Localization of a Plasmodium surface antigen epitope by Tn5 mutagenesis mapping of a recombinant cDNA clone.

A recombinant complementary DNA clone from Plasmodium knowlesi makes a beta-lactamase fusion polypeptide in Escherichia coli that reacts with a monoclonal antibody to a Plasmodium surface antigen. An epitope of the surface antigen was localized by transposon Tn5 mutagenesis mapping of the complementary DNA clone. The Tn5 mutation having the farthest 5' insert into the complementary DNA portion of the chimeric gene, giving the shortest truncated protein that maintained the ability to bind monoclonal antibody, defined the location of the epitope.

The scavenger receptor MARCO is involved in Leishmania major infection by CBA/J macrophages.

CBA/J mice are resistant to Leishmania major infection but are permissive to L. amazonensis infection. In addition, CBA/J macrophages control L. major but not L. amazonensis infection in vitro. Phagocytosis by macrophages is known to determine the outcome of Leishmania infection. Pattern recognition receptors (PRR) adorning antigen presenting cell surfaces are known to coordinate the link between innate and adaptive immunity. The macrophage receptor with collagenous structure (MARCO) is a PRR that is preferably expressed by macrophages and is capable of binding Gram-positive and Gram-negative bacteria. No research on the role of MARCO in Leishmania-macrophage interactions has been reported. Here, we demonstrate, for the first time, that MARCO expression by CBA/J macrophages is increased in response to both in vitro and in vivo L. major infections, but not to L. amazonensis infection. In addition, a specific anti-MARCO monoclonal antibody reduced L. major infection of macrophages by 30%-40% in vitro. The draining lymph nodes of anti-MARCO-treated mice displayed a reduced presence of immunolabelled parasite and parasite antigens, as well as a reduced inflammatory response. These results support the hypothesis that MARCO has a role in macrophage infection by L. major in vitro as well as in vivo.

Specificity of Tn5 insertions into a 36-bp DNA sequence repeated in tandem seven times.

The target junction sequences of six independent Tn5 insertions into a 36-bp tandemly repeated DNA segment have been determined. In all instances Tn5 preferentially inserts near one end of the tandem repeat, but in four out of six cases the insertion is between different nucleotides. The target sequence shares some similarity (8 out of 11 bp) with the ends of Tn5. All six insertions are accompanied by duplication of 9 bp of target DNA. The data imply that, even though Tn5 appears to insert randomly on a macro scale, at the nucleotide sequence level insertion into target DNA, which has limited similarity to the Tn5 end reactive sequences, may be a preferred event.

A novel ColE1::Tn3 plasmid vector that allows direct selection of hybrid clones in E. coli.

A plasmid, named pKY2289, consists of a whole ColE1 DNA molecule and a complete ampicillin transposon (Tn3). When induced, E. coli K-12 cells which carry pKY2289 promote synthesis of colicin E1, but they are not immune to colicin E1. Inserting a DNA fragment into the EcoRI or the XmaI site of this plasmid abolishes its ability to produce active colicin E1. Thus, cells carrying one of these in vitro recombinant pKY2289 plasmids are able to form normal colonies in the presence of 0.1 microgram/ml of mitomycin C and 50 microgram/ml of ampicillin, while cells carrying the parental pKY2289 form very tiny colonies under the same conditions. This allows a positive selection for an in vitro recombinant pKY2289 molecule carrying a foreign DNA insertion. The properties of cells carrying the original pKY2289 are described and its potential usefulness as a cloning vehicle is demonstrated by cloning all the EcoRI and XmaI fragments of lambda DNA.

Expression of the Plasmodium knowlesi circumsporozoite antigen in Escherichia coli directed by Plasmodium bacterial-like promoter sequences.

