a search of translated DNA and protein sequence databases. Thus the function of this family of proteins remains a matter for speculation.

Bovine babesiosis: failure to induce interferon gamma production in response to Babesia bovis antigens in cattle.

The immune response to Babesia bovis infection or vaccination was evaluated by measuring antibody and interferon gamma (IFN-gamma) production to protective recombinant and crude native B. bovis antigens. Cells from vaccinated or infected cattle failed to produce detectable IFN-gamma when stimulated with B. bovis antigens in vitro. In contrast, antibody was induced by protective recombinant B. bovis antigens. These findings are consistent with the argument that immunity to B. bovis infection is correlated most strongly with humoral rather than cell-mediated immune responses.

Anaplasma marginale: detection of carrier cattle by PCR-ELISA.

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of the minute levels of Anaplasma marginale present in the blood of long-term carrier cattle was developed. A simple lysis method was used to remove most of the haemoglobin from the blood to facilitate direct input of samples into the PCR reactions without prior purification of the DNA. PCR product was detected by enzyme-linked immunosorbent assay (ELISA) to simplify the processing of large numbers of samples. The sensitivity limit of the PCR-ELISA was 0.00015% parasitaemia (24 infected erythrocytes per microlitre of blood). No cross-reactivity of the assay was observed when A. marginale-negative blood infected with Babesia bovis or Theileria orientalis was tested. The PCR-ELISA was shown to be 92% efficient in the detection of long-term A. marginale carrier cattle. No false-positive results were obtained. These results compared favourably with 2 serological assays for detection of A. marginale carrier cattle (card agglutination test and ELISA) which were applied to the same experimental animals.

Anaplasma marginale: effect of challenge of cattle with varying doses of infected erythrocytes.

Three groups, each of 6 Hereford cattle, were infected by the i.v. inoculation of 10(10), 10(8) or 10(6) Anaplasma marginale-infected erythrocytes. The mean time taken to reach a 1% parasitaemia was 7.3, 13.9 and 19.9 days in the 10(10), 10(8)and 10(6) infection dose groups, respectively. The rates of increase in parasitaemias during the exponential phase of parasite multiplication were similar for the 3 groups (doubling time 0.9 days). The exponential increase of the parasitaemia in the 10(10) dose group extended to a higher level or 10(6) dose groups (to approximately 10% compared with 3%). The mean maximum parasitaemia attained in the 10(10), 10(8), and 10(6) infection dose groups was 23.7, 14.7 and 8.7%, respectively> The time taken to reach the treatment criterion (packed cell volume decrease to 15% or lower) from a 1% parasitaemia was similar for the 3 groups. These results showed that the pathological outcome (anaemia) of anaplasmosis were similar over the 10,000-fold infective dose range tested.

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.

A DNA probe to distinguish the species Anopheles quadriannulatus from other species of the Anopheles gambiae complex.

DNA probes used previously to distinguish the species Anopheles gambiae sensu stricto, An.arabiensis, An.melas and An.merus were tested against An.quadriannulatus. Using these DNA probes, An.gambiae s.s. and An.quadriannulatus were indistinguishable. A genomic library was constructed for An.quadriannulatus. Differential screening of this genomic library with An.gambiae s.s. and An.quadriannulatus genomic DNAs identified a species-specific, repeated DNA sequence. When used as a hybridization probe, this DNA sequence clearly distinguished An.gambiae s.s. from An.quadriannulatus. A simplified protocol for the use of DNA probes is described which may be used to identify material squashed directly on to nitrocellulose filter paper.

Increased resistance to Anaplasma marginale infection in cattle chronically infected with Theileria buffeli (syn. T. orientalis).

Calves chronically infected with the benign haemoprotozoan parasite Theileria buffeli (syn. T. orientalis) and T. buffeli-free calves were experimentally infected with virulent Anaplasma marginale. The daily mean maximum parasitaemia in the T. buffeli-carrier calves was lower and delayed relative to that of the Theileria-free calves. Anaemia was less marked in the Theileria infected calves, although this difference was not statistically significant. The susceptibility of Theileria-carrier and Theileria-free older cattle to virulent A. marginale infection was also investigated. The mean maximum parasitaemia observed in the Theileria-infected cattle was significantly lower than that of the Theileria-free cattle and the time to maximum parasitaemia was increased significantly in the Theileria-infected relative to the Theileria-free cattle. Of the Theileria-carrier cattle, 33% exhibited maximum parasitaemias of less than 0.1% infected erythrocytes and no clinical anaemia as a result of A. marginale infection. In contrast, the lowest maximum parasitaemia observed in the Theileria-free cattle was 7%. The percentage of cattle requiring treatment to prevent mortality due to anaemia was 50% and 91% in the Theileria-infected and Theileria-free cattle respectively. For the duration of increasing A. marginale parasitaemia, the level of Theileria in carrier cattle was significantly depressed or undetectable. Following the resolution of peak A. marginale parasitaemia, the level of Theileria parasites increased rapidly to become significantly higher than that prior to infection and then decreased gradually to a level similar to that prior to infection. The mechanism of the increased resistance to A. marginale infection conferred by T. buffeli-carrier state is unknown, but is likely to involve non-specific cell-mediated immunity, as no serological cross-reactivity exists between these two highly divergent parasite species. The susceptibility of relatively mature cattle to clinical anaplasmosis under field conditions is likely to be significantly affected by the widespread distribution and common occurrence of T. buffeli throughout the range of A. marginale in Australia, Africa and southeast Asia.

