captured near Merida, Yucatan, Mexico, led to the isolation in 3–4 days of small ... All Bartonella-positive rodents were Yucatán deer mice from San José Pituch.
VECTOR-BORNE AND ZOONOTIC DISEASES Volume XX, Number XX, 2016 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2016.1981
ORIGINAL RESEARCH
Identification of Bartonella Species Isolated from Rodents from Yucatan, Mexico, and Isolation of Bartonella vinsonii subsp. yucatanensis subsp. nov. Frederique B. Schulte Fischedick,1,2 Matthew J. Stuckey,1 Alvaro Aguilar-Setie´n,3 Hayde Moreno-Sandoval,3 Guillermo Galvez-Romero,3 Mo´nica Salas-Rojas,3 Nidia Arechiga-Ceballos,4 Paul A. M. Overgaauw,2 Rickie W. Kasten,1 and Bruno B. Chomel1
Abstract
Bartonella species are highly endemic among wild rodents in many parts of the world. Blood and/or blood clot cultures from 38 rodents, including 27 Yucatan deer mouse (Peromyscus yucatanicus), 7 Gaumer’s spiny pocket mouse (Heteromys gaumeri), 2 black rats (Rattus rattus) and 2 big-eared climbing rats (Ototylomys phyllotis) captured near Merida, Yucatan, Mexico, led to the isolation in 3–4 days of small gram-negative bacilli, which were identified as Bartonella spp. based on colony morphology. DNA extraction and PCR testing were also performed from heart samples of 35 of these 38 rodents. Overall, Bartonella spp. were isolated from the blood/blood clots of 22 (58%) rodents. All Bartonella-positive rodents were Yucata´n deer mice from San Jose´ Pituch. Sequencing of a fragment of the gltA gene showed that all but one rodent isolates were closest to B. vinsonii subsp. vinsonii and one isolate was intermediate between B. vinsonii subsp. berkhoffii and B. vinsonii subsp. arupensis. Further analysis of concatenated housekeeping genes (gltA, ftsZ, rpoB, and 16S rRNA) suggests that this outlier isolate is a new subspecies within the B. vinsonii genogroup, for which we proposed the name B. vinsonii subsp. yucatanensis. Keywords: Bartonella vinsonii subsp. arupensis—Bartonella vinsonii subsp. vinsonii—Mexico—Peromyscus yucatanicus—Yucatan
prolonged fever to endocarditis, such as Bartonella elizabethae, Bartonella grahamii, Bartonella washoensis, Bartonella vinsonii subsp. arupensis, or Candidatus Bartonella volans (Daly et al. 1993, Kerkhoff et al. 1999, Welch et al. 1999, Kosoy et al. 2003, Fenollar et al. 2005, Breitschwerdt et al. 2009, Bai et al. 2012). Little is known on the distribution of Bartonella species in wild rodents in Mexico as only a limited number of studies were conducted in that country, mainly in northeastern Mexico (Bai et al. 2009, Rubio et al. 2014, Ferna´ndezGonza´lez et al. 2015), and no investigation of the presence of Bartonella in mammals has been conducted yet in the specific ecosystem of Yucatan. The objective of the present study was to evaluate the prevalence of Bartonella spp. in small rodents captured near Merida, Yucatan, and determine which Bartonella species were infecting these mammals.
Introduction
B
artonella are emerging pathogens of increasing human and veterinary medical importance. Members of this genus are small, curved, gram-negative, vector-borne rod-shaped bacteria that parasitize erythrocytes of their hosts (Chomel and Kasten 2010). Bartonella are isolated from a wide range of mammalian species, including humans. New species, many of them not yet fully described, have been isolated from a wide range of rodents (Kosoy et al. 1997, Ellis et al. 1999). A common feature of most of these bacteria is transmission by arthropod vectors, especially fleas, as shown for cats and various rodent species (Chomel et al. 1996, Bown et al. 2004, Gutie´rrez et al. 2015). Several rodent-borne Bartonella species are zoonotic, causing a wide range of clinical manifestations from 1
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California. Division of Veterinary Public Health, School of Veterinary Medicine, Institute for Risk Assessment Sciences, University of Utrecht, Utrecht, the Netherlands. 3 Unidad de Investigacio´n Me´dica en Inmunologı´a, Hospital de Pediatrı´a, Centro Me´dico Nacional Siglo XXI, IMSS, Mexico City, Mexico. 4 Instituto de Diagno´stico y Referencia Epidemiolo´gicos, Mexico City, Mexico. 2
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SCHULTE FISCHEDICK ET AL.
Materials and Methods Study area and sampling procedures
The rodents were captured at three different sites, *100– 150 km east and southeast of the city of Merida, Yucatan, in early December 2013 (Table 1). Locations were specifically Peto (20 7¢ 33† North, 88 55¢ 22† West); Bokoba´ (21 0¢ 27† North, 89 10¢ 49† West); and San Jose´ Pituch (20 56¢ 59† North, -88 40¢ 29† West). The vegetation is characteristic of tropical savannah. Small rodents were trapped with Sherman traps (7.6 · 8.9 · 22.9 cm; H.B. Sherman Traps, Inc., Tallahasse, FL) baited with a mix of oat and peanut butter and placed every 10 meters on each side of a transect. The traps were placed at dusk and checked and emptied at dawn. The live trapped rodents were transported to a laboratory in Merida where they were anesthetized before euthanasia. Rodents were identified to species based on external morphology. Rodents were weighed, measured, and species, sex, and age (adult, juvenile) were recorded. Blood samples were collected by intracardiac puncture before euthanasia and disposed in 2.0-mL plastic EDTA tubes (Becton Dickinson, Forest Lakes, NJ). Blood clots were also collected after euthanasia. Unfortunately, the presence of ectoparasites was not consistently recorded. Blood samples were aliquoted for the present study and for an investigation on the presence of hantaviruses performed by the Pasteur Institute, Paris, France (Noe¨l Tordo, personal communication). Blood and blood clot samples were stored in a -80C freezer in one of the investigators’ laboratory in Mexico City, D.F., Mexico, until shipment to California. Ethics statement
This study and sampling protocol were approved by the Institutional Animal Care and Use Committee at the School of Veterinary Medicine of the University of California, Davis (approval no. 18367), and permit from SEMARNAT Ministry of Environment and Natural Resources (Secretarı´a del Medio Ambiente y Recursos Naturales) permiso No-SGPA/DGVS/ 08283/12. Scientific collection permits were issued by the Mexico Ministry of Agriculture, Livestock, Rural Development, Fisheries, and Food (SAGARPA License FAUT 0250) and the Veterinary and Zootechnical School at the National University of Mexico (UNAM Permit B00.02.02/1026 1708). Culture and isolation of Bartonella
Whole blood as well as blood clot samples and hearts available from the previous study were stored in a -80C freezer at the University of California, Davis, until thawed and plated. For each rodent, both whole blood and blood clots were cultured when available. For culture of rodent blood, 200 lL of thawed EDTA blood and M199 growth medium
(1:1) was plated onto fresh (