The Plasmodium knowlesi circumsporozoite (CS) gene is expressed in Escherichia coli directly from a parasite genomic DNA fragment, using promoter and ribosome-binding site (RBS) sequences present in this fragment. Transcription of the CS gene in E. coli is directed by tandem Plasmodium bacterial-like promoter elements located within the 0.5-kb EcoRI-HindIII fragment roughly 2.5 kb 5' from the CS gene within the 11-kb EcoRI parasite genomic DNA fragment. No readthrough from vector promoters or fortuitous promotion from plasmodial A + T-rich sequences was observed. The endogenous Plasmodium promoter of the CS gene does not seem to be recognized by E. coli RNA polymerases. Two tandem E. coli-recognized promoters are relatively strong judging by their ability to drive the bacterial chloramphenicol acetyl-transferase (CAT) gene. Translation of the message must be achieved by utilising an AAGAA sequence 4 bp 5' from the ATG initiation codon as RBS.

Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium falciparum antigens.

A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.

Organization and expression of the Plasmodium knowlesi circumsporozoite antigen gene.

To investigate the mechanisms regulating the stage specific expression of the Plasmodium knowlesi circumsporozoite (CS) antigen gene, the sporozoite genomic DNA copy of the CS gene has been isolated and compared with the blood stage genomic DNA and the sporozoite cDNA copies. The genomic DNA sequences of the two developmental stages are identical across 4 kilobase pairs of the chromosome containing the entire CS gene transcriptional unit. From restriction enzyme mapping no DNA rearrangements over 15 kilobase pairs of the chromosome containing the CS gene appear to be involved in its stage-specific expression and its regulation appears to be at the level of transcription or RNA stability. S1 nuclease and primer extension transcript mapping studies suggest that the CS mRNA has multiple start sites, that the leader sequence is devoid of introns, and is approximately 270 bases long. Consensus eukaryotic TATA and CAAT box sequences and potential regulatory elements, including sequences highly homologous to the reiterated and core enhancer sequences of SV40 precede the gene.

A putative RNA virus in Babesia bovis.

Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.

The polymorphic 11.1 locus of Plasmodium falciparum.

Clone pPF11.1 encodes a Plasmodium falciparum antigen expressed during the intraerythrocytic cycle and containing tandem repeats of a 9 amino acid unit. We report here an analysis of the genomic region specific for 11.1, which extends over 30 kb. It contains two blocks of repeats, spanning 13 kb and 9 kb. The restriction map suggests that the locus may result from a gene duplication. The 11.1 region is present in all P. falciparum strains examined so far. Southern analysis of 8 distinct isolates indicates that the locus is highly polymorphic. Thus the pPF11.1 repeats constitute a sensitive and discriminating probe to type P. falciparum strains.

Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme.

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.

ColE1 vectors for direct selection of cells carrying a hybrid plasmid.

pKY2289, a ColE1::Tn3 derivative, useful for direct selection of cells carrying a hybrid plasmid, was deleted of its mobility functions and parts of the transposon including the left inverted repeat. This deletion mutant, named pKY2700 expresses low levels of colicin E1 synthesis even in recA cells. A nitrosoguanidine mutant of pKY2289 which shows a high level of constitutive colicin E1 synthesis was also deleted of the same sequences as pKY2700. The second plasmid, named pKY2800, has the same molecular weight (3.8 megadaltons) and almost the same structure as pKY2700, but produces colicin E1 at much higher levels and has a copy number 10 times higher. pKY2800 requires no colicin E1 induction for the direct selection of hybrid clones, while pKY2700 requires mitonmycin C at a concentration of 10 ng per ml. These two colE1 derivatives are useful as safe and convenient vectors for cloning DNA fragments at the cleavage sites of EcoRI, XmaI, and SstII of plasmid.

Hepatitis C virus genotypes in Southern Brazil.

The prevalence of hepatitis C virus (HCV) genotypes in Southern Brazil was studied in the plasma of 100 HCV-RNA-positive patients attended in Porto Alegre, South of Brazil. Reverse transcription-polymerase chain reaction (RT-PCR) products from the 5' noncoding region were double digested with RsaI-HaeIII and BstNI-HinfI and analyzed by restriction fragment length polymorphism (RFLP). Three genotypes (1, 2 and 3) were demonstrable, the most prevalent being HCV type 1 (55 of 100 patients, 55%), followed by HCV type 3 (37 of 100 patients, 37%) and HCV type 2 (8 of 100 patients, 8%). There was an unusual high prevalence of genotype 3, in contrast to the majority of published data from the Southeast region.

