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).

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 (online Technical Appendix Figure 1, http:// wwwnc.cdc.gov/EID/article/21/3/14-0721-Techapp1.pdf) 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.
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 (online 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.   To the Editor: Thirty-eight patients with Mycobacterium bovis BCG-associated osteomyelitis/osteitis, including 8 who were previously reported (1), were identified during Taiwan's vaccine injury compensation program during 1989-2012; a total of 30 (79%) patients applied for compensation during 2009-2012 ( Figure). In Taiwan, a laboratory program to differentiate BCG from other species of the M. tuberculosis complex, using a kit for the Tokyo-172 vaccine strain spoligotyping, was established in 2004 (1). Since 2008, the isolated extrapulmonary tuberculosis strains and pathologic specimens collected from children <5 years of age have been sent to the national reference mycobacterial laboratory for BCG detection (2). The Parents or guardians signed written consent forms on behalf of the children when they submitted claims for the vaccine injury compensation program. After consent, children's hospital information was stored in the Taiwan Centers for Disease Control database and used for research.
As in previous reports (3,4), extremity bones were more commonly involved than axial bones. For 30 (79%) children, extremity bones were involved: 14 right lower limbs, 7 left lower limbs, 6 left upper limbs, and 3 right upper limbs. The tibia was the most common site (9 patients), followed by ankle bones (8 patients), femur (4 patients), radius and thumb (3 patients each), humerus and knee (2 patients each), and ulna (1 patient). Of these, 2 patients had 2 bony lesions. In 8 (21%) children, axial bones were involved: 5 sternums, 2 thoracic vertebrae, and 1 right rib. Eight (53%) of 15 patients had positive tuberculin skin test results. No specific abnormalities were found with regard to blood cell counts and inflammation markers or to chest radiographs, except for 1 child with rib