RESERVOIRS OF THE EMERGING PATHOGENS BARTONELLA SPP. AND YERSINIA PESTIS IN A. Martín-Alonso1; M. Soto2; P. Foronda1; E. Aguilar2; G. Bonnet1; R. Pacheco2; B. Valladares1; M. A. Quispe-Ricalde1,2. 1University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, La Laguna, Spain. 2Faculty of Biological Sciences, National University of San Antonio Abad of Cusco, Peru.

INTRODUCTION AND PURPOSES METHODS

Bartonella infections in have been reported worldwide and close contacts between humans and A total of 28 rodents were captured during 2010-2011 from three villages (Alto Ivochote, Aguas Calientes and Yomentoni) belonging to populations around the world have been found to create excellent conditions for transmission of Echarate District, La Convención Province (Figure 1). Spleens of the were collected in lysis buffer and DNA was extracted using Bartonella spp. from animals to humans (1). On the other hand, plague is a deadly rodent-associated flea- Illustra tissue and cells genomic Prep Mini Spin kit (GE Healthcare, UK). borne zoonosis caused by the bacterium Yersinia pestis, which has shaped the course of human history, Rodents were examined for the presence of Bartonella DNA by PCR targeting a 767-bp fragment of the citrate synthase gene (gltA) (5), killing millions of people in three major pandemics, and sylvatic plague remains enzootic (endemic) in which encompasses a 327-bp-specific zone of the gltA gene that was used for further phylogenetic analysis. multiple rodent foci around the world (2). On the other hand, the screening for plague was performed by using a PCR targeting the pla gene (6), a plasminogen activator protein In spite of the high number of plague and Bartonella-related human cases reported each decade in Peru (3-5), encoded by the Y. pestis-specific pPLA plasmid. Sequencing reactions were performed for both strands at Macrogen Inc. (Korea). New there are no published data about the identification of rodent reservoirs of these pathogens in this country. and previously published Bartonella and Yersinia pestis sequences were aligned with the multiple alignment program ClustalW in Therefore, the objectives of this study were to determine the possible infection of several rodent species from MEGA5.2 (7), and the neighbor-joining (NJ) method by Kimura´s two-parameter distance method and bootstrap calculation was carried Peru with Bartonella spp. and Y. pestis, evaluate the genetic heterogeneity of the Bartonella strains in this out with 1,000 resamplings. country and estimate the importance of our results from the point of view of the public health. Figure 1. Sampling sites in Echarate District, Peru

