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Am. J. Trop. Med. Hyg., 75(6), 2006, pp. 1069-1073
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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LEISHMANIA AMAZONENSIS INFECTIONS IN ORYZOMYS ACRITUS AND ORYZOMYS NITIDUS FROM BOLIVIA

SARA F. KERR*, LOUISE H. EMMONS, PETER C. MELBY, CHANG LIU, LUIS E. PEREZ, MARIA VILLEGAS, AND ROBERT MIRANDA
Biology Department, University of the Incarnate Word, San Antonio, Texas; Smithsonian Institution, Division of Mammals, Washington, DC; South Texas Veterans Health Care System and University of Texas Health Science Center, San Antonio, Texas


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three of thirteen Oryzomys acritus, Emmons and Patton 2005 (Rodentia: Muridae: Sigmodontinae) and 3 of 17 Oryzomys nitidus, Thomas 1884, collected from Noël Kempff National Park, Bolivia, from 2002 to 2005, tested positive for Leishmania (Leishmania) amazonensis or L. (L.) mexicana and negative for Leishmania (Viannia) spp. using the polymerase chain reaction (PCR). Based on previous records of L. (L.) amazonensis in humans, rodents, and sand flies from Bolivia, and the geographic distributions of L. (L.) amazonensis and L. (L.) mexicana, it was concluded that the Oryzomys were infected with L. (L.) amazonensis. These results identify two additional species of Oryzomys as hosts of L. (L.) amazonensis, and identify an ecological region of Bolivia where L. (L.) amazonensis is enzootic.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cutaneous and mucocutaneous leishmaniases are endemic in both the Andean highland and the Amazon basin of Bolivia, but visceral leishmaniasis is rare.1 Although most human infections have been attributed to L. (Viannia) spp., L. (Leishmania) amazonensis also has been reported.2–5 The rice rat Oryzomys capito Olfers (1818) was reported to be a host in Cochabamba, Bolivia.6 The taxonomy has been revised, and the currently accepted epithet for Western Bolivian specimens of this taxon is Oryzomys perenensis J.A. Allen, 1901.7 Leishmania (L.) amazonensis also was detected in Akodon sp. Meyen, 1833 (Rodentia: Muridae: Sigmodontinae) and Oligoryzomys sp. Bangs, 1900 (Rodentia: Muridae: Sigmodontinae) and the sand fly Lutzomyia nuneztovari anglesi (Le Pont & Desjeux, 1984).8,9 The purpose of our investigation was to test the hypothesis that Oryzomys sp. are reservoirs of L. (L.) amazonensis in Bolivia.10,11


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Field methods. Rodents were captured at two localities in Bolivia, Santa Cruz, Parque Nacional Noël Kempff Mercado: El Refugio Huanchaca (UTM 20L 0712187; 8368228, WGS 84, 220 m elevation) each September from 2002–2005, and Los Fierros (UTM 20 L 0724182; 8387597, 240 m elevation) in October 2002 and September 2003. The vegetation and ecology of the region has been described in detail.12 Small mammals were captured in live traps (H.B. Sherman, Tallahasee, FL) as part of a project by Emmons to study the faunal communities within the park. Procedures have been approved by Dr. Emmons’ institutional animal care and use protocol. Animals were transferred from the trap to a cloth bag. One person who was wearing leather gloves restrained the animal by gripping it firmly behind the head while another performed other procedures. The area to be biopsied was sprayed with benzocaine first-aid spray; with this procedure the subjects showed no signs of distress. Because long-term monitoring is being continued, most captured animals were marked and released, although a few were preserved as vouchers. Series of up to 20 voucher specimens per species, with tissue samples, have been collected for the localities, and are preserved in the Museo de Historia Natural Noël Kempff Mercado, Santa Cruz, with duplicates in the United States National Museum.

In 2002, animals with scars on the basal, dorsal surface of the tail were chosen for biopsy (Figure 1Go). A 2-mm punch biopsy was taken from the edge of the scarred area and from the rim of each ear, placed in 1.5-mL conical bottom tubes with screw tops (PGC Scientifics, Frederick, MD), containing 95% EtOH. Pooled samples of skin from each ear and the base of the tail were screened by the polymerase chain reaction (PCR) as described below.


