Characterization of spotted fever group rickettsiae in flea and tick specimens from northern Peru

Abstract

Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.

FIG. 1.

Representative example of htrA gene PCR denoting a positive sample from FL105. Evident are both a TG Rickettsia-specific (A) and SFG Rickettsia-specific band (B). The 129-bp product of the ompB gene amplified from DNA from FL105 with R. felis-specific primers is also shown (C). A 123-bp ladder (Gibco Life Sciences, Gaithersburg, Md.) was used as a guide for molecular sizes in all three gel slices.

FIG. 2.

PCR of the 540-bp ompA gene from tick samples. Lane 1, negative extraction control; lanes 2 and 3, positive controls (R. akari and R. rickettsii, respectively); lanes 4 to 6, ticks 124, 127, and 163, respectively. Samples were electrophoresed in 2% agarose gels.

FIG. 3.

Dendrogram of the sequence alignment of rickettsia species based on the nucleotide sequences of a 2,487-bp segment of the ompB gene. The analysis, including maximum parsimony and neighbor joining, was performed with the PAUP program. Bootstrap values are indicated at the nodes.