Am. J. Trop. Med. Hyg., 75(6), 2006, pp. 1069-1073 Copyright ? 2006 by The American Society of Tropical Medicine and Hygiene LEISHMANIA AMAZON EN SIS 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. Three of thirteen Oryzomys acritus, Emmons and Patton 2005 (Rodentia: Muridae: Sigmodontinae) and 3 of 17 Oryzomys nitidus, Thomas 1884, collected from Noel 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 Cutaneous and mucocutaneous leishmaniases are endemic in both the Andean highland and the Amazon basin of Bo- livia, 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 re- vised, and the currently accepted epithet for Western Boliv- ian 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: Sigmodon- tinae) 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 Field methods. Rodents were captured at two localities in Bolivia, Santa Cruz, Parque Nacional Noel 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 ecol- ogy of the region has been described in detail.12 Small mam- mals were captured in live traps (H.B. Sherman, Tallahasee, FL) as part of a project by Emmons to study the faunal com- munities within the park. Procedures have been approved by Dr. Emmons' institutional animal care and use protocol. Ani- mals were transferred from the trap to a cloth bag. One per- son 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 * Address correspondence to Sara F. Kerr, Biology Department, Uni- versity of the Incarnate Word, 4301 Broadway, San Antonio, TX 78209. E-mail: kerr@uiwtx.edu 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 Noel 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 1). 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 reac- tion (PCR) as described below. 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 cul- ture Leishmania promastigotes from the tissue samples col- lected in September 11-13, 2003. They were placed in IX 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 hypox- anthin (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-|xm 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 se- quences that were uniquely conserved among L. (L.) ama- zonensis and L. (L.) mexicana parasites, minicircle DNA se- quences 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 1069 1070 KERR AND OTHERS FIGURE 1. Tail of Oryzomys acritus from Noel Kempff National Park, Bolivia, showing lesions that were positive for Leishmania. was the genus specific primer 13A (5'-GTGGGGGAGGGG- CGTTCT-3'), and the reverse primer (Ml.l; 5'-CCAGTTT- CGACCGCCGGAGC-3') targeted a sequence that was con- served in the L. (L.) amazonensis and L. (L.) mexicana se- quences 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 spe- cific primer 13B (5'-ATTTTACACCAACCCCCAGTT-3'). Parasites and parasite DNA. Sixty-three Leishmania iso- lates 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 de- scribed in another report (Guarin N, Melby PC, unpub- lished). Reference isolates were generously provided by Dr. Nancy Saravia, Centro Internacional de Entrenamiento e In- vestigaciones Medicas-CIDEIM, Cali, Colombia (L. (Vian- nia) 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 subge- nus or species level had been determined previously by analy- sis of isoenzyme profile and/or reactivity to specific monoclo- nal antibodies. Leishmania spp. promastigotes were cultured in M199 me- dium supplemented with 15% heat inactivated fetal calf se- rum (HIFCS), 0.1 mM adenine, 5 jJLg/ml hemin, 1 jJLg/ml bi- otin, 2 mM L-glutamine, 100 U/ml penicillin, and 100 |xg/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 |xL 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 para- sites. Primers were re-suspended in nuclease-free water to a stock concentration of 100 pmol/pi and stored at -70?C. The DNA was amplified by PCR in a master mix containing 1.5U Taq DNA polymerase, 200 JJLM dNTPs, 0.4 |xM 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 72CC x 7 minutes for 1 cycle. DNA equivalent to 20 parasites was added to each reaction mix. Detection of amplified Leishmania DNA. The amplifica- tion products were detected by 1.8% (w/v) agarose gel elec- trophoresis 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 1). 