Figure 1
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  		    FIGURE 1. Tail of Oryzomys acritus from Noel Kempff National Park, Bolivia, showing lesions that were positive for Leishmania.

 
During September 11–13, 2003, rodents were captured at El Refugio Huanchaca and biopsies were taken only from the base of the tails from 25 rodents with or without scars or lesions, except that a biopsy from one ear was taken in the case of three Proechimys longicaudatus that were missing tails. These were screened by both PCR and culture. In 2004 biopsies were taken only from the tails of rodents with scars; in 2005 they were taken from all rodents captured in forests. These biopsies were only screened by PCR.

Culture methods. Unsuccessful attempts were made to culture Leishmania promastigotes from the tissue samples collected in September 11–13, 2003. They were placed in 1X medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (GIBCO), 1M Hepes buffer (pH 7.4) (Sigma Chemical Co., St. Louis, MO), 100X penicillin/streptomycin (GIBCO), 10 mM hypoxanthin (SIGMA), 0.25% (v/v) bovine hemin (Sigma), and 5 mL human urine cleared at 600 g for 20 minutes in a Centra-CL2 benchtop centrifuge. The cultured medium was sterilized by passage through a 0.2-µm filter and adjusted to pH 7.4 by 1M Sodium Hydroxide (Fisher Scientific Company, Fair Lawn, NJ). The cultures were kept at 25°C and monitored at least three times a week for the presence of promastigotes of Leishmania.

Primers and probe. DNA was amplified with the universal Leishmania primers 13A and 13B.13 To identify kDNA sequences that were uniquely conserved among L. (L.) amazonensis and L. (L.) mexicana parasites, minicircle DNA sequences from L. (Viannia) spp., L. (L.) amazonensis, and L. (L.) mexicana that had been deposited in GenBank were aligned using the CLUSTAL software. To detect DNA from L. (L.) amazonensis or L. (L.) mexicana the forward primer was the genus specific primer 13A (5'-GTGGGGGAGGGG-CGTTCT-3'), and the reverse primer (M1.1; 5'-CCAGTTT-CGACCGCCGGAGC-3') targeted a sequence that was conserved in the L. (L.) amazonensis and L. (L.) mexicana sequences but differed from the L. (Viannia) spp. sequences. To specifically amplify L. (Viannia) spp. the forward primer was specific to L. (Viannia) spp. (B4; 5'-TCGTACTCCCCGA-CATGCCTC-3') and the reverse primer was the genus specific primer 13B (5'-ATTTTACACCAACCCCCAGTT-3').

Parasites and parasite DNA. Sixty-three Leishmania isolates from both the Old and New Worlds were used to define the amplification specificity of the M1.1/13A PCR primers. The specificity of the B4/13B (Viannia subgenus) will be described in another report (Guarin N, Melby PC, unpublished). Reference isolates were generously provided by Dr. Nancy Saravia, Centro Internacional de Entrenamiento e Investigaciones Medicas-CIDEIM, Cali, Colombia (L. (Viannia) spp.); Dr. Fernando Andrade-Narvaez, Universidad Autonoma de Yucatan, Merida, Mexico (L. (L.) mexicana); and Dr. Hechmi Louzir, Institut Pasteur, Tunis, Tunisia (L. major). Identification of the reference isolates to the subgenus or species level had been determined previously by analysis of isoenzyme profile and/or reactivity to specific monoclonal antibodies.

Leishmania spp. promastigotes were cultured in M199 medium supplemented with 15% heat inactivated fetal calf serum (HIFCS), 0.1 mM adenine, 5 µg/ml hemin, 1 µg/ml biotin, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.14 For isolation of parasite DNA the cultured promastigotes were washed in PBS and 1 x 103 were pelleted in a microcentrifuge tube. The parasite DNA was isolated by adding 20 µL of 10 mM Tris, 10 mM EDTA and placing the sample in boiling water for 5 minutes. The sample was then diluted in nuclease-free water and an aliquot used for the PCR reaction.

