Molecular characterization of a novel fastidious mycobacterium causing lepromatous lesions of the skin, subcutis, cornea, and conjunctiva of cats living in Victoria, Australia

Abstract

Between 1999 and 2006, 15 cats were diagnosed with disease attributable to a novel mycobacterial species. The infections consisted of granulomatous lesions in the skin, subcutis, and ocular or periocular tissues with an indolent but progressive clinical course. Lesions typically were found in facial regions or on the distal limbs. Cats of all ages and both sexes were affected. Infections often were challenging to treat, although they could be cured using surgery in concert with combination antimicrobial therapy. Microscopically, lesions were granulomatous to pyogranulomatous and contained numerous acid-fast bacilli. Scanty cultures of the causal microorganisms occasionally could be obtained in mycobacterial broth, but subculture to solid media failed. When cultures were not available, DNA was extracted from fresh tissue, lyophilized material, and formalin-fixed, paraffin-embedded tissues from lesions. PCR amplification of the 5' end of the 16S rRNA gene and regions within four additional loci (ITS1, hsp65, rpoB, and sodA) was performed with various efficiencies using mycobacterial primers. Nucleotide sequences were unique for each locus tested. Nucleotide sequences obtained from individual cases were identical for each locus for which the amplification was successful. Phylogenetic analysis performed using concatenated partial 16S rRNA and hsp65 gene sequences indicated that this novel mycobacterial species from Victoria is a member of the Mycobacterium simiae-related group, taxonomically related to the mycobacterium causing leproid granulomas in dogs throughout the world. Based on the clustering of cases, we refer to this novel species as Mycobacterium sp. strain Tarwin.

FIG. 1.

Geographical distribution of cases associated with Mycobacterium sp. strain Tarwin. Case numbers refer to those in Table 1. The single case from New South Wales (case 11) is not illustrated on this map of Victoria.

FIG. 2.

Mycobacterial granulomas due to Mycobacterium sp. strain Tarwin on the periocular skin of case 9. The lesions take the form of multiple nodules in the subcutis. Lesions are neither hot nor painful, and in contrast to M. lepraemurium lesions, ulceration was not a feature. The distribution is consistent with the introduction of bacilli on the claws of a perpetrator as a result of a cat scratch. Images A and B show different perspectives to illustrate the extent and number of the lesions.

FIG. 3.

Mycobacterial keratitis and conjunctivitis due to Mycobacterium sp. strain Tarwin. The gross appearance of the lesions are illustrated (A), while the photomicrograph stained with ZN illustrates granulomatous inflammation with epithelioid macrophages containing enormous numbers of AFB (B).

FIG. 4.

Nucleotide sequence alignment within helix 18 of the V3 region of the 16S rRNA gene that compares the sequence determined for M. tuberculosis to the sequences determined for the Mycobacterium species associated with feline leprosy and disseminated disease in cats. Note that Mycobacterium sp. strain Tarwin (M. sp. Tarwin) has the insertion-free version, relative to the other slow-growing mycobacteria, characteristic of the M. simiae-related Mycobacterium species. Note also the C-T substitution (in boldface) at nt 467, relative to the other M. simiae-related species. Superscript letters a to f indicate the following GenBank accession numbers: a, BX842576; b, AJ279017; c, AJ294740 to AJ294746; d, AY061986; e, AY061985; f, AY061984. M. simiae-related refers to the following species: M. simiae, M. genavense, M. lentiflavum, M. triplex, M. interjectum, M. intermedium, M. heidelbergense, M. parascrofulaceum, M. kubicae, M. palustre, M. montefiorense, M. florentinum, M. sherrisii, Mycobacterium sp. strain IP20010510, and Mycobacterium sp. strain CLGS (M. sp. CLGS).

FIG. 5.

Unrooted phylogenetic tree of the M. simiae-related mycobacterial species, including Mycobacterium sp. strain Tarwin and Mycobacterium sp. strain CLGS, computed from the concatenated 16S rRNA gene and hsp65 sequences by the neighbor-joining method.