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, Valen- cia, CA), and sequenced at the Advanced Nucleic Acid Core Facility, Department of Microbiology at University of Texas Health Science Center at San Antonio (UTHSCSA). Se- quences were compared and aligned using the National Cen- ter for Biotechnology Information, U.S. National Library of Medicine, Basic Local Alignment Search Tool (BLAST).15 RESULTS Using a large number of Old World and New World Leish- mania isolates the M1.1/13A primers were determined to spe- TABLE 1 PCR detection of Leishmania DNA from cultured promastigotes* Species/WHO International Code 13A/13B 13A/M1.2 L. (V.) braziliensis (MHOM/CO/87/1277) + L. (V.) braziliensis (MHOM/CO/88/1407 + L. (V.) braziliensis (MHOM/BR/75/M2903) + L. (V.) guyanensis (MHOM/BR/75/M4147) + L. (V.) guyanensis (MHOM/CO/88/1390) + L. (V.) guyanensis (MHOM/BR/75/M4147) + L. (V.) panamensis (MHOM/CO/86/1166) + L. (V.) panamensis (MHOM/PA/71/LS94) + L. (V.) panamensis (MHOM/CO/84/2122) + L. (L.) mexicana (MHOM/MX/91/390) + + L. (L.) mexicana (MHOM/MX/87/67) + + L. (L.) mexicana (MNEO/US/90/WR972) + + L. (L.) mexicana (MOTO/MX/97/0p5) + + L. (L.) mexicana (MHOM/MX/98/847) + + L. (L.) mexicana (MHOM/MX/94/663) + + L. (L.) mexicana (MHOM/MX/98/840) + + L. (L.) mexicana (MHOM/MX/98/830) + + L. (L.) mexicana (MHOM/MX/94/681) + + L. (L.) mexicana (MHOM/MX/98/838) + + L. (L.) mexicana (MHET/MX/97/Hdl8) + + L. (L.) mexicana (MHOM/MX/94/758) + + L. (L.) mexicana (MPER/MX/97/Py4) + + L. (L.) mexicana (MHOM/MX/97/820) + + L. (L.) mexicana (MHOM/MX/97/823) + + L. (L.) amazonensis (IFLA/BR/67/PH8) + + L. (L.) amazonensis (MHOM/BR/73/M2269) + + L. (L.) major (MHOM/TN/88/TN435) + L. (L.) major (MHOM/IL/80/Friedlin) + L. (L.) major (MHOM/SY/94/Abdou) + L. (L.) donovani (MHOM/SD/00/1S-2D) + Negative control - * DNA equivalent to 20 parasites was used in each PCR reaction. Amplification products were detected by agarose gel electrophoresis and ethidium bromide staining. LEISHMANIA AMAZONENSIS IN BOLIVIAN ORYZOMYS 1071 TABLE 2 Number of individual mammals positive for Leishmania spp./Number screened by PCR using primers 13A/13B12 Locality Species Sept-Oct 2(102 Sept 2003 Sept 2004 Sept 2005 El Refugio Huanchaca Los Fierros Both Akodon dayi Day's Grass Mouse Didelphis marsupialis Southern Opossum Marmosops ocellatus Bolivian Slender Mouse Opossum Mesomys hispidus Spiny Tree Rat Oecomys bicolor Bicolored Arboreal Rice Rat Oligoryzomys microtis Small-eared Pygmy Rice Rat Oryzomys acritus Rio Itenez Rice Rat Oryzomys nitidus Elegant Rice Rat Proechimys longicaudatus Long-tailed Spiny Ray Total Juscelinomys huanchacae Huanchaca Juci Kunsia tomentosus Wooly Giant Rat Necromys lenguarum Bay Mouse Oryzomys acritus Rio Itenez Rice Rat Oryzomys nitidus Elegant Rice Rat Total Total 0/3 0/3 0/1 0/3 0/1 0/3 0/1 0/1 0/1 0/1 0/2 0/1 0/1 0/2 1/1 1/5 1/1 0/3 3/10 0/4 1/2 1/9 2/15 0/5 0/10 0/1 0/1 0/17 1/9 2/17 2/4 1/14 5/54 0/1 0/1 0/3 0/1 0/3 0/1 0/3 0/3 1/2 1/2 1/9 0/1 1/10 2/18 1/28 2/4 1/14 6/64 cifically amplify DNA from members of the L. (L.) amazon- ensis or L. (L.) mexicana (Table 1). Most notably, under the standardized reaction conditions, DNA from members of the Viannia subgenus was not amplified by these primers. Fur- thermore, the Viannia subgenus-specific B4/M13 primers did not amplify DNA from L. (L.) amazonensis or L. (L.) mexi- cana. Ultimately 64 individual mammals, 10 species of rodent and two species of opossum, were screened for Leishmania (Table 2). Three of thirteen O. acritus (Figure 2) and 2 of 17 O. nitidus (Figure 3) 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 Leish- mania was detected in any other mammal species. Both trap- lines where Leishmania positive rodents were collected were in semideciduous tall forest formations. The sequence of the DNA amplified from O. acritus (field no. 444) and O. nitidus (field no. 575) compared with a ref- erence kDNA sequence [(L. (L.) amazonensis, MHOM/BR/ 00/Raimundo ( = MHOM/BR/1983/M1132), NCBI Accession number M21326)] is shown below. The primer (13A and Ml.l) sequences are underlined and a vertical line indicates identity between the cloned and reference sequences. Each of the sequenced kDNA fragments was 102 nucle- otides in length, including the primer sequences. The se- quence 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.) ama- zonensis 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. 4 4 4 GTGGGGGAGGGGCGTTCTGCGGAATCCTCAAAAATGAGTGCAGAAACCCCGTTCATATTTTGGGGAATTTTG iiiiiintii m11IIIii111 III11111miiII11111HIII111inininni 111 n La GTGGGGGAGGGGCGTTCTGCGGAAACCTCAAAAATGAGTGCAGAAACCCCGTTCATATTTTGGGGGATTTTT I I I II I I 1 I I I I I I II I I I I I ! I II I IIIIUI.II llllllll III I I I 11 I I I I I I I II I II 57 5 GTGGGGGAGGGGCGTTCTGCGGGGAAGCCAAAAATGAGTGCAGAAACCCCGTTCATAATTTGGGGGAATTCC 4 4 4 GGGAATTCCGGCTCCGGCGGTCGAAACTGG I I I I I H III II I I I I I llllllll La GGGAATTTCGGTTCGGACGGTGGAAACTGG II I III II I I I I I llllllll 575 TCAAAATCCGGCTCCGGCGGTCGAAACTGG 1072 KERR AND OTHERS FIGURE 2. Three of thirteen Oryzomys acritus collected from Noel Kempff National Park, Bolivia, from 2002-2005, tested positive for Leishmania. DISCUSSION 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 com- parison 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 FIGURE 3. Three of seventeen Oryzomys nitidus collected from Noel Kempff National Park, Bolivia, from 2002-2005, tested positive for Leishmania. kDNA-PCR profiles to that of the human isolates.8 Isoen- zyme profiles of three isolates from the sand fly Lu.(n.) anglesi collected at this focus also indicated L. (L.) amazon- ensis.9 PCR using SSU rRNA was used to compare a large number of L. (L.) amazonensis strains from broadly distrib- uted 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 con- fined 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 dis- tributions of L. (L.) amazonensis and L. (L.) mexicana, we concluded that the rice rats were infected with L. (L.) ama- zonensis. These results identify two additional species of Oryzomys as hosts of L. (L.) amazonensis, and identify Noel Kempff National Park as an ecological region of Bolivia where L. (L.) amazonensis is enzootic. The fact that infections were de- tected 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 geo- graphic 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 Bar- bara Phillips for their inestimable help there. This work was part of Emmons' studies of the biodiversity of Parque Nacional Noel Kempff Mercado (PNNKM), in collaboration with the Museo de Historia Natural Noel Kempff Mercado. The authors thank Fundacion Ami- gos de la Naturaleza for their continuing support of research at Los Fierros, and Damian 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 Associa- tion (LHE), and Center for Disease Control and Prevention Grant H75/CCH615041 (PCM). The American Society of Tropical Medi- cine and Hygiene (ASTMH) assisted with publication expenses. 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 1. Davies CR, Reithinger R, Campbell-Lendrum D, Feliciangeli D, Borges R, Rodriguez N, 2000. The epidemiology and control of LEISHMANIA AMAZONENSIS IN BOLIVIAN ORYZOMYS 1073 leishmaniasis in Andean countries. Cad Saude Publica 16: 925- 950. 2. World Health Organization, 1990. Control of the leishmaniases. Technical Report Series 793. Geneva, Switzerland, 140 p. 3. Lainson R, Shaw JJ, Silveira FT, deSouza AAA, Braga RR, Ish- ikawa EAY, 1994. The dermal leishmaniases of Brazil, with special reference to the eco-epidemiology of the disease in Amazonia. Mem Inst Oswaldo Cruz 89: 435-443. 