Rickettsia rickettsii in Amblyomma patinoi Ticks, Colombia

To the Editor: Rickettsia rickettsii is the etiologic agent of Rocky Mountain spotted fever (RMSF), a highly lethal tick-borne rickettsioses restricted to the Western Hemisphere (1,2). In Colombia, R. rickettsii was first reported during the 1930s, when 62 (95%) of 65 affected persons died of RMSF in Tobia town (Cundinamarca Department) (3), from where highly virulent strains of R. rickettsii were isolated through the inoculation of patient blood or of Amblyomma cajennense sensu lato (s.l.) extracts into guinea pigs (4). Thereafter, RMSF remained unnoticed in Colombia until the 21st century, when new outbreaks with high case-fatality rates were reported in different regions, including Villeta, a nearby locality of Tobia (1).

Recent studies have shown that A. cajennense s.l., widely distributed from the southern United States to Argentina, is actually a complex of 6 different species: A. cajennense sensu stricto (Amazonian region), A. mixtum (from Texas, USA, to western Ecuador), A. sculptum (northern Argentina, Bolivia, Paraguay, Brazil), A. interandinum (inter-Andean valley of Peru), A. tonelliae (dry areas of northern Argentina, Bolivia, and Paraguay), and A. patinoi (eastern cordillera of Colombia) (5). With this new classification, A. patinoi, originally described from Villeta, is the only species of this complex known to occur in the RMSF-endemic area of Cundinamarca, Colombia (5).

In August 2013, we collected 15 A. patinoi adult ticks from cattle in Naranjal village (5°3′31.52′′N, 74°26′50.24′′W), Villeta town, an area of Cundinamarca, Colombia, to which RMSF is endemic. Ticks were taken alive to the laboratory, where they were frozen at –80°C for further analysis. The 15 ticks were defrosted, surface sterilized with iodine alcohol, and processed individually by the shell vial technique for isolation of rickettsiae in Vero cells, as described (6). Infected cells were always incubated at 28°C. Rickettsiae were observed by Gimenez staining within cells (Technical Appendix Figure 1) from only 1 (inoculated from a female tick) of the 15 inoculated shell vials. This isolate was subjected to at least 7 Vero cell passages, each achieving >90% infected cells.

DNA was extracted from an aliquot of first passage–infected cells and tested by a battery of PCR protocols targeting fragments of the rickettsial genes gltA, ompA, and ompB and the intergenic regions RR0155-rpmB, RR1240-tlc5^b, and cspA-ksgA (Table). We sequenced 1,106 bp, 512 bp, and 799 bp of the gltA, ompA, and ompB genes, respectively. By BLAST analyses (http://www.ncbi.nlm.nih.gov/blast), these sequences were 100% identical to corresponding sequences of R. rickettsii from Colombia and Brazil (GenBank accession nos. CP003306, CP003305). Generated sequences for 2 intergenic regions, RR0155-rpmB (228 bp) and RR1240-tlc5^b (306 bp), were 100% identical to corresponding sequences of the same 2 R. rickettsii isolates from Colombia and Brazil. A 337-bp sequence of the cspA-ksgA intergenic region was 100% (337/337 nt) identical to R. rickettsii from Brazil (CP003305) and 99.7% (336/337) to R. rickettsii from Colombia (CP003306). Partial sequences from R. rickettsii generated in this study were deposited into GenBank and assigned nucleotide accession nos. KJ735644–KJ735649.

Table. Primer pairs used for amplification of rickettsial genes or intergenic regions, Colombia, August 2013.

Target, primer pairs, primers	Primer sequences, 5′ → 3′	Fragment size, bp	Reference
gltA
1
CS-78	GCAAGTATCGGTGAGGATGTAAT	401	(6)
CS-323	GCTTCCTTAAAATTCAATAAATCAGGAT		(6)
2
CS-239	GCTCTTCTCATCCTATGGCTATTAT	834	(6)
CS-1069	CAGGGTCTTCGTGCATTTCTT		(6)
ompA
3
Rr190.70p	ATGGCGAATATTTCTCCAAAA	530	(7)
Rr190.602n	AGTGCAGCATTCGCTCCCCCT		(7)
ompB
4
120-M59	CCGCAGGGTTGGTAACTGC	862	(8)
120–807	CCTTTTAGATTACCGCCTAA		(8)
RR0155-rpmB
5
Forward	TTTCTAGCAGCGGTTGTTTTATCC	290	(9)
Reverse	TTAGCCCATGTTGACAGGTTTACT		(9)
RR1240-tlc5b
6
Forward	CGGGATAACGCCGAGTAATA	357	(10)
Reverse	ATGCCGCTCTGAATTTGTTT		(10)
cspA-ksgA
7
Forward	CATCACTGCTTCGCTTATTTT	405	(9)
Reverse	ATTTCTTTTCTTCCTCTTCATCAA		(9)

Whole-body remnants of the 15 ticks used to inoculate shell vials were also subjected to DNA extraction and processed by PCR for the rickettsial gltA gene (Table); only 1 tick (the one that provided the rickettsial isolate) contained rickettsial DNA, indicating a 6.6% (1/15) infection rate. We confirmed the taxonomic identification of this tick as A. patinoi by generating mitochondrial 16S rRNA partial sequences from it and from an A. patinoi paratype that is deposited in the tick collection of the University of São Paulo (Brazil) (accession no. CNC-1585) (5). Both sequences were 100% identical to each other (deposited into GenBank under accession nos. KP036466–KP036467).

A 3 mL-aliquot of the second infected cell passage was inoculated intraperitoneally into an adult male guinea pig, in which high fever (rectal temperature >40.0°C) developed during days 5–8 days post inoculation. A total of 3 guinea pig passages were performed, always followed by high fever. A second passage animal survived; scrotal necrosis developed (Technical Appendix Figure 2), and this animal seroconverted to R. rickettsii with 32,768 endpoint IgG titer through the immunofluorescence assay, as described (2).

A highly pathogenic strain of R. ricketsii was isolated from an A. patinoi specimen collected at Villeta, where recent human cases of RMSF have been reported (1). More than 70 years ago, the only previous R. rickettsii tick isolates in Colombia were obtained from A. cajennense s.l. in Tobia, only 20 km from Villeta (4). At that time, ticks of the A. cajennense complex were considered natural vectors of R. rickettsii in Tobia (4). Because the A. cajennense s.l. complex was recently found to be represented in the eastern cordillera of Colombia (which includes Tobia and Villeta) by the species A. patinoi (5), the tick isolates obtained >70 years ago also are highly likely to have been obtained from A. patinoi. Therefore, A. patinoi ticks should be considered the main vector of R. rickettsii to humans in this region of Colombia.

Technical Appendix

Testing of Rickettsia rickettsii isolated from an Amblyomma patinoi tick, Villeta, Colombia, August 2013.