Ticks and Tick-borne Diseases 6 (2015) 155–157
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Detection of human pathogenic Ehrlichia muris-like agent in Peromyscus leucopus Caroline G. Castillo a,1 , Marina E. Eremeeva b,2 , Susan M. Paskewitz c , Lynne M. Sloan a , Xia Lee c , William E. Irwin d , Stefan Tonsberg d , Bobbi S. Pritt a,∗ a
Mayo Clinic, Rochester, MN 55905, USA Centers for Disease Control and Prevention, Atlanta, GA, USA c University of Wisconsin, Madison, WI, USA d US Army Public Health Command Region-West, Joint Base Lewis-McChord, WA, USA b
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 11 November 2014 Accepted 18 November 2014 Available online 4 December 2014 Keywords: Ehrlichia muris Ehrlichia muris-like (EML) agent Ehrlichiosis Peromyscus
a b s t r a c t An Ehrlichia muris-like (EML) bacterium was recently detected in humans and Ixodes scapularis ticks in Minnesota and Wisconsin. The reservoir for this agent is unknown. To investigate the occurrence of the EML agent, groEL PCR testing and sequencing was performed on blood from small mammals and whitetailed deer that were collected in areas where human and tick infections were previously demonstrated. DNA of the EML agent was detected in two Peromyscus leucopus of 146 small mammals (1.4%); while 181 O. virginianus tested negative. This report provides the first evidence that DNA from the EML agent is found in P. leucopus, the same animal that is a reservoir for Anaplasma phagocytophilum in this region. The role of white-tailed deer remains inconclusive. Further sampling is warranted to understand the spatial and temporal distribution, transmission and maintenance of this pathogen. © 2014 Elsevier GmbH. All rights reserved.
Introduction Ehrlichioses are emerging tick-borne zoonoses caused by obligate intracellular gram-negative bacteria in the genus Ehrlichia. These organisms infect hematopoietic cells in humans and may cause a febrile illness associated with headache, malaise, muscle aches, dizziness and occasional rash (Chapman et al., 2006). Leukopenia, thrombocytopenia, and elevated liver function tests are common laboratory findings (Ismail et al., 2010). Healthy individuals usually recover without complication when treated with doxycycline; however, immunocompromised individuals may have a more severe illness with poor outcomes including multiorgan system failure and death (Chapman et al., 2006). Ehrlichiosis, most commonly caused by Ehrlichia chaffeensis and E. ewingii in humans, was historically reported primarily from the South, Southeast and mid-Atlantic regions of the US, reflecting the distribution
∗ Corresponding author at: Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA. Tel.: +1 507 538 8128; fax: +1 507 284 4272. E-mail address: [email protected] (B.S. Pritt). 1 Current address: University of Michigan Health System, Ann Arbor, MI, USA. 2 Current address: Georgia Southern University, Statesboro, GA, USA. http://dx.doi.org/10.1016/j.ttbdis.2014.11.006 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
of the tick vector, Amblyomma americanum (Demma et al., 2005; Childs and Paddock, 2003). In 2009, 4 cases of human ehrlichiosis caused by an unnamed Ehrlichia species were diagnosed in Minnesota and Wisconsin (Pritt et al., 2011). DNA from this organism was also detected in at least 17 of 697 Ixodes scapularis ticks collected in Wisconsin and Minnesota (Pritt et al., 2011). Genetically, this Ehrlichia sp. is most closely related to E. muris, which is present in Ixodes ticks in the Asia subcontinent and Japan (Eremeeva et al., 2007; Spitalska et al., 2008; Rar et al., 2005). The taxonomic status of this organism has not yet been determined and it hereto be referred to as the “Ehrlichia murislike” or EML agent. Vector surveillance conducted in proximity to the human cases has detected the EML agent in Ixodes scapularis ticks (tested individually or in pools of five), suggesting a potential role for this tick in the life cycle of this organism (Pritt et al., 2011). The natural animal reservoir of the EML agent is unknown. However, recent work has shown that lethal and persistent infections can be successfully established in laboratory murine models (Saito et al., 2014; Karpathy et al., 2010). Therefore, the purpose of this study was to investigate the occurrence of the EML agent in wild rodents and deer from areas where human and tick infections were previously demonstrated.
