Abstract.
Etiology of acute undifferentiated febrile syndrome (AUFS) is often unknown, leading to inaccurate diagnosis and treatment. Villeta town has been identified as an endemic area for spotted fever group (SFG) rickettsioses but little is known about possible amplifier hosts and other Rickettsia species different from Rickettsia rickettsii. Besides, few studies have approached other AUFS etiologies in the region. We investigated the role of dengue, leptospirosis, rickettsioses, human anaplasmosis, and Q fever as possible causes of AUFS in patients from Villeta. Sera specimens and ticks from animals as well as ticks from vegetation were studied for the presence of different Rickettsia spp. Among 104 sera from patients with AUFS, 16.4%, 24.0%, and 2.9% patients seroconverted to dengue, Leptospira, and SFG Rickettsia, respectively, with a case of probable coinfection or cross-reaction with Anaplasma phagocytophilum. None of the samples were reactive for Coxiella burnetii. Sera samples from 74 horses, 118 dogs, and 62 bovines were collected and showed 33.8%, 14.4%, and 50.0% of seroprevalence for SFG Rickettsia, respectively. A total of 1,287 ixodid ticks were collected from animals/vegetation and processed in pools for polymerase chain reaction. Among them, 1.7% was positive for Rickettsia genes, and Rickettsia amblyommii, R. rickettsii, and Rickettsia spp. were found. These results confirm the circulation of dengue, different SFG Rickettsia species and the relevance of other etiologies like leptospirosis and human anaplasmosis. Further studies must identify different epidemiological variables to establish proper surveillance and control programs.
INTRODUCTION
Acute undifferentiated febrile syndrome (AUFS) is defined as fever without a focus of infection on initial physical examination or in basic laboratory tests.1 Etiologic nature of AUFS has been a challenge everywhere. Although AUFS is common in tropical regions, the specific etiology is often unknown, making accurate diagnosis, treatment, and surveillance very difficult. Villeta town in Colombia has been considered an endemic area for dengue, spotted fever group (SFG) rickettsioses, and recently, for chikungunya and Zika fever.2,3 Since the first descriptions in 1937, high fatality rates of Rocky Mountain spotted fever (RMSF) have been reported in this region. In the beginning of the 21st century, fatal cases of RMSF and significant seroprevalences against SFG Rickettsia spp. in humans and domestic animals have been recorded.3–7 The relationship and connection between vertebrate species, including humans, through the human–animal–ecosystem interface, could have an impact on human health and should be considered.8 Nowadays, rickettsioses have generated a serious concern in public health because Rickettsia spp. are expanding their geographical distribution, hosts, and vectors leading to zoonotic processes that might affect humans.9 Recent studies have confirmed the presence of Rickettsia rickettsii in Amblyomma patinoi ticks collected from cattle in Naranjal village, Villeta.10 Despite this new evidence, since 2004, there are no new reported cases of SFG rickettsioses in this region. Several authors have confirmed the important role of domestic animals, mainly horses and dogs, as sentinels for monitoring the circulation of rickettsiae in urban areas.8 A previous study in Villeta analyzed seroprevalence against Rickettsia spp. in dogs and horses as a first approach to the dynamics of the infection in domestic animals from this geographic area, showing values of 18.2% and 16.3%, respectively.7 On the other hand, the role of the small wild mammals as amplifiers of rickettsioses has not been studied in the area. Besides, the circulating Rickettsia species in ticks and its possible relationship with the recent disease epidemiology for this area is unknown. In addition, no studies have evaluated the etiology of AUFS, except dengue and recent epidemiological surveys of chikungunya and Zika.2
The aim of this study was to approach the probable etiologies of AUFS. Since dengue can be misdiagnosed and/or overlapped with rickettsial syndromes,11 we investigated (among others) the role of rickettsioses as possible causes of AUFS in patients with presumptive diagnostic of dengue in Villeta, including clinical characteristics, demographics, and epidemiology from a One Health perspective. In addition, sera specimens and ticks from animals as well as ticks from vegetation were studied for the presence of different Rickettsia spp.
MATERIALS AND METHODS
Study area.
The study area was Villeta, Cundinamarca, Colombia (5°0′53″N, 74°28′29″W), located at 842 m above sea level and with an annual mean temperature of 26°C, a relative humidity varying between 80% and 97%, and a total area of 140 km2. According to the 2014 data from the National Department of Statistics (DANE), the estimated population is 25,061 inhabitants, distributed in 22 villages. Agriculture and ecotourism are the predominant economic activities (www.villeta-cundinamarca.gov.co).