Small extrachromosomal nucleic acid segments in protozoan parasites.

Viruses have been described in the following protozoa: Babesia spp., Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis and Eimeria spp. In order to study the Babesia bovis virus, merozoites have been prepared from the blood of infected cattle. Agarose gel electrophoresis of nucleic extracts from the bovine protozoa B. bovis and Babesia bigemina were separated into genomic DNA and at least two additional nucleic acids. One molecule with a relative mobility of 5.5 kilobase pairs (kbp) was identified as a double-stranded RNA virus-like particle. Another 6.2 kbp DNA molecule had sequences related to mitochondrial genome.

Serum of Boophilus microplus infested cattle reacts with different tick tissues.

Five cattle were each experimentally infested with 30,000 Boophilus microplus larvae and their humoral immune response to salivary gland, gut, embryo and larval extracts during infestation were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Serum antibodies to embryo extract were detected by ELISA 21 days after infestation and antibodies to the other extracts 28 days following infestation. Using Western blot analysis, new bands were recognized and others disappeared during infestation. One low molecular weight band (10,000) was detected in embryo extract 14 days after infestation and in other extracts 28 days after infestation; another band (12,000 mol. wt.) in embryo, larval and salivary gland extracts was detected at the same time as the 10,000 mol. wt. band. These results suggest that common antigens are present in different tissues of B. microplus.

Detection of genomic variability in different populations of the cattle tick Boophilus microplus in southern Brazil.

DNA from seven isolates of the cattle tick Boophilus microplus was analyzed by restriction fragment length polymorphism (RFLP). Three different cDNA clones, named P-9, P-25 and CP-12, isolated from a B. microplus cDNA library, were used as DNA probes. DNA sequences of P-9 have high similarity to ribosomal genes, whereas P-25 does not show significant homology with known sequences within databases. CP-12 is a cDNA clone encoding a cysteine endopeptidase gene. A limited degree of polymorphism was detected with P-9 and P-25, while CP-12 showed a different pattern of bands for each tick isolate. These findings suggest the existence of a complex genotypic diversity of the tick B. microplus population in endemic regions.

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.

Cryptosporidium parvum in diarrheic calves detected by microscopy and identified by immunochromatographic and molecular methods.

Cryptosporidium is an important protozoan parasite that causes diarrhea in neonates and young bovines. The objective of the present study was to determine the frequency of Cryptosporidium infection in animals of dairy farms of the Metropolitan Region (Santiago), Chile. Fecal samples of 205 newborn calves with diarrhea were studied and used for comparing the efficiency of two microscopic staining methods for diagnosis of the parasite, the auramine (AU) and a modified Ziehl-Neelsen (ZN) procedure. Out of the 205 fecal samples, we detected oocysts in 115 (56.1%) with AU and 102 (49.8%) with ZN. Comparison of results obtained with the two microscopic techniques showed significant difference (p<0.05), AU being more sensitive. On the other hand, concordance between the two methods was almost perfect (kappa value of 0.83). The results with these two operator dependent methods were confirmed using an operator independent immunochromatographic (IC) method. The IC method also enabled us to determine the identity of the parasite species as that of Cryptosporidium parvum. Identification of the parasite species was further corroborated by performing a Cryptosporidium species-specific polymerase chain reaction (PCR) test on few samples taken at random. Overall, the results showed a high number of infected animals suggesting the parasite C. parvum as a major parasitic disease agent of neonatal calves with diarrhea in dairy farms of the Metropolitan Region (Santiago) of Chile.

Different sensitivities of avian- and mammalian-haemoglobin synthesis to elevated temperatures.

Avian- and mammalian-haemoglobin synthesis show different sensitivities to elevated temperatures. Temperature-dependent, reversible polyribosome disaggregation in avian cells occurs only at 45 degrees C, which is 3 degrees higher than the temperature for mammalian cells, and seems to be due to a block in the initiation of new polypeptide chains. The implications of these findings are discussed.