Amplification of Leishmania DNA from cultured parasites. Primers were re-suspended in nuclease-free water to a stock concentration of 100 pmol/µl and stored at –70°C. The DNA was amplified by PCR in a master mix containing 1.5U Taq DNA polymerase, 200 µM dNTPs, 0.4 µM of each primer, and 1.5 mM of MgCl2. The PCR reaction conditions were: 94°C x 5 minutes, followed by 94°C x 1 minute, 65°C x 1 minute, 72°C x 1.5 minute for 30 cycles, followed by 72°C x 7 minutes for 1 cycle. DNA equivalent to 20 parasites was added to each reaction mix.

Detection of amplified Leishmania DNA. The amplification products were detected by 1.8% (w/v) agarose gel electrophoresis with ethidium bromide staining under UV light. The 13A/M1.1 primers gave a ~105 bp amplification product from L. (L.) amazonensis and L. (L.) mexicana DNA, the B4/13B primers gave a ~135 bp amplicon from DNA from Viannia subgenus parasites, and the universal primers 13A/ 13B gave a ~120 bp amplicon for all Leishmania strains used to test the specificity of the primers (Table 1Go).


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TABLE 1
PCR detection of Leishmania DNA from cultured promastigotes*
 
Polymerase chain reaction screening of Bolivian rodents. The rodent tissue was lysed and the conserved region of kDNA amplified with the primers described previously in this article. PCR products amplified from two of the animals whose test results were positive were cloned into the plasmid vector pCR®2.1-TOPO® (TOPO TA Cloning® kit, Invitrogen Corporation, Carlsbad, CA). The plasmids were purified by QIAGEN® Plasmid Maxi Purification kit (QIAGEN, Valencia, CA), and sequenced at the Advanced Nucleic Acid Core Facility, Department of Microbiology at University of Texas Health Science Center at San Antonio (UTHSCSA). Sequences were compared and aligned using the National Center for Biotechnology Information, U.S. National Library of Medicine, Basic Local Alignment Search Tool (BLAST).15


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Using a large number of Old World and New World Leishmania isolates the M1.1/13A primers were determined to specifically amplify DNA from members of the L. (L.) amazonensis or L. (L.) mexicana (Table 1Go). Most notably, under the standardized reaction conditions, DNA from members of the Viannia subgenus was not amplified by these primers. Furthermore, the Viannia subgenus-specific B4/M13 primers did not amplify DNA from L. (L.) amazonensis or L. (L.) mexicana.

Ultimately 64 individual mammals, 10 species of rodent and two species of opossum, were screened for Leishmania (Table 2Go). Three of thirteen O. acritus (Figure 2Go) and 2 of 17 O. nitidus (Figure 3Go) from El Refugio Huanchaca, and 1 of 2 O. nitidus from Los Fierros tested positive for Leishmania spp. and negative for L. (Viannia) spp., using PCR.16 Efforts to culture the parasite were unsuccessful. No positive for Leishmania was detected in any other mammal species. Both trap-lines where Leishmania positive rodents were collected were in semideciduous tall forest formations.


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TABLE 2
Number of individual mammals positive for Leishmania spp./Number screened by PCR using primers 13A/13B12
 

Figure 2
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  		    FIGURE 2. Three of thirteen Oryzomys acritus collected from Noël Kempff National Park, Bolivia, from 2002–2005, tested positive for Leishmania.

 

Figure 3
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  		    FIGURE 3. Three of seventeen Oryzomys nitidus collected from Noël Kempff National Park, Bolivia, from 2002–2005, tested positive for Leishmania.

 
The sequence of the DNA amplified from O. acritus (field no. 444) and O. nitidus (field no. 575) compared with a reference kDNA sequence [(L. (L.) amazonensis, MHOM/BR/ 00/Raimundo (=MHOM/BR/1983/M1132), NCBI Accession number M21326)] is shown below. The primer (13A and M1.1) sequences are underlined and a vertical line indicates identity between the cloned and reference sequences.