Patients.
Patients included in the study were those who attended Salazar Hospital (Villeta) with presumptive diagnosis of dengue from October 2011 to March 2013. Sera specimens from acute and convalescent phase (> 15 days and < 2 months from the onset of symptoms) were obtained. Only those patients who had paired samples were included in the study. All of them accepted the informed consent that was approved by the ethical committee from Pontificia Universidad Javeriana.
Serological assays in humans.
Sera samples were centrifuged and stored at −20°C for subsequent analyses. The following diagnostic tests were performed: 1) capture enzyme-linked immunosorbent assay (Panbio Diagnostic®, San Diego, CA) for the detection of IgM antibodies against dengue virus in the acute illness phase; 2) microagglutination test (MAT) for five Leptospira pathogenic serovars (Icterohaemorrhagiae, Hardjo, Pomona, Grippotyphosa, and Canicola) for the detection of IgG antibodies with a cutoff value of 1:25; 3) indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against R. rickettsii (strain Taiaçu) and Rickettsia amblyommii (strain Ac37) with a cutoff value of 1:6412; 4) IFA using the commercial kit “Rickettsia IFA IgG” (Focus Diagnostics, Cypress, CA) for the detection of IgG antibodies of Rickettsia typhi, with a cutoff value of 1:64; 5) IFA using the commercial kit “Anaplasma phagocytophilum IFA IgG” (Focus Diagnostics) for the detection of IgG antibodies against A. phagocytophilum, with a cutoff value of 1:64; 6) IFA using the commercial kit “Q Fever IgG” (Focus Diagnostics) for the detection of IgG antibodies against Coxiella burnetii (phase I and II antigens), with a cutoff value of 1:64. Throughout the text, for IFA and MAT, seroconversion was defined as a difference of 4-fold titers between acute and convalescent phases or when a negative sample turned into positive.
Serum samples from domestic mammals.
From November to December 2011, domestic animals (dogs, horses, and cattle) were sampled for sera. Sera samples were analyzed by in-house IFA identifying IgG antibodies against R. rickettsii (strain Taiaçu). Samples with IgG titers ≥ 64 were considered positive for Rickettsia.7 Reactive sera (R. rickettsii antigen) obtained from a dog and a horse in the same town in 20087 and a reactive serum for SFG Rickettsia spp. (Rickettsia africae antigen) obtained from cattle from Caribbean Islands, were used as positive controls.
Ticks from domestic, wild mammals, and vegetation.
From November to December 2011, ticks were removed with tweezers from the above-mentioned domestic animals and during July 2012, from wild mammals captured (by Sherman and Tomahawk traps) in 17 villages, previously selected for high seroprevalence against SFG Rickettsia in domestic animals and humans.6,7 Ticks from vegetation were also captured by flagging and dragging methods. Collected specimens were kept in 70% ethanol and further classified by taxonomic keys.13,14
Molecular detection of Rickettsia species in ticks collected from Villeta.
Ticks were grouped by species, stage, locality, and host as main criteria. Females were processed individually and males, nymphs, and larvae in pools of 2–4, 7–12, and 10–20 individuals, respectively. They were processed for DNA extraction using a modified protocol of the commercial kit DNeasy Blood and Tissue (QIAGEN Inc., Valencia, CA) with the addition of guanidine thiocyanate (DNAzol; Invitrogen™, Life Technologies Corp., Grand Island, NY).15
DNA extracts were used for molecular detection of Rickettsia spp. by conventional and nested polymerase chain reaction (PCR) assays, ensuring 150 ng/μL of DNA per reaction. An initial PCR targeting 16S rRNA mitochondrial tick gene (as internal control) was performed.16 Positive pools were further tested for rickettsial infection using ompA (seminested assay with primers Rr190.70p-Rr190.701n/Rr190.602n)17,18 and ompB (nested assay with primers rompB OF-rompB OR/rompB SFG IF-rompB SFG IR)19 as PCR target genes. DNA of Rickettsia slovaca strain S14ab (from the Center of Rickettsiosis and Arthropod-borne Diseases, CIBIR, La Rioja-Spain) and water were used as positive and negative controls, respectively. All PCR products with expected sizes (532 and 420 base pairs for partial ompA and ompB genes, respectively) were sequenced, and nucleotide sequences were compared with GenBank data by BLAST analysis. Minimum infection rates for positive pools were calculated as the percentage of the ratio between the number of pools positive for SFG Rickettsia, and the total number of individual ticks included in the specific sample.
