Novel Corynebacterium diphtheriae in Domestic Cats

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identifi ed. Molecular methods distinguished the feline isolates from known C. diphtheriae.
T he clinical relevance of Corynebacterium diphtheriae recovered from a cat with otitis is poorly understood. Historically, humans have been thought to be its sole reservoir, and the few human cases reported annually in the United States are generally associated with international travel (1). Therefore, when C. diphtheriae was isolated from the ears of a cat, an investigation was initiated to evaluate potential sources of the cat's infection and potential public health risks and to preliminarily characterize the C. diphtheriae isolate.
The cat, an 8-month-old female domestic shorthair, was examined at a West Virginia veterinary hospital on 5 occasions during January-June 2007. Pertinent fi ndings included severe bilateral otitis, vestibular signs, mild ataxia, anorexia, and failure to gain weight; the cat had a history of ear, eye, and lung infections. Results of diagnostic tests showed no evidence of systemic disease and were nega-tive for feline immunodefi ciency and leukemia viruses and feline infectious peritonitis. Culture of an otic swab collected from the cat in May 2007 yielded 4 organisms: C. diphtheriae, Streptococcus equi zooepidemicus, Staphylococcus spp., and Achromobacter xylosoxidans. The cat was treated with oral clindamycin, otic enrofl oxacin, and an ear-fl ushing solution.

The Study
In June 2007, investigators visited the veterinary clinic and the household of the index cat and conducted a contact investigation and carrier study. Interviews of 2 household members and 8 veterinary staff members indicated no recent respiratory illness, skin infection, or risk factors for diphtheria (e.g., travel to countries to which diphtheria is endemic or contact with known case-patients). Half of these 10 contacts had received diphtheria vaccination within the previous 5 years. Cultures of oropharyngeal swab samples obtained from each person were negative, including cystine tellurite blood agar, which is selective for C. diphtheriae. Household members also were interviewed about medical history of a convenience sample of household animals (4 cats, including the index cat; 2 dogs; and 1 horse). Each animal was briefl y examined, and oropharyngeal, otic, or ocular swab samples were collected. Otitis was observed in all 4 cats and 1 dog. The horse reportedly had had an eye infection ≈5 years earlier. No other abnormal fi ndings were noted. Animal specimens yielded 3 additional isolates of C. diphtheriae: 1 from each ear of the index cat and 1 from the left ear of a 2-year-old domestic medium-hair cat. Both cats had been born on the premises and had remained with the same household since birth.
Feline C. diphtheriae and reference isolates used are described in the Table. Tinsdale agar plate growth (Remel, Lenexa, KS, USA) gave rise to black colonies with a brown halo, typical of cysteinase-producing C. diphtheriae, C. ulcerans, or C. pseudotuberculosis. After 24 hours on blood agar, 1-2-mm grey-white or opaque, rounded, convex colonies with no hemolysis were observed. Microscopically, the bacteria were gram-positive, club-shaped rods, 1 μm in diameter, arranged singly or at angles. Biochemical profi les to determine species and biotype were done by using an API Coryne strip (bioMérieux, Durham, NC, USA, and St-Laurent, Quebec, Canada). Query of API Coryne code 0010304 obtained for all isolates by APIWEB (https://apiweb.biomerieux.com) indicated a decreased level of confidence of C. diphtheriae biotype mitis or belfanti (89.5%) because of a maltose-negative result. Isolates were further characterized morphologically and biochemically by using tube substrates (2) and were identifi ed by using a standard taxonomic scheme (3). Feline isolates were biochemically identical with each other and phenotypically consistent with C. diphtheriae biotype belfanti, except for the lack of maltose fermentation, which was considered an unusual fi nding (3).
Results from use of the modifi ed Elek test (6) indicated that all feline isolates were negative for production of diphtheria toxin; however, an atypical precipitation was observed after 36 h of incubation. Lack of toxin expression was corroborated by negative Vero cell assay results (7) and confi rmed by using Western blot. Real-time PCR selective for the C. diphtheriae and C. ulcerans toxin gene (tox) (8) was positive for all feline isolates. However, realtime PCR for A and B subunits of tox (9) amplifi ed subunit A but not subunit B. Sequence analysis of the tox gene was performed as previously outlined (10) and compared with a reference tox gene, GenBank accession no. K01722. The 4 feline tox sequences were identical to each other but con-  (11) and partial rpoB (12) gene sequencing. By 16S rRNA gene sequence analysis, the feline strains had 100% identity with each other and >99.1% identity with various reference sequences for C. diphtheriae biotype gravis and belfanti sequences, including NCTC 11397 T . Partial rpoB sequence analyses indicated 100% identity among the feline isolates and 97.7% identity with C. diphtheriae NCTC 11397 T . Neighbor-joining phylogenetic trees based on both 16S rRNA ( Figure 1) and partial rpoB gene sequencing ( Figure 2) positioned the feline isolate sequences within the C. diphtheriae clade but clearly distinguished them from the other C. diphtheriae isolates. Comprehensive molecular analyses to characterize differences between biotype belfanti strains, including these feline isolates, with other C. diphtheriae biotypes, are the subject of a separate publication (C.G. Dowson, pers. comm.).

Conclusions
We identifi ed a potentially novel biotype of C. diphtheriae recovered from domestic cats in West Virginia but found no evidence of zoonotic transmission. Although rare, isolation of C. diphtheriae from animals has been reported, including C. diphtheriae biotype belfanti from a skin lesion of a cow (13) and toxigenic C. diphtheriae biotype gravis from a wound of a horse (14). C. ulcerans is a known animal pathogen, and zoonotic transmission of toxigenic C. ulcerans from companion animals has been reported, often associated with predisposing concurrent illnesses (15).
The feline strains isolated during this investigation differed phenotypically from previously described biotypes but were otherwise regarded as typical of C. diphtheriae. However, isolates were nontoxigenic and harbored a modi-fi ed tox gene with sequence differences from Corynebacterium spp. capable of expressing diphtheria toxin. On the basis of published criteria (11), the feline strain might represent a novel subspecies of C. diphtheriae because it shares <98% sequence homology to the type strain within the rpoB gene. Potential for zoonotic transmission of this novel, cat-associated C. diphtheriae and associated public health implications are unknown. Additional studies are needed to further characterize these isolates and determine their appropriate taxonomy. Large-scale screening of domestic cat populations is recommended to determine the prevalence of C. diphtheriae and its pathogenic potential and to identify additional isolates for more formal description and classifi cation.