Taxon 25
(Mannheimia cavia sp. nov.)

Several taxa of Pasteurellaceae, including taxon 25, were reported to be associated with guinea pigs by Bisgaard (1993). The ecology and significance of these taxa, however, remains to be investigated, just as a final classification and naming is needed to allow unambiguous identification.

Phenotypically identical Pasteurella-like organisms were reported by Mannheim et al., (1978) from an outbreak of epidemic conjunctivitis in a large colony of guinea pigs. Isolates from guinea pigs, previously reported as [Pasteurella] gallinarum (later reclassified as Avibacterium gallinarum), were subsequently identified as members of the SP-group (Necropsobacter rosorum), taxon 6 or taxon 25 (Boot and Bisgaard, 1995). Two isolates recovered from guinea pigs suffering from otitis media and classified as taxon 25 have subsequently been shown to share the phenotypical characteristics of a group of organisms represented by  strain T138021-75^T as reported by Mannheim et al. (1978) (Bisgaard, unpublished results).

A single lung isolate, P244, obtained from a rabbit and phenotypically related to M. granulomatis clustered with the genus Mannheimia in all phylogenetic trees based on the rrs gene, the consensus tree based on rpoB, infB and recN genes, and individual trees based on these three genes (Kuhnert et al., 2007), without demonstrating a clear species association.

A polyphasic approach was applied to classify and name the four strains mentioned above (Christensen et al., 2011). The type strains of the five species of Mannheimia and the type strain of P. multocida were included for comparison based on their published DNA sequences.

Phenotypic results obtained were in accordance with those previously reported for members of the genus Mannheimia (Angen et al., 1999). Four to nine different characters separated the strains investigated from recognized species of the genus Mannheimia, while differences in ornithine decarboxylase, growth on MacConkey agar, β-glucosidase (NPG), α-galactosidase, β-xylosidase (ONPX), and production of acid from maltose, D (+) melibiose, raffinose, dextrin and glycosides separated the guinea pig isolates from the rabbit isolate (Christensen et al., 2011).

Strains T138021-75^T and Pg20 of taxon 25 shared identical 16SrRNA gene sequences and were distantly related to other species of Mannheimia. The highest 16S rRNA gene sequence similarity was to strains of M. glucosida (97.8%), while the type strain of M. varigena showed 97.3% 16S rRNA gene sequence similarity. All other type strains of the genus Mannheimia, including the type strain of the type species (96.9%) demonstrated less than 97% similarity. In the maximum-likelihood analysis of 16S rRNA gene sequences the rabbit strain, P224, clustered with T138021-75^T demonstrating a bootstrap value of 68%. These two strains demonstrated 98.6% 16S rRNA gene sequence similarity.

All three strains of taxon 25 shared identical rpoB gene sequences. The closest related strains included the type strains of M. varigena and M. haemolytica biovar 8, both of which showed 93.9% similarity. The highest rpoB gene sequence similarity observed for P224 was 95.1%, which was observed with the type strains of M. haemolytica and M. glucosida. The rabbit isolate, P224, only demonstrated 89.9% sequence similarity with taxon 25 strains

The three guinea pig isolates of taxon 25 also shared identical recN gene sequences, and the close relationship of the rabbit strain and the three guinea pig strains in the 16S rRNA gene sequence based phylogenetic tree was confirmed in the tree based on maximum-likelihood analysis of partial recN gene sequences.

DNA reassociation values between the guinea pig strain T138021-75^T and the rabbit strain P224 were 82.6% and 80.5% (mean 81.6%), while the DNA relatedness values between strain T138021-75^T and M. glucosida DSM 19638^T were 41.8 and 38.7% (mean 40.3%), indicating that the rabbit isolate should be classified with the guinea pig isolates, since species of the family Pasteurellaceae mainly have been defined on the basis of DNA-DNA reassociation values of 80-85% as measured by the spectrophotometric method (Mutters et al., 1989: Christensen et al., 2005).

Finally, all three guinea pig strains of taxon 25 shared the same recN gene sequence, which showed 98.2% similarity with strain P224, while only 78.3-81.3% similarity was observed for species of Mannheimia. Converted to whole genome similarities (Kuhnert and Korczak, 2006) these values correspond to 0.93 between strains T138021-75^T and P224 and 0.46-0.62 between strain T138021-75^T and recognized species of Mannheimia. According to Kuhnert and Korczak (2006) strains of the same species showed similarity values of 0.9 and higher, while species of the same genus had similarity values above 0.4. Similarity values between 0.85 and 0.9 were considered intermediate, and could indicate different species or subspecies. In addition, threshold similarity values for recN alone, as used above, were comparable to MLSA with very few exceptions.

In conclusion, results obtained with the guinea pig strains of taxon 25 clearly documents that they represent a novel species of the genus Mannheimia, for which the name M. caviae sp. nov. is proposed. The type strain is T138021-75^T (CCUG 59995^T), and the G+C content of DNA from the type strain is 41.4 mol%.

The differences in hosts, genotypic- and phenotypic characteristics between the rabbit isolate P224 and the guinea pig isolates T138021-75^T, Pg19 and Pg20 have parallels in both M. varigena for which two genotypic populations associated with pigs and ruminants, respectively, have been demonstrated (Angen et al., 1999) and M. granulomatis for which three populations have been demonstrated associated with leporine, bovine and cervine hosts, respectively (Bojesen et al., 2007). For the same reason, additional leporine, isolates should be investigated before final taxonomic conclusions can be drawn.