Data analysis.
Official Colombian report documents for dengue from patients included in the study were evaluated. Gender, age, village, symptoms, laboratory analysis (hematocrit, platelets, and leukocytes), were analyzed.
RESULTS
AUFS cases from Salazar Hospital.
A total of 104 paired samples (31%) were obtained from 335 patients who attended Salazar Hospital with presumptive diagnoses of dengue during the inclusion period. Seventeen of 104 patients (16.4%) presented only IgM positive for dengue in the acute illness phase. Twenty-five (24%) presented unique seroconversion to one or more pathogenic serovars of Leptospira. Individually, the Icterohaemorrhagiae serovar was the most representative one (15 patients). Three patients (2.9%) showed unique seroconversion to SFG Rickettsia (one patient for R. rickettsii [< 1:64/1:128] and two for R. amblyommii [1:64/1:256 and < 1:64/1:128]).
We detected probable coinfection or cross-reaction in 17/104 (16.4%) for dengue/leptospirosis, 6/104 (5.7%) leptospirosis/SFG rickettsioses, 5/104 (4.8%) leptospirosis/SFG rickettsioses/dengue, and 1/104 (0.9%) for SFG rickettsioses/dengue. Likewise, a probable case of human anaplasmosis/dengue coinfection was detected in one sample (0.9%). Curiously, 70/104 (67.3%) and 7/104 patients (6.7%) presented seropositivity to SFG Rickettsia (R. rickettsii and/or R. amblyommii) and A. phagocytophilum, respectively, in at least one of the two sera specimens (in acute or convalescent phase), without evidence of seroconversion in paired samples.
None of the paired samples were reactive for C. burnetii (phase I and II antigens) or for R. typhi. Finally, for 29/104 samples (27.8%), all the tests were negative. No patients developed clinical complications or death.
A total of 88 Official Colombian report documents for dengue were evaluated from the 104 patients with paired serum samples. For each etiology, we analyzed all patients that had shown unique probable diagnosis for dengue, leptospirosis, and rickettsioses (N = 45) and sociodemographic features like gender, age, and area of origin (Table 1). The most common symptoms for the three etiologies were fever, myalgia, headache, arthralgia, and retro-orbital pain. Specifically, two-thirds of patients with SFG rickettsioses (66.67%) showed rash (Table 2). Patients with unique seroconversion for SFG Rickettsia presented the following clinical signs: fever, myalgia, nonpurulent conjunctival injection, vomit, and headache, for the R. rickettsii-seropositive case; and fever, myalgia, arthralgia, retro-orbital pain, abdominal pain, rash, and headache, for both R. amblyommii-seropositive cases.
Table 1.
Sociodemographic features of patients with acute febrile syndrome by etiology attended in Villeta town (October 2011–March 2013)
| Variables | Dengue n (%) | Leptospirosis n (%) | SFG rickettsioses n (%) |
|---|---|---|---|
| Gender | |||
| Male | 9 (53) | 11 (44) | 2 (66.7) |
| Female | 8 (47) | 14 (56) | 1 (33.3) |
| Age (years) | |||
| < 10 | 6 (35.3) | 6 (24) | 1 (33.3) |
| 10–20 | 7 (41.2) | 5 (20) | – |
| 21–30 | 1 (5.9) | 5 (20) | 1 (33.3) |
| 31–40 | 3 (17.6) | 4 (16) | – |
| 41–50 | – | 3 (12) | – |
| 51–60 | – | 1 (4) | – |
| > 60 | – | 1 (4) | 1 (33.3) |
| Area | |||
| Urban | 11 (64.7) | 14 (56) | 2 (66.7) |
| Rural | 4 (23.5) | 2 (8) | 1 (33.3) |
| Undetermined | 2 (11.8) | 9 (36) | – |
| No. of patients | 17 (100) | 25 (100) | 3 (100) |
SFG = spotted fever group.
Table 2.
