A multilocus VNTR analysis (MLVA) scheme for Chlamydia felis genotyping: 1 comparison with MLST. 2

Chlamydia felis is an important ocular pathogen in cats worldwide. A multilocus VNTR analysis (MLVA) system for 16 detection of tandem repeats across the whole genome of C. felis strain Fe/C-56 was developed. Nine selected genetic 17 loci were tested by MLVA on 17 C. felis isolates, including the vaccine C. felis Baker strain, and 122 clinical samples 18 from different geographical origins. Analysis of the results identified 25 distinct C. felis MLVA patterns. In parallel, a 19 MLST scheme recently described for typing of Chlamydia was applied to 13 clinical samples belonging to 12 different C. 20 felis MLVA patterns. Rare sequence differences were observed. Thus, the newly developed MLVA system provides a 21 highly sensitive, high resolution test for the differentiation of C. felis isolates from different origins that is suitable for 22 molecular epidemiological studies. MLVA and Analysis (MLVA) is based on the detection of tandem repeat polymorphisms (38) and has been used successfully for typing many pathogens including Chlamydia trachomatis (27), Chlamydia psittaci (19) and Chlamydia abortus (20). The typing method termed Multilocus sequence typing (MLST) is often based on the sequence analysis of internal fragments 61 of seven housekeeping genes. A MLST scheme previously described has been successfully used to understand the population genetic structure of C. trachomatis, C. pneumonia and C. psittaci (24, 25). The aim of the present study was to develop a novel multilocus VNRT analysis (MLVA) scheme specific for C. felis genotyping and to compare it with the previously described MLST typing method. The clinical samples selected from the large (multitudinous) MLVA C. felis patterns [2], [3], [5], [6], [7], [11], [12], [14], [15], [18], [22] and [23] were successfully amplified with the MLST scheme. Identical sequences (ST_21) were identified within this clinical sample panel for the seven targeted housekeeping genes except for one point mutation in the hfl X gene for the sample 11E3-4. In addition, two non-synonymous mutations detected in the eno A gene of the vaccine C. felis Baker strain. differences in clinical behaviour. In the present study, it was not possible to determine if C. felis isolates with different MLVA genotypes might be associated with variations in disease pathogenesis or clinical outcomes as detailed clinical information from individual cases was not available except for cats observed within the Italian catteries. In these latter cases, cats infected with different C. felis MLVA genotypes, including the cat bearing the hct B-deleted C. felis isolate, showed broadly similar clinical signs typical of C. felis infection. Investigation conducted in catteries revealed the diversity of C. felis strains that can be harboured by cats within a confined environment. Interestingly, from two cats from two different catteries, and where both eyes were sampled, two different genotypes were identified, which in one case differed by only one tandem repeat. These findings provide the first evidence to suggest that mixed infections with strains of different C. felis genotypes occurs in cats, similar to that described for C. trachomatis in humans (39). The possibility that the different genotypes identified in these two cases may represent divergent populations resulting from instability in the MLVA loci cannot be excluded, however analysis of two C. felis isolates passaged ten times through chicken eggs suggests that MLVA genotype is highly stable. such in vitro methods reflect in vivo selective pressures from factors such as host immune responses. Future experiments analysing the stability of MLVA genotypes during the course of C. felis infection and antibiotic treatment, and studies of C. felis superinfection with two or more MLVA genotypes in individual cats and cat communities, would be of considerable interest. Multi-locus sequence typing (MLST) based on the partial sequences of seven housekeeping genes was recently used to understand the population genetic structure of C. trachomatis, C. pneumoniae (24) and C. psittaci (25). Results of cluster analyses of concatenated sequences of the seven housekeeping genes validated the proposed typing system for all chlamydial species. In our study, twelve conjunctival samples belonging to twelve different C. felis MLVA patterns as well as the unique placental sample collected from an abortive feline tissue, and the Baker vaccine strain, were submitted to the MLST scheme. Sequences were identical with only two mutations detected in the eno A gene of the Baker vaccine strain and one point mutation in the hfl X gene for a clinical sample. Almost all clinical samples were of the same sequence type (ST_21) described for the reference Fe/C-56 C. felis strain (http://pubmlst.org/chlamydiales/) the strong conservation of these genes among C. felis except for 1 mutation only identified in the Fe/C-56 hem N gene. based on differences in numbers of tandem repeats in multiple loci on the chromosome of bacteria is on the sequence analysis of housekeeping genes. In this case, the discriminatory power of MLVA was superior to MLST. No specific pattern, neither by MLVA nor by MLST, was identified for the single C. felis sample collected from an abortion tissue and those collected from conjunctival samples. However, before any formal conclusion can be drawn, it will be necessary to analyse a larger cohort C. felis samples recovered feline mutations in the eno A gene, however the biological significance of these mutations remains unknown. our findings show that novel MLVA is a genotyping method providing enhanced discrimination for C. felis Preliminary work on clinical samples MLVA