Yersinia pestis Bartonella spp. RESULTS Area of study Host species (N) + (P%) + (P%) Prevalence data are summarized in Table. Coinfections with both bacteria were found in three rodents (10.7%). According to Bartonella spp., the prevalence found in H. perenensis was higher than that observed in R. rattus (P‹0.001). Infected rodents were Alto Ivochote Rattus rattus (20) 3 (15) 0 (0) found in the three studied villages, although the prevalence in Aguas Calientes was higher than in Alto Ivochote (P‹0.001). One out of the eight rodents trapped inside the houses and one out of the two captured at peridomestic sites were positive for Bartonella perenensis (1) 0 (0) 1 (100) spp. Aguas Calientes Hylaeamys perenensis (2) 2 (100) 2 (100) Sequence analysis allowed for the identification of three gltA sequences, B259, B273 and B280 (GenBank accession nos. KF021602-KF021604), which exhibited 98% and 99% sequence similarity to the genotypic variant A3 of undescribed Bartonella genogroup A, previously cultured from Oryzomis palustris in the southeastern United States (8). The NJ method (Figure 2) grouped our isolates B259, B273 and B280 together in one cluster with a relatively high support (83% bootstrap support). All the spp. (1) 1 (100) 1 (100) genotypic variants belonging to the genogroup A, including those obtained in our study, were placed in a monophyletic clade, with 100% support for separation of this clade from the B. vinsonii complex. Yometoni Rattus rattus (4) 0 (0) 1 (25) With regard to Y. pestis, the pla amplicons showed 98% sequence identity with Y. pestis reference sequences. Two out of the three H. perenensis specimens and the only Oecomys spp. specimen were coinfected with Y. pestis and Bartonella spp. The prevalence Total (28) 6 (21.4) 5 (17.9) of Y. pestis was higher in H. perenensis than in R. rattus (P‹0.05). According to the areas of study, infected rodents were found in all the studied villages except Yomentoni and the prevalence observed in Aguas Calientes was higher than in Alto Ivochote (P‹0.01). Two out of the eight rodents (25%) trapped inside the houses were infected with Y. pestis. Table. Prevalence of Bartonella spp. and Yersinia pestis in rodents from Echarate District, Peru (N, sample size; P%, prevalence) B273 - A6 83 B280 B259 - A7 84 Bartonella sp. (U84373.1) - A2 DISCUSSION Bartonella sp. (AF082321) - A4 100 Bartonella sp. - (U84374.1) - A3 98 Bartonella sp. (U84372.1)- A1 The results of this study indicate that infections of rodents with Bartonella spp. and Y. pestis are common and widespread throughout the Echarate District. To our knowledge, this study constitutes the first finding of Bartonella Bartonella sp. (AF082322.1) - A5 B. vinsonii subsp. arupensis (AF214557.1) spp. and Y. pestis in H. perenensis and Oecomys spp. Two new Bartonella variants belonging to the genogroup A (A6 and A7) have been found in this work. Considering the phylogeny obtained by NJ method, we propose the 77 B. vinsonii subsp. berkhofii (U28075) B. vinsonii subsp. vinsonii (Z70015) genotype found in isolates B273 and B280 to be considered as the variant A6, whereas the isolate B259 would correspond to the genotypic variant A7. B. alsatica (AF204273) The epidemiological importance of rodent-borne Bartonella spp. as a cause of disease in animals and humans is emerging in America, as suggested for a novel rodent Bartonella-associated febrile illness in the rural B. birtlesii (AF204272) 100 B. queenslandensis (JX158354) southwestern USA (9,10) and the finding of a likely strain of the pathogenic B. elizabethae, an agent of human endocarditis, in Huayllacallán Valley (Peru) (3). Because most identified Bartonella species have been reported as 99 B. tribocorum (AJ005494) human infectious agents elsewhere, our results could be of public health concern. B. grahamii (Z70016) B. elizabethae (JX158352) The detection of new rural zoonotic foci of plague in Peru must be taken into account in order to enlarge the distribution of plague in Peru, which was thought to be limited to the northern part of the country. In conclusion, the B. rattimassiliensis (JX158360) high prevalence observed in our study and the finding of plague and bartonellosis in intradomestic and peridomestic areas where workers remain in close contact with rodents could indicate that the actual incidence of both 99 B. coopersplainsensis (EU111803) 99 B. japonica (AB242289) diseases in humans from Echarate District may be underestimated. B. rattaustraliani (EU111797) B. phoceensis (AY515126) B. silvatica (AB242287) B. taylorii (AF191502) BIBLIOGRAPHY ACKNOWLEDGEMENTS 98 B. washoensis (AF470616) B. washoensis subsp. cynomysii (DQ834440.1) 72 B. quintana (Z70014) 1. Welc-Faleciak R, Paziewska A, Bajer A, Behnke JM, Sinski E. Vector Borne Zoonotic Dis. 2008;8:467-74. doi: 10.1089/vbz.2007.0217. This study was funded by AECID under PCI program (A1/037176/11), 95 75 B. henselae (JQ009406.1) B. koehlerae (AF176091) 2. Gage KL, Kosoy MY. Annu Rev Entomol. 2005;50:505–28. doi: 10.1146/annurev.ento.50.071803.130337. Spanish Ministry of Foreign Affairs and Cooperation. This work was also B. doshiae (AF207827) 3. Birtles RJ, Canales J, Ventosilla P, Alvarez E, Guerra H, Llanos-Cuentas A, et al. Am J Trop Med Hyg. 1999;60:799-805. supported by the project “Red de Investigación Colaborativa de Centros B. bacilliformis (HQ416691) 99 B. clarridgeiae (KCBeijerinckia331017) indica (CP001016.1) 4. 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Iralu J, Bai Y, Crook L, Tempest B, Simpson G, McKenzie T, Koster F. Emerg Inf Dis. 2006;12:1081-6. Figure 2. Phylogenetic relationships of the Bartonella spp. genotypes detected in this study. GenBank accession numbers are indicated between parentheses. The sequences obtained in this study are indicated in bold letters.