More frequent symptoms presented in dengue fever, leptospirosis, and SFG rickettsioses from patients attended in Villeta town (October 2011–March 2013)
| Symptoms | Dengue fever (%) | Leptospirosis (%) | SFG rickettsioses (%) |
|---|---|---|---|
| Fever | 16/17 (94.12) | 25/25 (100) | 3/3 (100) |
| Myalgia | 13/17 (76.5) | 18/25 (72) | 3/3 (100) |
| Headache | 11/17 (64.7) | 15/25 (60) | 3/3 (100) |
| Arthralgia | 10/17 (58.8) | 15/25 (60) | 1/3 (33.3) |
| Retro-orbital pain | 9/17 (52.9) | 13/25 (52) | 1/3 (33.3) |
| Rash | 2/17 (11.8) | 7/25 (28) | 2/3 (66.7) |
| Vomit | 4/17 (23.5) | 8/25 (32) | 1/3 (33.3) |
| Abdominal pain | 3/17 (17.6) | 8/25 (32) | 1/3 (33.3) |
| Red eye illness | 3/17 (17.6) | 4/25 (16) | 1/3 (33.3) |
| Diarrhea | 3/17 (17.6) | 4/25 (16) | ND |
| Jaundice | 2/17 (11.8) | ND | ND |
| Tachycardia | 1/17 (5.8) | 3/25 (12) | 1/3 (33.3) |
| Hypotension | 1/17 (5.8) | ND | ND |
ND = no data; SFG = spotted fever group.
According to the laboratory results, the initial average hematocrit was 37.2% ± 12.0 for dengue, 39.7% ± 10.2 for leptospirosis, and 31% ± 16.5 for SFG rickettsioses (normal range: 39–50); the average leukocyte counts were 3,282.3 ± 714.3, 4,420 ± 2,626.3 and 3,300 ± 1,473.0 cells/mm3 (normal range: 4,600–10,600) and platelet counts were 134,352 ± 26,542, 124,300 ± 34,563.6, and 154,666 ± 17,214 cells/mm3 (normal range: 160,000–380,000), respectively.
Seroprevalence against SFG Rickettsia in domestic mammals.
Sera samples were collected from 74 horses, 118 dogs, and 62 cows. Among the sera samples from domestic animals, 25/74 from horses (33.8%), 17/118 from dogs (14.4%), and 31/62 from cattle (50%) showed titers equal to or higher than 64, and three of them reached titers of 8,192 (Table 3).
Table 3.
Seropositive dogs, horses, and cattle from Villeta town against Rickettsia rickettsii antigen by IFA (IgG titer ≥ 64); November–December 2011
| Village | Horses | Dogs | Cattle | |||
|---|---|---|---|---|---|---|
| No. positive /no. tested | Maximum titer | No. positive /no. tested | Maximum titer | No. positive /no. tested | Maximum titer | |
| Alto de Paja | 1/2 | 64 | 0/5 | – | – | – |
| Alto de Torres | 0/3 | – | 0/5 | – | – | – |
| Bagazal | 0/1 | – | 0/9 | – | 2/3 | 64 |
| Balsal | 1/3 | 128 | 0/2 | – | – | – |
| Chapaima | 3/8 | 64 | 4/5 | 4,096 | 1/4 | 64 |
| Chorrillo | 1/4 | 64 | 1/8 | 64 | 0/5 | – |
| Cune | 0/5 | – | 1/5 | 8,192 | 3/4 | 64 |
| El Puente | 1/4 | 512 | 0/6 | – | – | – |
| Ilo Grande | 1/6 | 64 | 0/6 | – | – | – |
| La Bolsa | 3/4 | 64 | 0/2 | – | 0/1 | – |
| La Esmeralda | – | – | 0/5 | – | 0/2 | – |
| La Mazata | 0/1 | – | 1/3 | 64 | – | – |
| Maní | 1/1 | 128 | 0/3 | – | 7/8 | 64 |
| Mave | 1/5 | 64 | 3/8 | 64 | 0/1 | – |
| Naranjal | 2/4 | 128 | 1/3 | 512 | 6/12 | 64 |
| Payandé | 2/2 | 64 | 0/4 | – | – | – |
| Potrero Grande | – | – | 1/4 | 64 | 2/2 | 64 |
| Quebrada Honda | – | – | 0/4 | – | – | – |
| Río Dulce | – | – | 1/3 | 64 | 1/1 | 64 |
| Salitre Blanco | 0/3 | – | 2/11 | 8,192 | 2/9 | 64 |
| Salitre Negro | 2/7 | 128 | 0/1 | – | 0/2 | – |
| San Isidro | 1/4 | 128 | 0/2 | – | 7/8 | 128 |
| Urban area | 5/7 | 8,192 | 2/14 | 64 | – | – |
| Total (%) | 25/74 (33.8) | 17/118 (14.4) | 31/62 (50.0) | |||
IFA = immunofluorescence assay.