An ELISA for the diagnosis of Babesia ovis infection utilizing a synthetic, Babesia bovis-derived antigen.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Babesia ovis antibodies is described. In an initial study, a crude Babesia bovis antigen and a synthetic B. bovis-derived antigen (designated 11C5) were used to screen 46 B. ovis-positive and 55 negative sheep sera. A 95% correlation between the two antigenic preparations was found with the positive sera; no negative sera gave positive reactions. The synthetic antigen was then used in the screening of 1466 sera collected from sheep from 18 regions of Turkey. A high incidence of B. ovis-positive reactions was found from all regions (60-80%) in sheep over 1 year old, while from two smaller samples the incidence in young sheep was much less (28 and 52%). This test is superior to existing ones because the synthetic antigen can be produced in a highly reproducible state, is specific and is stable over extended periods of time.

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.

Characterization of three low-molecular-weight Glu-D3 subunit genes in common wheat.

Low-molecular-weight glutenins (LMW-GS) in common wheat (Triticum aestivum L.) are of great importance for processing quality of pan bread and noodles. The objectives of this study are to identify LMW-GS coding genes at GluD3 locus on chromosome 1D and to establish relationships between these genes and GluD3 alleles (a, b, c, d, and e) defined by protein electrophoretic mobility. Specific primer sets were designed to amplify each of the three LMW-GS chromosome 1D gene regions including upstream, coding and downstream regions of eight wheat cultivars containing GluD3 a, b, c, d and e alleles. Three LMW-GS genes, designated as GluD3-1, GluD3-2 and GluD3-3, were amplified from the eight wheat cultivars. The allelic variants of these three genes were analysed at the DNA and protein level. GluD3-1 showed two allelic variants or haplotypes, one common to cultivars containing protein alleles a, d and e (designated GluD3-11) and the other was present in cultivars with alleles b and c (designated GluD3-12). Comparing with GluD3-12, a 3-bp deletion was found in the coding region of the N-terminal repetitive domain of GluD3-11, leading to a glutamine deletion at the 116th position. GluD3-2 had three variants at the DNA level in the eight cultivars, which were designated as GluD3-21, GluD3-22 and GluD3-23. In comparison to GluD3-21, a single nucleotide polymorphism (SNP) was detected for GluD3-22 in the signal peptide region, resulting in an amino acid change from alanine to threonine at the 11th position; and 11 mutations were found at GluD3-23, with five in upstream region, four in coding region and two in downstream region, respectively. GluD3-3 had two haplotypes, designated as GluD3-31 and GluD3-32, both belonging to LMW-s glutenin subunits though their first amino acids in N-terminal region are different. Compared with the GenBank GluD3 genes, nucleotide sequences of GluD3-21 and GluD3-23 were the same as X13306 and AB062875, respectively. GluD3-22 and GluD3-11 had only one-base difference from U86027 and AB062865. GluD3-12 was not found in the GenBank database, indicating a newly identified GluD3 gene variation. GluD3-3 was a new gene different from any other known GluD3 genes. Analyses of the relationship between Glu-D3 alleles defined by protein electrophoretic mobility and different GluD3 gene variations at the DNA or protein level provided molecular basis for DNA based identification of glutenin alleles.

Genetic characterisation of dough rheological properties in a wheat doubled haploid population: additive genetic effects and epistatic interactions.