156
C.G. Castillo et al. / Ticks and Tick-borne Diseases 6 (2015) 155–157
Table 1 Primers and probes used in the groEL heat-shock protein operon PCR assay used for testing of animal and tick specimens. These represent a slightly modified version of the originally published assay (Bell and Patel, 2005). Name
Sequence (5 to 3 )
EHR1 primer EHR2 primer EHR3 probea EHR3a probea EHR4 probeb
TAC TCA GAG TGC TTC TCA ATG T GCA TAC CAT CAG TTT TTT CAA C ATT TCA GCT AAT GGA GAT AAG AAT ATA CAT TGT CTG CGA ATG GAG ACA AGA ACA TAG GA GTA AGA TTG CAC AGT GTG TTC AAG AAG TCG GTA
a b
Labeled with fluorescein on 3 end. Labeled with LC640 on 5 end and a phosphate on 3 end.
Materials and methods Blood collection Blood was sampled from small mammals in Fall Creek, Eau Claire County, Wisconsin (June 2010) and Camp Ripley, Morrison County, Minnesota (August 2010) following approved Institutional Animal Care and Use Committee protocols from the University of Wisconsin and the U.S. Army. Small mammals were trapped using Sherman traps (Tallahassee, FL) baited with bread and peanut butter, anesthetized using 5% isoflurane, and blood was obtained using retroorbital sampling. Blood (20–60 L) was spotted onto WhatmanTM FTATM DNA filter cards (GE Healthcare Life Sciences, Pittsburg, PA) and stored at room temperature prior to DNA extraction. The Whatman FTA technology allows for cells lysis and protein denaturation upon application of the blood to the card, allowing DNA to remain intact in the card fiber matrix for long term storage at room temperature (GE Healthcare Life Sciences, 2014). After the sample was obtained, small mammals were released at the site of capture. Blood was also collected onto Whatman FTA cards from available wounds on white-tailed deer (Odocoileus virginianus) killed during Minnesota archery season (September 14–December 31, 2010) at Camp Ripley, MN. DNA processing and extraction Two spots containing approximately 10–20 L of blood each were obtained from each filter card using a standard ¼-inch singlehole punch and washed in sterile water. The sample was then eluted into 500 L of sterile water by heating at 95 ◦ C for 30 min followed by vigorously vortexing. The DNA was extracted from 200 L of each sample using the Total DNA kit on the MagNA Pure LC 2.0® System (Roche Applied Science® , Indianapolis, IN, Total DNA kit, 100 L final volume). Extracted DNA was stored at 4 ◦ C prior to the testing. The hole punch was cleaned between filter cards with 10% bleach followed by 70% ethanol. DNA amplification Extracted DNA from each blood spot was individually tested for the EML agent, E. chaffeensis, E. ewingii and Anaplasma phagocytophilum using a multiplex PCR assay targeting a conserved region of the groEL heat-shock protein operon (Bell and Patel, 2005). Modifications were made to the mastermix components and thermocycling profile to standardize with other clinical assays in the diagnostic laboratory, with minor changes to the FRET probe sequences (Table 1) to produce well-defined melting peaks. The reaction mix was prepared using the LC FastStart® DNA Master Hybridization Probes Kit (Roche Applied Science) with the following final concentrations of reagents: 3 mM MgCl2 , 0.5 M each of primers ESP-F and ESP-R, 0.2 mM each of probes APHPF and ECHPF, and 0.4 mM of probe ESPHPR. Amplification parameters included 40 ◦ C × 10 min at 20 ◦ C/s, denaturation at
Fig. 1. Genetic relationships of Ehrlichia sp. Wisconsin and selected members of the Family anaplasmataceae. Sequence comparison was conducted with MEGA version 5 (www.megasoftware.net). The phylogenetic optimal tree was inferred by using the Neighbor-Joining method, and distances were evaluated by implementing the Kimura 2-parameter model of substitution (sum of branch length = 0.83048643). In total, 315 nt sites GroEL gene fragment were evaluated; primer sequences and sites containing gaps and deletions were excluded from the analysis. Statistical reliability of the tree is based on 1000 bootstrap replicates; only bootstrap values >90 are shown above the branches. The corresponding sequences of reference species and isolates were obtained from the National Center for Biotechnology Information GenBank database; their accession numbers are shown next to the sequence names. Corresponding groEL sequences from human blood (Wisconsin, HM543746) and rodent blood (Hull54, KF986479) are identical and shown on the same branch.
