Molecular Epidemiology of Feline and Human Bartonella henselae Isolates

Multiple locus variable number tandem repeat analysis was performed on 178 Bartonella henselae isolates from 9 countries; 99 profiles were distributed into 2 groups. Human isolates/strains were placed into the second group. Genotype I and II isolates shared no common profile. All genotype I isolates clustered within group B. The evolutive implications are discussed.

Ninety-nine different MLVA profi les were observed (Table 1), corresponding to an average number of isolates per profi le of 1.8 (Table 2). Sixty-nine of these profi les were found in only 1 isolate or strain (67%), and 30 were observed in >1 isolate. Among these, none was shared by genotype I and genotype II isolates. Diversity index (DI) was 0.98 (Table 1). Diversity was observed in both genotypes because genotype-specifi c DIs were almost identical (Table 1).
MLVA profi les appeared location-specifi c because only 4 (13%) of the 30 profi les observed in >1 isolate/strain were present in >1 continent (Table 2). Within continents, no marked dominance of a given profi le was observed, and continent-specifi c DIs were similar (Table 1).
Of the 99 B. henselae profi les, 12 were obtained from the 21 human isolates/strains and 1 from the dog, whereas 92 profi les were obtained from the 156 feline isolates. Five profi les were common to 5 human and 11 feline isolates. Among the 30 profi les observed in >2 isolates, 23 were observed only in feline isolates ( Table 2). The proportion of genotype I profi les was signifi cantly higher in humanspecifi c profi les than in cat-specifi c profi les (p = 0.01, by Fisher test).
For BHV-A, only 2 alleles (14 and 15 copies) were found in isolates from humans, whereas all 8 identifi ed alleles were observed in cat isolates. The number of repeats differed signifi cantly between sick humans and healthy cats (p = 0.02, by Fisher test).
Relationships between the 99 MLVA profi les were analyzed by unweighted pair group method with arithmatic mean (UPGMA), using a categorical distance, with a B. koehlerae isolate used as an outgroup. To take into account that UPGMA is sensitive to taxa entry order, we computed the majority-rule consensus tree of 500 dendrograms built with random taxa entry order. MLVA profi les were grouped into 2 main groups named A and B (online Appendix Figure, available from www.cdc.gov/EID/ content/15/5/813.htm). Group A (26 profi les), was exclusively constituted by genotype II feline isolates. Group B (73 profi les), to which all human isolates belonged, further divided in 2 subgroups, Ba and Bb. Subgroup Ba (38 profi les) was exclusively composed of genotype I isolates, including the reference strain Houston I and a homogenous subgroup, Ba1, containing 84% of the Asian isolates. Finally, 83% of subgroup Bb isolates belonged to genotype II (29/35 profi les).
The utility of MLVA for molecular epidemiologic analysis of clusters was tested using isolates from California cats and their owners (14). Five human-cat groups of B. henselae isolates were analyzed. For 1 cat-human pair of isolates, which belonged, respectively, to genotype II and genotype I, major profi le differences were observed, as expected. The 4 other cat-human groups, which possessed the same genotype, also had the same MLVA profi le with the 5 tested BHV, as well as with the 6 additional BHV (F-K) and variant alleles for BHV-A and/or B (6). Sequencing confi rmed these results.

Conclusions
Our results confi rm that VNTRs are excellent molecular markers for confi rming or excluding the responsibility of a given cat in the transmission of B. henselae to a human. In California, the profi le identity observed within 4 clusters further supports the hypothesis that all these humans acquired infection from their respective domestic cat contacts.
MLVA enabled a clear separation between genotypes I and II, because no profi le was shared between both genotypes. The dendrogram showed a high level of discrimination between 16S rDNA genotypes in the B. henselae population tested. Interestingly, the groups and subgroups delineated by MLVA were the same as those defi ned by MLST, a standard method for phylogenetic analysis (12). The same was observed with MST (13). The isolates of the subgroup Bb appeared divergent and distant from each other and from subgroup Ba that contains almost all genotype I profi les (98%). Moreover and despite possible clustering for some of the isolates, none of the 21 human isolates was present in group A. Interestingly, as for most of the human patients, the isolate obtained from the ill dog also belonged to genotype I.
These observations suggest that all genotype I isolates could be phylogenetically derived from genotype II isolates located in group B but not in group A, as already suggested using MLST (15). This observation could mean that genotype II isolates belonging to group B are closer to genotype I isolates than to genotype II isolates belonging to group A; it also raises an important clinical question: Are feline genotype II isolates belonging to group A nonpathogenic for humans? Genotype I isolates could represent the most pathogenic isolates for humans within a group of potentially zoonotic isolates, all belonging to group B and could represent an ultimate evolutionary step toward human infection. Additionally, within group B, the differences in the number of BHV-A repeat units observed between isolates from patients (humans, dog) versus cat isolates suggest that this specifi c VNTR could constitute a marker for the ability to cross the species barrier from reservoir cats to susceptible species, independent of the 16S rDNA genotype.  -NZ  10  14  2  2  1  14  14  14  14  9  15  2  1  1  8  8  8  5  3  10  15  2  2  1  8  8  8  8  14  34  2  7  4  8  8  1  7  8  14  22  10  5  3  7