Figure 4

Each of the sequenced kDNA fragments was 102 nucleotides in length, including the primer sequences. The sequence of amplified DNA (excluding the primer sequences) from O. acritus (field no. 444) and O. nitidus (field no. 575) showed 93% and 78% identity to the reference L. (L.) amazonensis kDNA sequence, respectively. A similar level of identity was observed with other L. (L.) amazonensis and L. (L.) mexicana sequences. Notably, the BLAST search did not identify homology to kDNA sequences of parasites of the Viannia subgenus, even though more than 40 such sequences of the conserved region of the minicircle DNA are present in the NCBI database.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results indicate that both O. acritus and O. nitidus were infected with either L. (L.) amazonensis or L. (L.) mexicana. It is noteworthy that both PCR products that were sequenced showed high homology with DNA from parasites collected from humans in Brazil. Previously, eight human stocks from the sub-Andean region of LaPaz were identified as L. (L.) amazonensis based on isoenzyme electrophoresis and comparison with reference strains.4 Leishmania isolates from one Akodon and two Oligoryzomys collected at this focus were identified as L. (L.) amazonensis based on the similarity of kDNA-PCR profiles to that of the human isolates.8 Isoenzyme profiles of three isolates from the sand fly Lu.(n.) anglesi collected at this focus also indicated L. (L.) amazonensis.9 PCR using SSU rRNA was used to compare a large number of L. (L.) amazonensis strains from broadly distributed geographical areas.5 Results indicated that of these two species, L. (L.) amazonensis is the main species occurring in South America, whereas L. (L.) mexicana is primarily confined to North and Central America. Therefore, based on the previous identification of L. (L.) amazonensis from humans, rodents, and sand flies from Bolivia, and the geographic distributions of L. (L.) amazonensis and L. (L.) mexicana, we concluded that the rice rats were infected with L. (L.) amazonensis.

These results identify two additional species of Oryzomys as hosts of L. (L.) amazonensis, and identify Noël Kempff National Park as an ecological region of Bolivia where L. (L.) amazonensis is enzootic. The fact that infections were detected in either O. acritus or O. nitidus each year from 2002–2005, and the lack of infections in other potential reservoirs such as Proechimys longicaudatus, suggest that O. acritus and O. nitidus are reservoirs of L. (L.) amazonensis in this area. Seemingly, Oryzomys is a host, and possible reservoir, of L. (L.) amazonensis or L. (L.) mexicana over a broad geographic range, including Mexico, Central America, and South America.4,17–22 Since Oryzomys spp. also occur in the eastern United States where sand flies (Lutzomyia sp.) are abundant, it may also be a reservoir there.23 According to Eisenberg (1989), oryzomyines are close relatives of the neotomine-peromyscine group of North America, and the tylomyine and nyctomyine of Central America.24 Based on the information that reservoirs of Leishmania spp. are known from all of these closely related groups, Kerr (2000) suggested that oryzomyine rodents might be reservoirs of Leishmania in South America.11 Our results support this hypothesis.

Received November 10, 2005. Accepted for publication July 27, 2006.

Acknowledgments The authors thank the Weedon Foundation for its support of field research at El Refugio Huanchaca, and Ian and Barbara Phillips for their inestimable help there. This work was part of Emmons’ studies of the biodiversity of Parque Nacional Noël Kempff Mercado (PNNKM), in collaboration with the Museo de Historia Natural Noël Kempff Mercado. The authors thank Fundacion Amigos de la Naturaleza for their continuing support of research at Los Fierros, and Damián Rumiz and Kathia Rivero for help with logistics and permits. Permits: U.S. Public Health Importation Permit No. 2002-10-190, scientific permit from Bolivia Ministerio de Desarollo Sostenible y Planificacion.

Financial support: This work received financial support from U.S. National Institutes of Health Grant GM55337 (SFK, PCM), the Sr. Joseph Marie Armer Chair fund, the Amazon Conservation Association (LHE), and Center for Disease Control and Prevention Grant H75/CCH615041 (PCM). The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.

* Address correspondence to Sara F. Kerr, Biology Department, University of the Incarnate Word, 4301 Broadway, San Antonio, TX 78209. E-mail: kerr{at}uiwtx.edu Back

Authors’ addresses: Sara F. Kerr, Chang Liu, Maria Villegas, and Robert Miranda, Biology Department, University of the Incarnate Word, 4301 Broadway, San Antonio, TX 78209. Louise Emmons, Smithsonian Institution, Division of Mammals NB390 MRC108, PO Box 37012 Washington, DC 20013-7012. Peter C. Melby and Luis E. Perez, South Texas Veterans Health Care System and University of Texas Health Science Center, 7400 Merton Minter, San Antonio, TX 78229.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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