95 ◦ C × 10 min followed by 45 cycles of: 95 ◦ C × 10 s at 20 ◦ C/s slope, 55 ◦ C × 15 s at 20 ◦ C/s slope, and 72 ◦ C × 15 s at 20 ◦ C/s slope. Melting curve analysis was performed as follows: 95 ◦ C × 0 s at 20 ◦ C/s slope, 40 ◦ C × 60 s at 20 ◦ C/s slope, and 85 ◦ C × 0 s at 0.2 ◦ C/s slope with continuous fluorescence acquisition. Amplicons with a melting temperature consistent with Ehrlichia sp. Wisconsin (51.5–53.5 ◦ C) were sequenced using a 3730 DNA Analyzer (Applied Biosystems® , Life TechnologiesTM , Grand Island, NY) and analyzed using Sequencher® DNA sequence analysis software, version 4.2 (Gene Codes Corporation, Ann Arbor, MI). Further confirmation was performed upon detection and sequencing of the variable fragment of Anaplasmataceae 16S ribosomal RNA gene as described previously (Eremeeva et al., 2007). Results Blood specimens were obtained and tested by PCR and sequencing from 59 Peromyscus leucopus, one P. maniculatus, one Microtus pennsylvanicus, and one unidentified rodent at the Wisconsin location, from which EML agent DNA was detected in one P. leucopus sample (1.6% of small mammals tested). Specimens were also obtained and tested from 80 P. leucopus, one P. maniculatus, one Myodes gapperi, one Blarina brevicauda and one Tamias striatus at the Minnesota location. DNA of one P. leucopus blood sample (1.2% of small mammals tested) was positive for the EML agent. Ehrlichia sp. sequences detected in rodent blood (NCBI accession #KF986479) were identical to homologous sequences of 16S rRNA gene previously detected in humans (NCBI accession #HM543746) and ticks from the same or adjacent areas (Fig. 1). A. phagocytophilum was also detected in blood from five P. leucopus and one T. striatus in the Minnesota rodents; no co-infections with A. phagocytophilum and the EML agent were detected. Ehrlichia muris-like agent DNA was not detected in 181 O. virginianus blood specimens. Conclusions The EML agent is a newly detected human pathogen that appears to be geographically restricted to Wisconsin and Minnesota based on human testing performed to date (Pritt et al., 2011). Prior to the initial description of the EML agent, ehrlichioses
C.G. Castillo et al. / Ticks and Tick-borne Diseases 6 (2015) 155–157
were recognized as human diseases mostly limited to the Southeast and central regions of the US, transmitted solely by the A. americanum. This study confirms the presence of the EML agent DNA in P. leucopus mice in Minnesota and Wisconsin, suggesting a potential role for this rodent in the natural life cycle of this organism. It is likely that the prevalence and distribution of the EML agent is larger and broader than reported here since only a small number of rodents and white-tailed deer were tested. In addition, it is unknown if detection of the EML agent in the blood of P. leucopus rodents represents productive infection or transient introduction by feeding nymphs. To better assess the spatial and temporal distribution of this pathogen in small mammals and deer to further understand the transmission cycle and maintenance of this pathogen, we recommend additional testing using PCR, we recommend additional testing using PCR, organism-specific serologic studies, culture and/or passage in animals. Testing whole blood or the buffy coat fraction of blood rather than samples collected on FTA paper may increase the sensitivity of detection. In summary, we provide the first evidence for EML agent DNA in two wild P. leucopus from Wisconsin and Minnesota, areas in which ehrlichiosis was previously thought to be rarely found. Health Care providers in these states should consider a diagnosis of ehrlichiosis in the appropriate clinical context and seek confirmatory PCR testing when available. Continued monitoring for the EML agent among geographically diverse collections of ticks, rodents and humans in the United States will help to further characterize the epidemiology and ecology of this pathogen. Acknowledgements The authors would like to thank Members of the Camp Ripley Environmental Office and the Central Lakes College (Natural Resources Club) for their assistance in collecting the deer blood samples at Camp Ripley, MN, and Ellison Stead for her laboratory assistance in mouse species identification.
