Biomedical subjects

S Morzaria

Publications and source records attributed to S Morzaria.

24 records · Page 2Linked to original sources

PubMed1995

Theileria: improved species discrimination using oligonucleotides derived from large subunit ribosomal RNA sequences.

Partial nucleotide sequences of large subunit ribosomal RNA (LSU rRNA) coding genes of Theileria parva and T. taurotragi were determined. Alignment of the T. parva and T. taurotragi sequences revealed species-specific regions within the LSU rRNA genes. A major species-variable region, located between the first and second conserved secondary structure domains of LSU rRNA, was identified. The sequence of the major species-variable region was determined for T. annulata, T. buffeli, and an unclassified Theileria species isolated from buffalo. Species-specific oligonucleotides complementary to LSU rRNA sequences were designed for five species of Theileria. The oligonucleotide probes discriminated between Theileria species when hybridized to amplified parasite LSU rRNA genes or directly to parasite rRNA. Three species-specific oligonucleotides derived from nonoverlapping sequences were developed for each of T. parva and T. taurotragi. A simplified procedure was developed in which T. parva- and T. taurotragi-specific oligonucleotides were used to discriminate the two species under nonstringent hybridization and washing conditions. Use of the nonstringent conditions also increased the sensitivity of target detection. Comparison of Theileria species-specific oligonucleotides derived from large subunit and small subunit rRNA genes demonstrated that, for closely related species, probes derived from LSU rRNA sequences had improved specificity.

Animals↗

PubMed1995

DNA measurements and ploidy determination of developmental stages in the life cycles of Theileria annulata and T. parva.

The relative DNA levels of different developmental stages of Theileria annulata and T. parva in the cow and the tick were measured by the cytophotometric DNA technique using the fluorochrome Hoechst 33258 as a staining dye. The results revealed that sporozoites, merozoites, gamonts, and gametes were haploid, whereas multinucleated intralymphocytic schizonts were polyploid. No difference was observed between T. parva and T. annulata in these stages. For both Theileria species, the DNA measurements revealed that fusion of gametes occurred in the gut of the final host, thus providing evidence of sexual reproduction. However, differences were observed between the two parasites in the tick. Whereas T. parva zygotes underwent a two-step meiotic division, a comparable reduction division could not be unequivocally detected in T. annulata. Differences could also be detected in the further development of kinetes, indicating that Theileria species are not characterized by only one life cycle, which is specific for this genus.

Animals↗

PubMed1995

Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle.

Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoite surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural protein of influenza virus, and a linker DNA sequence. NS1-p67 lacked reactivity with TpM 12, a monoclonal antibody to native p67, and had an estimated molecular mass of 110 kDa, as opposed to the calculated mass of 85,000 Da. We have used the baculovirus expression system in an attempt to express this parasite protein in a native form and thereby increase the protective capacity of the antigen. However, Spodoptera frugiperda SF21AE cells infected with recombinant virus expressed p67 as a 100-kDa molecule. The host cells exhibited a limited capacity to glycosylate this molecule to a 110-kDa form, and p67 was not exported to the surface membrane. TpM 12 did not bind to these recombinant forms but, at time points late during viral infection, reacted with a molecule of about 70 kDa. Since the bulk of insect cell-derived p67 was not expressed in an appropriate form, we tested the immunogenicity of these partially processed recombinant p67 forms in cattle. Two groups of three cattle were inoculated with antigen formulated either with saponin or Freund's adjuvant. As seen previously with NS1-p67, all animals developed high levels of anti-p67 antibodies that neutralized sporozoite infectivity in vitro, but antigen-specific T-cell proliferative responses were not detected in peripheral blood. Given the caveat of the small number of cattle analyzed, insect cell-derived p67 does not appear to be superior to NS1-p67 as an immunogen, and the latter remains the molecule of choice for the development of vaccines against East Coast fever.

Animals↗

PubMed1993

Theileria parva: detection of genomic polymorphisms by polymerase chain reaction amplification of DNA using arbitrary primers.

Genomic DNA fingerprints were generated from Theileria parva piroplasm DNA by polymerase chain reaction amplification using arbitrary decamers as primers. Four of 12 primers tested generated amplified fragments which were polymorphic between different T. parva stocks or clones. Hybridization of radiolabeled total amplification products to Southern blots of T. parva piroplasm and uninfected bovine DNA confirmed that none of the amplified fragments were derived from any contaminating bovine DNA which may have been present in small quantities. Polymorphic DNA fragments were reproducibly amplified from independent DNA preparations made from cloned parasites. Individual amplified DNA fragments were gel purified and shown to hybridize to size-polymorphic EcoRI fragments in T. parva genomic DNA. Hybridization of individual amplified fragments to total fingerprints, generated using the same arbitrary primer, demonstrated that a single primer detected at least three independent genomic polymorphisms in T. parva DNA.

Animals↗

PubMed1992

A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle.

At present immunization against Theileria parva is by infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has major limitations in that live organisms are used and the immunity engendered is parasite stock specific. In an attempt to develop an alternative immunization procedure, the gene encoding p67, a major surface antigen of sporozoites, has been expressed by using the plasmid expression vector pMG1. The gene, which has been characterized previously, encodes 709 amino acid residues, contains a single intron of 29 base pairs, and is only transcribed during sporogony. The recombinant p67 sequences were fused to the first 85 amino acid residues derived from a nonstructural gene (NS1) of influenza virus A. Immunization with a partially purified recombinant antigen emulsified in 3% saponin induced sporozoite neutralizing antibodies in cattle and provided protection in six of nine animals on homologous challenge with T. parva sporozoites. This recombinant antigen is therefore a candidate for development of a vaccine against T. parva.

Animals↗

PubMed1992

New methods for the diagnosis of Babesia bigemina infection.

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currently used methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These tests have been used in the fild but because they lack sensitivity and specificity, newer and improved methods of diagnosis are being developed. The quantitative buffy coat (QBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with greater sensitivity and specificity than previously described serological tests. Similarly, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of differentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.

Acridine Orange↗