Molecular detection of Rickettsia species in ticks.
A total of 516 ticks were collected from domestic animals and were identified as follows: Dermacentor nitens and Amblyomma cajennense sensu lato (s.l.) from horses; Rhipicephalus sanguineus s.l., A. cajennense s.l., and Amblyomma ovale from dogs; and Rhipicephalus microplus and A. cajennense s.l. from cattle (Table 4). Besides, 13 wild mammals (five Didelphis marsupialis, three Marmosa robinsoni, three Mus musculus, one Rattus rattus, and one Sigmodon hirsutus) were captured and tick samples were only collected from D. marsupialis and classified as Ixodes luciae and Ixodes spp. (Table 4). A total of 744 ticks were obtained from vegetation and identified as R. microplus, Amblyomma sp., A. cajennense s.l., and Dermacentor sp. (Table 4).
Table 4.
Number and species of ticks collected from animals and vegetation in Villeta town (November–December 2011 and July 2012)
| No. of individuals | Rickettsia infection* | ||||||
|---|---|---|---|---|---|---|---|
| Species of ticks | Source | M | F | N | L | Total | |
| Amblyomma cajennense s.l. | Horses | 35 | 26 | 7 | 1 | 69 | – |
| Dogs | 3 | – | – | – | 3 | – | |
| Cattle | 18 | 19 | 1 | – | 38 | Positive | |
| Vegetation | 2 | 2 | 158 | – | 162 | Positive | |
| Amblyomma ovale | Dogs | 1 | – | – | – | 1 | – |
| Amblyomma sp. | Vegetation | – | – | – | 222 | 222 | Positive |
| Dermacentor nitens | Horses | 61 | 120 | 50 | 10 | 241 | Positive |
| Dermacentor sp. | Vegetation | – | – | – | 64 | 64 | – |
| Ixodes luciae | Didelphis marsupialis | 14 | 11 | – | – | 25 | – |
| Ixodes sp. | D. marsupialis | – | 2 | – | – | 2 | – |
| Rhipicephalus microplus | Cattle | 9 | 31 | 2 | – | 42 | Positive |
| Vegetation | – | – | – | 296 | 296 | Positive | |
| Rhipicephalus sanguineus s.l. | Dogs | 66 | 47 | 9 | – | 122 | – |
F = female adults; L = larvae; M = male adults; N = nymphs.
Positive for Rickettsia ompA or ompB genes.
As it is showed in Table 5, ticks were grouped into 446 pools. Among them, 358 pools were positive for the 16S rRNA PCR and further screened for Rickettsia spp. (ompA and ompB genes). Rickettsia spp. was found in 6/358 pools (1.7%). Rickettsia amblyommii (GenBank accession no. KJ433807) was detected in larvae of R. microplus (pool M841). In addition, R. rickettsii (GenBank accession nos. KJ433802, KJ433806, and KJ433805, respectively) was found in male adults of D. nitens (pool M181), in nymphs of A. cajennense s.l. (pool M827), and in larvae of Amblyomma sp. (pool M822). Moreover, Rickettsia spp. were detected in male adults of A. cajennense s.l. (pool M235) and a female specimen of R. microplus (pool M196), showing 94.9% identity with Rickettsia conorii (GenBank accession no. KJ433804) and 99.7% identity with Rickettsia monacensis (GenBank accession no. KJ433803), respectively, as highest identities with validly published Rickettsia species (Table 5).
Table 5.
Tick pools evaluated for the presence of Rickettsia spp. in Villeta town (November–December 2011, and July 2012)
| Village | Tick species (source) | Positive pools*/total of pools | Total of ticks (% MIR) | BLAST analysis |
|---|---|---|---|---|
| Chapaima | Amblyomma cajennense s.l. (DA) | 0/5 | ||
| Dermacentor nitens (DA) | 0/81 | |||
| Rhipicephalus sanguineus s.l. (DA) | 0/3 | |||
| Rhipicephalus microplus (DA) | 0/1 | |||
| Cune | A. cajennense s.l. (DA) | 0/7 | ||
| D. nitens (DA) | 0/11 | |||
| R. sanguineus s.l. (DA) | 0/1 | |||
| R. microplus (DA) | 0/22 | |||
| Mani | R. microplus (V) | 1/26 | 229 (0.4) | Rickettsia amblyommii† |
| Naranjal | A. cajennense s.l. (DA) | 1/43 | 43 (2.3) | Rickettsia sp.‡ |
| A. cajennense s.l. (V) | 1/32 | 51 (2.0) | R. rickettsii§ | |
| Amblyomma ovale (DA) | 0/1 | |||
| Amblyomma sp. (V) | 1/23 | 104 (1.0) | R. rickettsii¶ | |
| Dermacentor sp. (V) | 0/8 | |||
| Ixodes luciae (WA) | 0/16 | |||
| Ixodes sp. (WA) | 0/2 | |||
| R. sanguineus (DA) | 0/1 | |||
| Urban area | A. cajennense s.l. (DA) | 0/12 | ||
| D. nitens (DA) | 0/58 | |||
| R. sanguineus s.l. (DA) | 0/69 | |||
| Salitre Blanco | A. cajennense s.l. (DA) | 0/5 | ||
| D. nitens (DA) | 1/4 | 10 (10) | R. rickettsii‖ | |
| R. microplus (DA) | 1/15 | 16 (6.3) | Rickettsia sp.** |
DA = domestic animals; MIR = minimum infection rate (no. of positive pools/total no. of individual ticks) × 100; V = Vegetation; WA = wild animals.
Positive pools for ompA or ompB.
Pool “M841”: 15 larvae from vegetation positive for ompA gene.
Pool “M235”: four males from cattle positive for ompA gene (94.9% identity with Rickettsia conorii).
Pool “M827”: 10 nymphs from vegetation positive for ompA gene.
Pool “M822”: 15 larvae from vegetation positive for ompA gene.
Pool “M181”: three males from a horse positive for ompB gene.
Pool “M196”: one female from a cow positive for ompB gene (99.7% identity with Rickettsia monacensis).
DISCUSSION
The probable etiology of AUFS for patients who attended Salazar Hospital from October 2011 to March 2013 was mainly leptospirosis, with 24% of seroconversion, especially against the Icterohaemorrhagiae serovar (60%). In a national study that showed the epidemiology of human leptospirosis from 2007 to 2011, the Icterohaemorrhagiae serovar represented 7.56% of the circulation.20 Our study evidences the circulation of Leptospira serovars and indicates that leptospirosis, a common worldwide zoonotic disease with a predominant presence in tropical regions,21 is one of the main causes of AUFS in Villeta. Our results of possible etiologies of AUFS are comparable with those from a recent study in the region of Urabá (Antioquia),22 where R. rickettsii cases were confirmed.23,24 In Antioquia, dengue was probably the main cause of AUFS (37.3%) with higher seroprevalence than the one found herein (16.4%). In addition, percentages of Rickettsia infection (2.7%) and coinfection of SFG rickettsiosis/dengue (0.5%) were comparable to those found in our study (2.9% and 0.9%, respectively).22
Herein, the continuous circulation of SFG Rickettsia spp. in humans and in domestic animals (cattle, horses, and dogs) from Villeta has been confirmed, showing 67.3%, 50.0%, 33.8%, and 14.4% of seroprevalence, respectively. These data suggest that pathogenic and nonpathogenic SFG Rickettsia species may cause human diseases or asymptomatic infections in the area. Also, the finding of three patients with unique SFG Rickettsia seroconversion is noteworthy; those with R. amblyommii seroconversion presented clinical signs previously reported from probable cases of R. amblyommii rickettsiosis (i.e., fever, headache, myalgia, and rash)25; unfortunately it was not possible to confirm (with molecular or Western Immunoblotting tests) the specific SFG Rickettsia species involved in these patients.