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References Bell, C.A., Patel, R., 2005. A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Diagn. Microbiol. Infect. Dis. 53, 301–306. Chapman, A.S., Bakken, J.S., Folk, S.M., Paddock, C.D., Bloch, K.C., Krusell, A., Sexton, D.J., Buckingham, S.C., Marshall, G.S., Storch, G.A., Dasch, G.A., McQuiston, J.H., Swerdlow, D.L., Dumler, S.J., Nicholson, W.L., Walker, D.H., Eremeeva, M.E., Ohl, C.A., 2006. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis—United States: a practical guide for physicians and other health-care and public health professionals. MMWR 55, 1–27. Childs, J.E., Paddock, C.D., 2003. The ascendancy of Amblyomma americanum as a vector of pathogens affecting humans in the United States. Annu. Rev. Entomol. 48, 307–337. Demma, L.J., Holman, R.C., McQuiston, J.H., Krebs, J.W., Swerdlow, D.L., 2005. Epidemiology of human ehrlichiosis and anaplasmosis in the United States, 2001–2002. Am. J. Trop. Med. Hyg. 73, 400–409. Eremeeva, M.E., Oliveira, A., Moriarity, J., Robinson, J.B., Tokarevich, N.K., Antyukova, L.P., Pyanyh, V.A., Emeljanova, O.N., Ignatjeva, V.N., Buzinov, R., Pyankova, V., Dasch, G.A., 2007. Detection and identification of bacterial agents in Ixodes persulcatus Schulze ticks from the north western region of Russia. Vector Borne Zoonotic Dis. 7, 426–436. GE Application Note, Whatman FTA® DNA Archiving. Analyze Blood and Buccal DNA after Years of Room Temperature Storage. http://www.whatman.com.cn/ upload/starjj 2009414132332.pdf (accessed 10.11.14). Ismail, N., Bloch, K.C., McBride, J.W., 2010. Human ehrlichiosis and anaplasmosis. Clin. Lab. Med. 30, 261–292. Karpathy, S.E., Levin, M.L., Munderloh, U.G., Goldsmith, C., Nelson, C.M., Pritt, B.S., Dasch, G.A., 2010. Mouse model and morphology of an Ehrlichia muris-like human pathogen. In: Poster: 24th Meeting of the American Society for Rickettsiology, Stevenson, WA. Pritt, B.S., Sloan, L.M., Johnson, D.K.H., Munderloh, U.G., Paskewitz, S.M., McElroy, K.M., McFadden, J.D., Binnicker, M.J., Neitzel, D.F., Liu, G., Nicholson, W.L., Nelson, C.M., Franson, J.J., Martin, S.A., Cunningham, S.A., Steward, C.R., Bogumill, K., Bjorgaard, M.E., Davis, J.P., McQuiston, J.H., Warshauer, D.M., Wilhelm, M.P., Patel, R., Trivedi, V.Z., Eremeeva, M.E., 2011. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N. Engl. J. Med. 365, 422–429. Rar, V.A., Fomenko, N.V., Dobrotvorsky, A.K., Livanova, N.N., Rudakova, S.A., Fedorov, E.G., Astanin, V.B., Morozova, O.V., 2005. Tickborne pathogen detection, Western Siberia, Russia. Emerg. Infect. Dis. 11, 1708–1715. Saito, T.B., Thirumalapura, N., Shelite, T.R., Rockx-Brouwer, D., Popov, V.L., Walker, D.H., 2014. An animal model of a newly emerging human ehrlichiosis. J. Infect. Dis. [Epub ahead of print]. Spitalska, E., Boldis, V., Kostanova, Z., Kocianova, E., Stefanidesova, K., 2008. Incidence of various tick-borne microorganisms in rodents and ticks of central Slovakia. Acta Virol. 52, 175–179.
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Detection of human pathogenic Ehrlichia muris-like agent in Peromyscus leucopus Caroline G. Castillo a,1 , Marina E. Eremeeva b,2 , Susan M. Paskewitz c , Lynne M. Sloan a , Xia Lee c , William E. Irwin d , Stefan Tonsberg d , Bobbi S. Pritt a,∗ a
Mayo Clinic, Rochester, MN 55905, USA Centers for Disease Control and Prevention, Atlanta, GA, USA c University of Wisconsin, Madison, WI, USA d US Army Public Health Command Region-West, Joint Base Lewis-McChord, WA, USA b
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 11 November 2014 Accepted 18 November 2014 Available online 4 December 2014 Keywords: Ehrlichia muris Ehrlichia muris-like (EML) agent Ehrlichiosis Peromyscus
a b s t r a c t An Ehrlichia muris-like (EML) bacterium was recently detected in humans and Ixodes scapularis ticks in Minnesota and Wisconsin. The reservoir for this agent is unknown. To investigate the occurrence of the EML agent, groEL PCR testing and sequencing was performed on blood from small mammals and whitetailed deer that were collected in areas where human and tick infections were previously demonstrated. DNA of the EML agent was detected in two Peromyscus leucopus of 146 small mammals (1.4%); while 181 O. virginianus tested negative. This report provides the first evidence that DNA from the EML agent is found in P. leucopus, the same animal that is a reservoir for Anaplasma phagocytophilum in this region. The role of white-tailed deer remains inconclusive. Further sampling is warranted to understand the spatial and temporal distribution, transmission and maintenance of this pathogen. © 2014 Elsevier GmbH. All rights reserved.
Introduction Ehrlichioses are emerging tick-borne zoonoses caused by obligate intracellular gram-negative bacteria in the genus Ehrlichia. These organisms infect hematopoietic cells in humans and may cause a febrile illness associated with headache, malaise, muscle aches, dizziness and occasional rash (Chapman et al., 2006). Leukopenia, thrombocytopenia, and elevated liver function tests are common laboratory findings (Ismail et al., 2010). