Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs.

Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein's upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Cowdria ruminantium DNA is unstable in a SuperCos1 library.

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.

Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium.

BACKGROUND: Epitopes can be mapped by comparing immunoaffinity-selected peptides from fragmented-gene display libraries with the target gene. With larger libraries derived from unsequenced genomes, this is not possible. Spurious epitope mimics may be created by expressing DNA in a variety of meaningless reading frames and orientations. OBJECTIVES: To determine empirically whether panning a large fragmented-genome phage display library with antibodies to MAP1, the major antigenic protein of the rickettsial parasite Cowdria ruminantium, would result in the selection of irrelevant, cross-reactive mimotopes. STUDY DESIGN: A gene III phage library displaying peptides derived from C. ruminantium was constructed using cloned DNA from a bacteriophage lambda genomic library. After in vivo excision, plasmids were cleaved with PvuII followed by PCR. Genes with a PvuII site, including MAP1 were therefore not amplified. DNA was sonicated, partially digested with DNase and cloned into the display vector fUSE2. Affinity-purified MAP1 antibodies were used for panning. Peptides expressed by panned phages were tested for recognition in Western blot and ELISA. Oligonucleotides representing antigenic sequences were used to locate their encoding DNA sequences in the original lambda library. The phage display library was also panned with two monoclonal antibodies (Mabs) against bluetongue virus (BTV). RESULTS: Five different peptide sequences were selected from the MAP1-deficient phage display library. None was identical to MAP1, but four peptides had regions that were similar, both to each other, and to the parasite protein. They produced strong signals in ELISA and Western blot. None could be located to any C. ruminantium open reading frame. Two BTV Mabs recognised a sequence similar to their authentic epitope. CONCLUSION: Large genome-targeted phage display libraries may be sufficiently diverse to allow the selection of peptides that mimic actual antigenic determinants. This diversity may be exploited in the search for useful epitopes.

Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes.

The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools. We have developed an immunoaffinity chromatographic method to purify C. ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA. This DNA has been used to construct what we believe to be the first fully representative C. ruminantium expression library. A clone containing the complete Cowdria map1 gene has been isolated and sequenced. This gene has been expressed in E. coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms. Parts of three other Cowdria genes have also been isolated and sequenced.

Detection of Babesia equi in infected horses and carrier animals using a DNA probe.

The ability of the Babesia equi repetitive probes, pSE2 and pSB20, to detect parasites in blood from experimentally infected, naturally infected and carrier animals was tested using a spot hybridization assay. The clinical course of the experimentally infected horses was monitored using microscopy, indirect fluorescent antibody tests, packed cell volume, temperature and the probe assay. The probes sensitively monitored the parasite level during the development of the disease and correlated well with the other parameters tested. The sensitivity of the probe assay was superior to that of light microscopy, and a parasitaemia equivalent to less than 0.0025% could be detected. Detection of B. equi DNA was possible in all natural cases tested and 20 of the 119 randomly selected horses were identified as carriers of B. equi parasites. Microscopy could identify parasites in only 8 of these carrier animals. These results show that the probes can detect B. equi parasites in carrier animals and that they are suitable for use in a laboratory-based assay for B. equi.

[The incidence pattern of certain cancers in the RSA]. / Die voorkomspatroon van sekere karsinome in die RSA.

The numbers of patients treated for seven types of carcinoma during 1977 at 10 hospitals in South Africa have been reviewed. The total number of patients admitted to the 10 hospitals in 1977 was 286 373. Slightly more than 1%, namely 3 409, of these patients suffered from carcinoma of the cervix, oesophagus, breast, lung, liver, stomach or colon. Carcinoma of the cervix was commonest among Black patients and carcinoma of the colon among Whites. The relative incidence of the different types of carcinoma among Whites was almost the opposite of the sequence among Blacks.

[Ischemic heart disease--a cause of unexpected sudden death in black people]. / Isgemiese hartsiekte--'n oorsaak van onverwagte skielike dood by swartmense.

Myocardial infarction has been found to be a significantly frequent cause of unexpected sudden death in Black males in Pretoria and its vicinity. A study of 758 autopsies performed for unexplained sudden death in Blacks revealed 39 fatal infarcts in 511 males but only 1 in 247 females. The condition became evident in the third decade, rose sharply in the fourth and declined gradually thereafter.