La detección de Bartonella spp. y Rickettsia spp. en pulgas, garrapatas y piojos recolectados en las zonas rurales de Perú

Bartonellosis and rickettsiosis are commonly reported in Peru. In order to detect Bartonella sp. and Rickettsia sp. in fleas, ticks and lice, specimens from five distinct locations in Peru (Marizagua, Cajaruro, Jamalca, Lonya Grande and El Milagro) were collected and screened for the presence of these bacteria using PCR and later confirmation by DNA sequencing. The specimens collected were distributed in 102 pools (76 Ctenocephalides felis, 2 Ctenocephalides canis, 16Pulex irritans, 5 Pediculus humanus, 2 Rhiphicephalus sanguineus, and 1 Boophilus spp.), whereBartonella was detected in 17 pools (6 of C. felis, 9 of P. irritans, 1 of C. canis, and 1 P. humanus). Also, Rickettsia was detected in 76 pools (62 C. felis, 10 P. irritans, 2 P. humanus, and 2 C. canis). Bartonella clarridgeiae was detected in C. felis, C. canis and P. irritans pools at 5.3%, 50% and 12.5%, respectively. Bartonella rochalimae was detected in one C. felis and two P. irritans pools at 1.3% and 12.5%, respectively. Furthermore, B. henselae was detected in one C. felis pool and one P. humanus pool corresponding to 1.3% and 20%, respectively; andBartonella spp. was also found in 5 pools of P. irritans at 31.3%. Additionally, R. felis was detected in C. felis, C. canis and P. irritans pools at 76.3%, 100% and 37.5%, respectively; and Rickettsia spp. was detected in C. felis, P. irritans and P. humanus pools at 5.3%, 25% and 40%, respectively. These results demonstrate the circulation of these bacteria in Peru.

Keywords: Bartonella ; Rickettsia; arthropods; PCR detection; Peru.

Resumen

La Bartonellosis y la Rickettsiosis son enfermedades comúnmente reportadas en Perú. Con el propósito de detectar Bartonella sp. y Rickettsia sp. especímenes de pulgas, garrapatas y piojos de cinco localidades del Perú (Marizagua, Cajaruro, Jamalca, Lonya Grande and El Milagro) fueron colectadas y analizadas. Para la detección se usó PCR y una posterior confirmación con secuenciamiento de DNA. Los especímenes colectados fueron agrupados en 102 pools (76 Ctenocephalides felis, dos Ctenocephalides canis, 16Pulex irritans, cinco Pediculus humanus, dos Rhiphicephalus sanguineus, y un Boophilus spp.). Bartonella fue detectada en 17 pools (seis de C. felis, nueve de P. irritans, uno de C. canis, y uno de P. humanus). Rickettsia fue detectada en 76 pools (62 de C. felis, 10 de P. irritans, dos de P. humanus, y dos de C. canis). Bartonella clarridgeiae fue detectada en C. felis (5.3% especímenes), C. canis (50%) y P. irritans (12.5%). Bartonella rochalimae fue detectada en C. felis (1.3%) y P. irritans (12.5%). Además, se detectó B. henselae en C. felis (1.3%) y P. humanus (20%). Bartonella spp. también se encontró en P. irritans (31,3%). Además, se detectó R. felis en C. felis (76.3%), C. canis (100%) y P. irritans (37.5%), y Rickettsia spp. se detectó en C. felis (5,3%), P. irritans (25%) y P. humanus (40%). Estos resultados demuestran la circulación de estas bacterias en el Perú.

Keywords: Bartonella ; Rickettsia; arthropods; PCR detección; Perú.

Introduction

Human bartonellosis is caused by various species of Bartonella. Nowadays, there are several species of this genus that can infect humans including B. bacilliformis, B. henselae, B. quintana, B. rochalimae, B. elizabethae, B. vinsonii subsp. arupensis, B. vinsonii subsp. berkhoffii, B. grahamii, B. koehlerae, B. washoensis, B. alsatica, and B. tamiae (Billeter et al. 2008, Daly et al. 1993, Kerkhoff et al. 1999, Roux et al. 2000, Eremeeva et al. 2007, Kosoy et al. 2008). Only B. bacilliformis, B. vinsonii, B. quintana, B. rochalimae, B. elizabethae and B. cladrrigeiae have been reported in Peru (Billeter et al. 2008, Eremeeva et al. 2007, Parola et al. 2002, Roux & Raoult 1999, Raoult et al. 1999). Bartonella is believed to be transmitted by an arthropod vector, and infection can result in Carrion’s disease, cat scratch disease, trench fever, endocarditis, neuroretinitis, bacillar angiomatosis, hepatic granulomatosis, arthritis, osteomelitis, and sepsis (Billeter et al. 2008, Breitschwerdt & Kordick 2000).