Doubled haploid lines (n = 160) from a cross between wheat cultivars 'Cranbrook' (high dough extensibility) and 'Halberd' (low dough extensibility) were grown at three Australian locations. The parents differ at all high- and low-molecular-weight glutenin loci. Dough rheological parameters were measured using small-scale testing procedures, and quantitative trait locus (QTL) mapping procedures were carried out using an existing well-saturated genetic linkage map for this cross. Genetic parameters were estimated using three software packages: QTLCartographer, Epistat and Genstat. Results indicated that environmental factors are a major determinant of dough extensibility across the three trial sites, whereas genotypic factors are the major determinants of dough strength. Composite interval mapping analysis across the 21 linkage groups revealed that as expected, the main additive QTLs for dough rheological properties are located at the high- and low-molecular-weight glutenin loci. A new QTL on chromosome 5A for M-extensibility (a mixograph-estimated measure of extensibility) was detected. Analysis of epistatic interactions revealed that there were significant conditional epistatic interactions related with the additive effects of glutenin loci on dough rheological properties. Therefore, the additive genetic effects of glutenins on dough rheological properties are conditional upon the genetic background of the wheat line. The molecular basis of the interactions with the glutenin loci may be via proteins that modify or alter the gluten protein matrix or variations in the expression level of the glutenin genes. Reverse-phase high performance liquid chromatography analysis of the molar number of individual glutenin subunits across the population showed that certain conditional epistases resulted in increased expression of the affected glutenin. The epistatic interactions detected in this study provide a possible explanation of the variable genetic effects of some glutenins on quality attributes in different genetic backgrounds and provide essential information for the accurate prediction of glutenin related variance in marker-assisted wheat breeding.

Use of a male-specific DNA probe to distinguish female mosquitoes of the Anopheles gambiae species complex.

A method has been developed to distinguish between mated female individuals of the sibling species Anopheles gambiae Giles sensu stricto and Anopheles arabiensis Patton. The DNA probe pAna1, reported by Gale & Crampton (1987a) to be useful for the specific identification of An. arabiensis males, is here shown to be sufficiently sensitive to deduce the species identity of inseminated females from the identity of the sperm contained within the spermatheca.

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex.

Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabelled DNA from different species of An.gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.

Characterisation and marker development for low molecular weight glutenin genes from Glu-A3 alleles of bread wheat (Triticum aestivum. L).

PCR was used to amplify low-molecular-weight (LMW) glutenin genes from the Glu-A3 loci of hexaploid wheat cultivars containing different Glu-A3 alleles. The complete coding sequence of one LMW glutenin gene was obtained for each of the seven alleles Glu-A3a to Glu-A3g. Chromosome assignment of PCR products using Chinese Spring nulli-tetrasomic lines confirmed the amplified products were from chromosome 1A. All sequences were classified as LMW-i-type genes based on the presence of an N-terminal isoleucine residue and eight cysteine residues located within the C-terminal domain of the predicted, mature amino acid sequence. All genes contained a single uninterrupted open reading frame, including the sequence from the Glu-A3e allele, for which no protein product has been identified. Comparison of LMW glutenin gene sequences obtained from different alleles showed a wide range of sequence identity between the genes, with between 1 and 37 single nucleotide polymorphisms and between one and five insertion/deletion events between genes from different alleles. Allele-specific PCR markers were designed based on the DNA polymorphisms identified between the LMW glutenin genes, and these markers were validated against a panel of cultivars containing different Glu-A3 alleles. This collection of markers represents a valuable resource for use in marker-assisted breeding to select for specific alleles of this important quality-determining locus in bread wheat.

Anaplasma marginale: failure of sera from immune cattle to confer protection in passive-transfer experiments.

High levels of immunity to Anaplasma marginale were induced in cattle either by vaccination using sonically disrupted A. marginale-infected erythrocytes or by repeated infection with different strains of the rickettsia. In both instances, high levels of anti-A. marginale antibody were detected in the sera of the immune cattle by immunoblotting. Serum from one animal that had been made immune by repeated infection was transferred intravenously to A. marginale-susceptible calves (three non-splenectomised and two splenectomised) undergoing initial A. marginale infection at serum doses of 2-10 ml/kg. Neither the course nor the outcome of infection as indicated by the parasite levels attained or the level of anaemia induced was altered in the calves that received the immune serum relative to the course or outcome of infection in control calves (two non-splenectomised and two splenectomised) that received serum from an two splenectomised) that received serum from an A. marginale-naive donor animal. In a similar experiment, a pool of sera from four steers that had been vaccinated with sonically disrupted A. marginale initial bodies was transfused into two intact A. marginale-susceptible calves during the early stage of A. marginale infection at a dose of 10 ml/kg. No difference was observed in the course or outcome of infection in these calves relative to the course or outcome of infection in the two non-splenectomised calves that were transfused with non-immune serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of SNPs and development of allele-specific PCR markers for gamma-gliadin alleles in Triticum aestivum.