4. Martinez E, Le Pont F, Torrez M, Telleria J, Vargas F, Mufioz M, DeDoncker S, Dujardin JC, Dujardin JP, 1998. A new focus of cutaneous leishmaniasis due to Leishmania amazonensis in a Sub Andean region of Bolivia. Ada Trop 71: 97-106. 5. Uliana SRB, Ishikawa E, Stemliuk VA, de Souza A, Shaw JJ, Floeter-Winter LM, 2000. Geographical distribution of neo- tropical Leishmania of the subgenus Leishmania analysed by ribosomal oligonucleotide probes. Trans R Soc Trop Med Hyg 94: 261-264. 6. Bermudez H, Torrico F, Rojas E, Balderrama F, Le Ray D, Guerra H, Arevelo J, 1993. Leishmaniasis in the lowlands of Bolivia, prevalence of the disease in two groups of localities with different settlement ages in Carrasco Tropical, Cocha- bamba. Archs Inst Pasteur Tunis 70: 443-453. 7. Wilson DE, Reeder DM, eds., 2005. Mammal Species of the World: A Taxonomic and Geographic Reference. Third edition. Baltimore: Johns Hopkins University Press, 2,142 pp. 8. Telleria J, Bosseno MF, Tarifa T, Buitrago R, Martinez E, Torrez M, Le Pont F, Breniere SF, 1999. Putative reservoirs of Leish- mania amazonensis in a Sub-andean focus of Bolivia identified by kDNA-Polymerase Chain Reaction. Mem Inst Oswaldo Cruz 94: 5-6. 9. Martinez E, LePont F, Torrez M, Telleria J, Vargas F, Du- jardin JC, Dujardin JP, 1999. Lutzomyia nuneztovari anglesi (LesPont & Desjeuz, 1984) as a vector of Leishmania amazon- ensis in a sub-Andean leishmaniasis focus of Bolivia. Am I Trop Med Hyg 61: 846-849. 10. Kerr SF, 2000. Palaearctic origin of Leishmania. Mem Inst Os- waldo Cruz 95: 75-80. 11. Kerr SF, MacKinnon C, Merkelz R, 2000. Further support for a Palaearctic origin of Leishmania. Mem Inst Oswaldo Cruz 95: 579-581. 12. Killeen T, Killeen ST, eds., 1998. A biological assessment of Parque Nacional Noel Kempff Mercado, Bolivia. RAP Work- ing Papers 10. Conservation International, Washington DC. 13. Rogers MR, Popper SJ, Wirth DF, 1990. Amplification of kine- toplast DNA as a tool in the detection and diagnosis of Leish- mania. Exp Parasitol 71: 267-275. 14. Sacks D, Melby P, 1998. Animal models for the analysis of im- mune responses to leishmaniasis. Strober W, ed. Current Pro- tocols in Immunology. New York: John Wiley and Sons, Inc., 19.2.1-19.2.20. 15. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basic local alignment search tool. / Mol Biol 215: 403-410. 16. Emmons LH, Patton JL, 2005. A new species of Oryzomys (Ro- dentia: Muridae) from eastern Bolivia. Am Mus Nov 3878: 1-26. 17. Nery-Guimares FN, Azevedo M, Damasceno R, 1968. Oryzomys goeldi, a wild rat from amazonia, as reservoir of Leishmania braziliensis. Mem Inst Oswaldo Cruz 66: 151-168. 18. Lainson R, Shaw JJ, 1968. Leishmaniasis in Brazil: I. observations on enzootic rodent leishmaniasis-incrimination of Lutzomyia flaviscutellata (Mangabeira) as the vector in the lower Amazon Basin. Trans R Soc Trop Med Hyg 62: 385-395. 19. Lainson R, Shaw JJ, 1970. Leishmaniasis in Brazil: V. Studies on the epidemiology of cutaneous leishmaniasis in Mato Grosso State, and observations on two distinct strains of Leishmania isolated from man and forest animals. Trans R Soc Trop Med Hyg 64: 654-667. 20. Tikasingh ES, 1974. Enzootic rodent leishmaniasis in Trinidad, West Indies. PAHO Bulletin VIII: 232-242. 21. Herrer A, Christensen HA, Beumer RJ, 1973. Reservoir hosts of cutaneous leishmaniasis among Panamanian Forest Mammals. Am J Trop Med Hyg 22: 585-591. 22. Chable-Santos JB, Van Wynsberghe NR, Canto-Lara SB, An- drade-Narvaez FJ, 1995. Isolation of Leishmania (L.) mexicana from wild rodents and their possible role in the transmission of localized cutaneous leishmaniasis in the state of Campeche, Mexico. Am I Trop Med Hyg 53: 141-145. 23. Young DG, Perkins PV, 1984. Phlebotomine sand flies of North America (Diptera: Psychodidae). Mosq News 44: 263-304. 24. Eisenberg JF, 1989. Mammals of the Neotropics. The Northern Neotropics. Volume I. Panama, Colombia, Venezuela, Guyana, Suriname, French Guiana. Chicago: The University of Chicago Press, 449 pp.