Rickettsia spp. are gram negative coco-bacilli and intracellular pathogens and most of them are transmitted transtadially by a wide variety of arthropod hosts including fleas, lice and ticks. Infections with these pathogens can cause diverse human diseases such as endemic typhus, scrub typhus, erhlichiosis and others rickettsiosis (Raoult & Roux 1997). In Peru, rickettsiosis occurs sporadically along the coast, highlands and jungle. The exposure to rickettsiosis was confirmed by immunofluorescence assay (IFA) in regions of Peru (Raoult et al. 1999, Blair et al. 2004a) and Rickettsia species were detected in ticks and fleas (Schoeler et al. 2005, Jiang et al. 2005).

Several arthropods belonging to the orders Diptera (Noguchi et al. 1929, Caceres et al., 1997), Anoplura (Ellis et al. 1999), Siphonaptera (Brouqui et al. 1999; Higgins et al. 1996) and Parasitiformes (Schouls et al. 1999, Angelakis et al. 2010) have been implicated in the transmission of Bartonella spp. Previous experimental studies have demonstrated that Lutzomyia verrucarum (sandfly) are competent vectors of B. bacilliformis, which causes Carrion’s disease (Breitschwerdt & Kordick 2000, Maguiña et al. 2009). In order to detect Bartonella spp. and Rickettsia spp. in fleas, lice and ticks, specimens were captured from humans, domestic and wild animals in five distinct locations in Peru, where Carrion’s disease is endemic. The presence of Bartonella spp. in these specimens was evaluated by polymerase chain reaction (PCR) and confirmed by DNA sequencing.

Materials and methods

Study sites and specimen collections. Samples were collected from random houses in different places: San Ignacio (Marizagua) of Cajamarca departament; Cajaruro, Jamalca, Lonya Grande and El Milagro of Amazonas departament in July 2000 to September 2001 (Table 1, Fig. 1).

These samples were obtained from different pets such as dogs, cats, and guinea pigs (considered as a food source); moreover, foxes found near the neighborhood were trapped in cages for later sample collection. Specimens such as fleas were collected by hand from animals; but lice were collected from people (head, body and clothes) and ticks were collected only from dogs. These were then stored in vials containing 70% ethanol, and organized according to host and place of collection.

The taxonomic classification was done in the Entomology Laboratory at the “Daniel A. Carrión” Tropical Medicine Institute, of the Universidad Nacional Mayor de San Marcos in Lima, using entomological keys (Jonson 1957, Lewis 1972).

After taxonomic classification, the specimens were grouped into pools of 6 to 20 specimens, each pool from one or two hosts, to reduce the processes for analysis (see Table 1).

DNA extractions from specimens. The whole specimens were grouped in pools and washed with 1X Phosphate Buffered Saline (PBS). They were then triturated with a micro-pestle in 100 µL of PBS. DNA was extracted using a Tissue DNA Purification Kit (QIAGEN) following the standard procedure recommended by the manufacturer.

DNA amplification of the gltA gene by PCR. The gltA gene of Bartonella was amplified using the BhCS.781p and BhCS.1137 primers (Norman et al. 1995). The same specimens were analyzed using the Rp877p and Rp1258n primers, theses primers amplified the gltA gene of Ricketsia (Regnery et al. 1991).

The amplification reactions consisted of 5 µL of amplification buffer 10X, 5 µL (10 µM) of each primer, 5 µL (25 mM) of magnesium chloride, 2 µL (10 mM) of nucleotide triphosphates, 0.2 µL (5 UµL) of AmpliTaq Gold DNA polymerase (Applied Biosystems Inc.) and 5 µL of purified DNA to each tube. The final volume of the amplification reaction was 50 µL. Purified DNA from B. bacilliformis strain ATCC 35685 and a Rickettsia felis characterized culture were used as positive controls. The amplifications were performed on a 9700 Applied Biosystems thermocycler, and the cycles were as follows: 1 cycle for 95 °C for 10 min; 35 cycles at 95 °C for 0.5 min, 50 °C for 0.5 min, and 72 °C for 1 min; 1 cycle at 72 °C for 5 min; and a final soaking at 4 °C until analysis.

The amplified products were analyzed by electrophoresis in agarose gels, stained with ethidium bromide and visualized in a UV transilluminator.

The amplified products were then purified using QIAQuick Gel extraction Kit (QIAGEN Inc.) and sequenced using a Big Dye DNA terminator Kit (Applied Biosystems Inc.) on an ABI310 sequencer (Applied Biosystems) at the Peruvian National Institute of Health and following standard procedures. The sequences obtained were analyzed with GenBank using BLAST software (www.ncbi.nlm.nih.gov/BLAST). Clustalw software (http://align.genome.jp/) was used for alignment and comparison of sequences.