A 398 bp fragment of the capsid protein (VP60) gene of 39 clinical samples of rabbit haemorrhagic disease virus (RHDV) and 17 of European brown hare syndrome virus (EBHSV), collected between 1981 and 1995 from 17 countries, was amplified by PCR and directly sequenced. The alignment of the nucleotide sequences and the subsequently constructed phylogenetic tree clearly separated RHDV from EBHSV as phylogenetic entities. The nucleotide homology rates between the RHDV and EBHSV groups ranged between 52.6% and 60.0%. The homology rates within the groups were much higher, 89.4% to 100% for the RHDV samples, and 89.4% to 100% for the EBHSV specimens. No intermediate viruses were found. Despite the high homology, three main branches could be identified in the phylogenetic tree of the RHDV samples, corresponding to the epizootiological data, while the EBHSV dendrogram did not show such well defined branches. The present results support the classification of RHDV and EBHSV as two distinct members of the Caliciviridae family. Nevertheless, a comparison with previously determined sequences of other caliciviruses shows that RHDV and EBHSV are more closely related to each other than to any other calicivirus.

Mycoplasma sp. (strain F38) is the causative agent of contagious caprine pleuropneumonia, which is a goat disease of great global concern. Strain F38 belongs to the so-called "Mycoplasma mycoides cluster," and the members of this cluster have many biochemical and serological properties in common, which makes it difficult to differentiate between them by conventional methods. Their phylogenetic interrelationship are thus uncertain. The 16S rRNA gene of the rrnB operon from strain F38 was cloned and sequenced. The sequence was compared with the 16S rRNA sequences of related mycoplasmas, and phylogenetic trees were constructed by parsimony analysis. A three-way ambiguity among strain F38, Mycoplasma capricolum, and Mycoplasma sp. strain PG50 was observed in the trees. This observation is in agreement with a recent proposal to reclassify strain F38 and M. capricolum. A primer set was designed for in vitro amplification by PCR of a fragment of the 16S rRNA genes from the M. mycoides cluster. The amplimers of strain F38 could be distinguished easily from the corresponding amplimers from other members of the M. mycoides cluster by restriction enzyme analysis with PstI. This observation was utilized to design an identification system for strain F38. Part of the 16S rRNA gene of the rrnA operon from strain F38 was also cloned, and several sequence differences between the two rRNA operons were discovered, revealing microheterogeneity between the two 16S rRNA genes of this organism.

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Contagious caprine pleuropneumonia (CCPP), one of the most serious and dramatic diseases of goats, is caused by Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae). This organism is very difficult to isolate and to correctly identify. In a previous report we described a method for the rapid detection and identification of M. capripneumoniae. This method is based on a PCR system by which a segment of the 16S rRNA gene from all mycoplasmas of the M. mycoides cluster can be amplified. The PCR product is then analyzed by restriction enzyme cleavage for the identification of M. capripneumoniae DNA. This system has now been further evaluated with respect to specificity and diagnostic efficacy for the identification and direct detection of the organism in clinical material. Identification by restriction enzyme analysis of amplified DNA from mycoplasmas of the M. mycoides cluster was verified for 55 strains, among which were 15 strains of M. capripneumoniae. The PCR was applied to clinical samples from the nose, ear, pharynx, pleural fluid, and lung tissue containing M. capripneumoniae or other mycoplasmas. As expected, mycoplasmas belonging to the M. mycoides cluster could be detected by the PCR. Restriction enzyme analysis of the PCR products could then be applied for the identification of M. capripneumoniae. Clinical samples and cultures containing M. capripneumoniae were dried on filter paper, to try an easier sample transport method, and were tested by PCR. M. capripneumoniae DNA could be detected in the dried specimens, but the sensitivity of the PCR test was reduced.

Two reverse transcription-PCR (RT-PCR) assays have been developed for the detection and differentiation of rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), two closely related caliciviruses. In order to select highly specific primers, comparative analysis was performed with a large number of RHDV and EBHSV genomic sequences. Regarding these data, primers were selected from similar regions of the VP60 genes to amplify a fragment of 316 nucleotides from the genome of RHDV and a fragment of 265 nucleotides from the genome of EBHSV. In sensitivity studies, as few as 10 copies of cloned viral genomic fragments were detected in each PCR assay, and no cross amplification was observed between the two viruses. The diagnostic value of the assays was confirmed with clinical material by testing fresh and formalin-fixed, paraffin-embedded liver and spleen specimens from a large number of geographically and temporally distant outbreaks. Thus, the two PCR assays provide highly specific and sensitive, novel means of direct detection of the two caliciviruses. In addition, by detecting the viruses in formalin-fixed, paraffinembedded tissues (PETs), the RT-PCR assays facilitate retrospective virological and epidemiological studies. For example, the identification of EBHSV in PET specimens collected in the 1970s indicates that this virus appeared in the hare populations several years before the first reports of European brown hare syndrome during the 1980s.