The coding regions of 28 entries of hexaploid wheat gamma-gliadin genes, gene fragments or pseudogenes in GenBank were used for nucleotide alignment. These sequences could be divided into nine subgroups based on nucleotide variation. The chromosomal locations of five of the seven unassigned subgroups were identified through subgroup-specific polymerase chain reactions (PCR) using Chinese Spring group-1 nulli-tetrasomic lines. Multiple single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified in each subgroup. With further mining from wheat expressed sequence tag databases and targeted DNA sequencing, two SNPs were confirmed and one SNP was discovered for genes at the Gli-A1, Gli-B1 and Gli-D1 loci. A modified allele-specific PCR procedure for assaying SNPs was used to generate dominant DNA markers based on these three SNPs. For each of these three SNPs, two allele-specific primer sets were used to test Chinese Spring and 52 commercial Australian wheat varieties representing a range of low-molecular-weight (LMW) alleles. PCR results indicated that all were positive with one of the primer sets and negative with the other, with the exception of three varieties containing the 1BL/1RS chromosomal translocation that were negative for both. Furthermore, markers GliA1.1, GliB1.1 and GliD1.1 were found to be correlated with Glu-A3 a, b or c, Glu-B3 b, c, d or e and Glu-D3 a, b or e LMW glutenin alleles, respectively. Markers GliA1.2, GliB1.2 and GliD1.2 were found to be correlated with the Glu-A3 d or e, Glu-B3 a, g or h and Glu-D3 c alleles, respectively. These results indicated that the gamma-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.

Characterization of low-molecular-weight glutenin genes in Aegilops tauschii.

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3' and 5' flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.

Anaplasma marginale: effect of the treatment of cattle with an interferon gamma-neutralizing monoclonal antibody or the nitric oxide synthetase inhibitor aminoguanidine on the course of infection.

Cattle undergoing initial infection with the rickettsia Anaplasma marginale were treated with either a monoclonal antibody (MoAb) with neutralizing activity for bovine interferon gamma (IFN-gamma) or aminoguanidine (AG), a specific inhibitor of the inducible form of nitric oxide synthetase (iNOS). Plasma levels of MoAb and AG were measured over the time of administration. The course of A. marginale infection was not altered in the MoAb-treated cattle relative to untreated controls. In cattle treated with AG however, A. marginale infection was significantly ameliorated, as judged by lower parasite levels and decreased anaemia in these cattle relative to the controls. The implications of these findings in relation to the basis for immunity against this economically important haemoparasite are discussed.

Peripheral blood lymphocyte proliferative responses in cattle infected with or vaccinated against Anaplasma marginale.

An assay was developed for measurement of the peripheral blood lymphocyte proliferative response (PBLPR) in cattle infected with or immunised against Anaplasma marginale. PBLPR was not evident in all cattle that had recovered from A. marginale infection. However, A. marginale-sensitised lymphocytes were detected in the spleens of all immune cattle tested in the absence of detectable PBLPR. During the course of initial infection, cattle exhibited detectable PBLPR for a period corresponding with and up to 2 weeks after patent parasitaemia, followed by a second, usually larger peak in PBLPR corresponding to the time of sub-clinical relapse of cattle. Analysis of the PBLPR of A. marginale chronically infected cattle demonstrated highly variable PBLPR between individuals and over time. A positive PBLPR was induced in cattle by vaccination using a crude A. marginale antigen preparation. The PBLPR of vaccinated cattle subsequently infected with A. marginale was markedly different from that of naive cattle, with reduced PBLPR being associated with the onset of parasitaemia. The antigen used in the PBLPR assay was inactivated by proteolysis. Proteolysis also abolished immunity that had been induced in cattle vaccinated using the antigen preparation. A marginale-sensitised PBL did not proliferate in response to antigen from the heterologous species A. centrale. A. centrale-sensitised PBL, however, responded to A. marginale antigen. Interferon-gamma (IFN-gamma) was detected in PBLPR-assay supernatants and was associated with a strong PBLPR.

An improved ELISA for the detection of antibodies against Babesia bovis using either a native or a recombinant B. bovis antigen.

Two new enzyme-linked immunosorbent assays (ELISA) for the diagnosis of Babesia bovis in cattle are described. The ELISA using a native antigen is more sensitive and less laborious than the assays described previously, because it does not require adsorption of sera with bovine erythrocytes. The second ELISA, using a recombinant B. bovis antigen expressed in Escherichia coli, was both sensitive and specific. It is suitable to replace the native antigen, thus avoiding large batch-to-batch variations in antigen preparations and the need to sacrifice experimental cattle.