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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 ) .
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 briefly examined , and oropharyngeal , otic , or ocular swab samples were collected . otitis was observed in all 4 cats and 1 dog .
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 .
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 , 12-mm grey - white or opaque , rounded , convex colonies with no hemolysis were observed .
microscopically , the bacteria were gram - positive , club - shaped rods , 1 m in diameter , arranged singly or at angles .
biochemical profiles to determine species and biotype were done by using an api coryne strip ( biomrieux , 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 identified 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 finding ( 3 ) .
* cd , centers for disease control and prevention identifier number ; atcc , american type culture collection ; nd , not deposited in this study ; nctc , national collection of type cultures , london , uk .
additional strains used as controls for specific assays : toxigenic c. diphtheriae biotype belfanti isolates used for real - time pcr of tox gene were 718 , g4182 , c59 , c60 , c75 , c76 , c77 ; toxigenic c. diphtheriae atcc 27012 used as positive control for elek ; c. diphtheriae nctc 10481 and c. ulcerans cd199 used as positive and negative controls for vero cell assay . 
nctc 10356 is described in the nctc catalogue as c. diphtheriae biotype mitis ; however , analyses in this study found this strain to be nitrate negative and therefore consistent with c. diphtheriae biotype belfanti .
antimicrobial drug susceptibility testing was performed according to the clinical and laboratory standards institute s recommended methods and interpretative criteria ( 4 ) .
all 4 feline isolates were sensitive to ampicillin , cefepime , cefotaxime , ceftriaxone , cefuroxime , chloramphenicol , ciprofloxacin , clindamycin , daptomycin , erythromycin , ertapenam , gatifloxacin , gentamicin , levofloxacin , linezolid , meropenem , moxifloxacin , penicillin , quinupristin / dalfopristin , rifampin , telithromycin , tetracycline , tigecycline , trimethoprim / sulfamethoxazole , and vancomycin .
cellular fatty acid composition analysis was performed as described ( 5 ) by using the sherlock system ( midi , inc . ,
newark , de , usa ) , except that version 4.5 of the operating software was used .
the cellular fatty acid composition profiles were consistent for c. diphtheriae , c. ulcerans , or c. pseudotuberculosis , including a substantial proportion ( 28%30% of total ) of c16:17c ( 5 ) .
all feline isolates produced 715 meq / l of propionic acid among fermentation products , a feature associated with c. diphtheriae ( 2 ) .
results from use of the modified 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 confirmed 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 , real - time pcr for a and b subunits of tox ( 9 ) amplified 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 .
the 4 feline tox sequences were identical to each other but contained multiple nucleotide substitutions and deletions compared with the reference gene . by ncbi blast search ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) , the feline tox had higher sequence identity ( 97%98% ) to the tox sequences of c. ulcerans , compared with those from c. diphtheriae ( 94%95% ) .
a deletion at nt  55 , coupled with a cytosine - to - thymine substitution at nt  74 , prematurely terminated the peptide at  aa  25 .
species characterization was corroborated by using 16s rrna ( 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 .
partial rpob sequence analyses indicated 100% identity among the feline isolates and 97.7% identity with c. diphtheriae nctc 11397 .
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 .
neighbor - joining phylogenetic tree based on 16s rrna gene sequence analysis of corynebacterium diphtheriae isolates , including 4 feline isolates from west virginia , 2008 ( atcc baa-1774 , cd 448 , cd 449 , cd 450 ) .
the tree was constructed from a 1,437-bp alignment of 16s rrna gene sequences by using the neighbor - joining method and kimura 2-parameter substitution model .
bootstrap values ( expressed as percentages of 1,000 replicates ) > 40% are illustrated at branch points .
feline isolates had 100% identity with each other and > 99.1% identity with c. diphtheriae biotypes gravis and belfanti .
atcc , american type culture collection ; cd , centers for disease control and prevention identifier number ; nctc , national collection of type cultures .
jukes - cantor  derived phylogenetic tree based on sequence analysis of a selected region of the rpob gene of corynebacterium isolates , including 2 feline isolates from west virginia , 2008 ( atcc baa-1774 , cd 450 ) .
feline isolates had 100% identity with each other and 97.7% identity with c. diphtheriae biotypes gravis and belfanti .
atcc , american type culture collection ; cd , centers for disease control and prevention identifier number ; nctc , national collection of type cultures .
we identified 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 modified 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 .
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 classification .
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 .