Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

The closely related ermB-ermAM genes from Clostridium perfringens, Enterococcus faecalis (pAM beta 1), and Streptococcus agalactiae (pIP501) are flanked by variants of a directly repeated sequence.

The Clostridium perfringens macrolide-lincosamide-streptogramin B resistance gene, ermBP, was sequenced and shown to be identical to the ermB-ermAM gene from the promiscuous Enterococcus faecalis plasmid pAM beta 1 and to have at least 98% nucleotide sequence identity to other ermB-ermAM genes. Flanking the ermBP structural gene were almost identical directly repeated 1,341-bp sequences (DR1 and DR2). These repeats potentially encoded a 298 (or 284)-amino-acid protein that had sequence similarity to chromosomal and plasmid partitioning proteins. The pAM beta 1 and Streptococcus agalactiae (pIP501) erm determinants appeared to have DR2 but had similar internal 973- or 956-bp deletions in DR1, respectively. Some of the other ermB-ermAM class determinants had small portions of DR1, but none had complete copies. It is postulated that the C. perfringens ermBP determinant was derived from an enterococcal or streptococcal determinant that had complete copies of both DR1 and DR2.

Cloning and sequence analysis of ermQ, the predominant macrolide-lincosamide-streptogramin B resistance gene in Clostridium perfringens.

The erythromycin resistance determinant from Clostridium perfringens JIR100 has been cloned, sequenced, and shown to be expressed in Escherichia coli. An open reading frame with sequence similarity to erm genes from other bacteria was identified and designated the ermQ gene. On the basis of comparative sequence analysis, it was concluded that the ermQ gene represented a new Erm hybridization class, designated ErmQ. Genes belonging to the ErmQ class were found to be widespread in C. perfringens, since 30 of 38 macrolide-lincosamide-streptogramin B-resistant C. perfringens strains, from diverse sources, hybridized to an ermQ-specific gene probe. The ermQ gene therefore represents the most common erythromycin resistance determinant in C. perfringens.

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.

Cloning and hybridization analysis of ermP, a macrolide-lincosamide-streptogramin B resistance determinant from Clostridium perfringens.

The erythromycin resistance determinant from Clostridium perfringens CP592 was cloned and shown to be expressed in Escherichia coli. The resultant plasmid, pJIR122 (7.9 kilobase pairs [kb]), was unstable since in both recA+ and recA E. coli hosts spontaneous deletion of 2.7 kb, including the erythromycin resistance determinant, was observed. Subcloning, as well as deletion analysis with BAL 31, localized the erythromycin resistance gene (ermP) to within a 1.0-kb region of pJIR122. A 0.5-kb fragment internal to ermP was 32P labeled and used as an ermP-specific probe in DNA hybridization experiments which used target DNA prepared from representatives of each of the known erm classes and also from erythromycin-resistant isolates of a variety of clostridial species. Hybridizing sequences were detected in DNA from several Clostridium difficile isolates and a Clostridium paraputrificum strain; however, ermP was not widespread in erythromycin-resistant C. perfringens isolates. The ermP determinant hybridized to, and shared significant restriction identity with, the ermB class gene from the streptococcal plasmid pAM beta 1. No hybridization was detected with the other six hybridization classes of erm determinants.

Hybridization analysis of the class P tetracycline resistance determinant from the Clostridium perfringens R-plasmid, pCW3.

The tetracycline resistance determinant from pCW3, a conjugative plasmid from Clostridium perfringens, has been identified and the structural gene localized to within a 1.4-kb region. Hybridization analysis, which utilized an internal 0.8-kb specific gene probe, showed that eight nonconjugative tetracycline resistant C. perfringens strains all carried homologous resistance determinants. No homology was detected in DNA prepared from tetracycline resistant isolates of Clostridium difficile or Clostridium sporogenes. However, the one strain of Clostridium paraputrificum that was tested did contain an homologous determinant. No homology was found to any of the recognized classes of tetracycline resistance determinants. The C. perfringens tetracycline resistance determinant represents a new hybridization group, Class P.