In addition, this study represents the second report of probable exposition to A. phagocytophilum in Colombia,26 based on the probable case of infection defined by seroconversion and the seroprevalence found in our study (6.7%), which is lower than that reported in Cordoba.26 Although A. phagocytophilum is typically transmitted by Ixodes ticks,27 which did not represent a huge concern in Latin America,28 this bacterium has been recently detected in A. cajennense s.l.29 This tick species is present in our area but the finding of A. phagocytophilum in Amblyomma ticks does not confirm their role as vectors. Besides, to confirm the circulating Anaplasma species and its vector, further studies must attempt to isolate it from vertebrate and/or invertebrate hosts.
Herein, A. cajennense s.l. was parasitizing the three species of domestic mammals sampled (horses, dogs, and cattle) with the highest levels of infestation in cattle (47.5%). The multihost feeding habits and the anthropophilic behavior of this tick28,30 represent a high risk of exposition to SFG Rickettsia for the human population of Villeta, even more considering that this species has been incriminated as vector of R. rickettsii in the region.4,10 Furthermore, the tick species A. ovale and I. luciae have been recorded for the first time in this geographical area of Colombia. Previous studies documented the presence of I. luciae parasitizing D. marsupialis in the Orinoquia region,31 and A. ovale parasitizing dogs in Sucre and Urabá (Antioquia).32,33
Molecular evidence of SFG Rickettsia spp. in ticks from animals has been previously reported in Colombia.33–36 In this study, R. amblyommii was found in R. microplus from vegetation and R. rickettsii in D. nitens from a horse and also in A. cajennense s.l. and Amblyomma sp. from vegetation. In the same region, A. cajennense s.l. harboring R. amblyommii and D. nitens infected with R. rickettsii as well as the isolation of R. rickettsii (strain Villeta) from A. patinoi have been previously published.10,36,37 The detection of R. microplus infected with R. amblyommii has been previously reported in Panama.38 The cofeeding phenomenon could explain the association of R. rickettsii with D. nitens since this tick species and A. cajennense s.l. are common parasites of horses in Latin America.13,39
Furthermore, our study shows the first detection of SFG Rickettsia spp. in cattle from this country, and it is the second report from South America.40 Specifically, A. cajennense s.l. and R. microplus removed from cows were found to be infected with Rickettsia spp. that showed the highest identities with R. conorii (94.9%) and R. monacensis (99.7%), respectively. New rickettsial genotypes related to R. monacensis had been found in other Colombian areas34 or in neighboring countries such as Ecuador.41 Further studies are necessary for a better characterization of these bacteria.
Despite the high lethality caused by R. rickettsii 80 years ago,3 the occurrence of human fatal cases of RMSF in 2003 and 2004,5 the high rates of seroprevalence for SFG Rickettsia spp. in humans and domestic mammals,5–7 and the recent isolation of R. rickettsii from A. patinoi ticks,10 there are no new reports of human cases caused by R. rickettsii in this endemic region of Villeta (Cundinamarca). This situation could be explained by misdiagnosis of this illness due to lack of clinical suspicions and of compulsory notification in Colombia, by possible cross-immune protection caused by less pathogenic rickettsiae,42–44 and by ecological transformations which modulate the epidemiological pattern of the disease.45 To better understand the latter scenario, further research must identify the vertebrate hosts which sustain the natural cycle of these pathogens (mainly as amplifier hosts) and comprehend the related epidemiological determinants and the effects of environmental changes on the tick–pathogen–vertebrate interface as has been studied for similar diseases.46
The results presented herein confirm the relevance of rickettsioses as a differential diagnosis in patients with AUFS and the importance of the clinical suspicion and laboratory confirmation of different etiologies with an early treatment. Nonetheless, other diseases (e.g., leptospirosis, anaplasmosis, and dengue), their interrelationships, and other epidemiological variables should be further studied in the region.
Acknowledgments:
We acknowledge all personnel from Hospital Salazar, UMATA, and local farms from Villeta for their invaluable help during all phases of the study. Additionally, we thank Patrick Kelly from Ross University School of Veterinary Medicine (Basseterre, St. Kitts) for kindly providing bovine sera for IFAT, Patricia Hernández (Universidad de La Salle) for kindly providing MAT-Leptospira diagnosis, and Marcelo Labruna from University of São Paulo for kindly providing antigens of R. rickettsii and R. amblyommii for IFAT. We also thank the Pontificia Universidad Javeriana (PUJ) research project: “Caracterización de factores climáticos y ecológicos de una especie de garrapata y su relación con la epidemiologia de las rickettsiosis en un área endémica” (ID Cod ppta 4344).
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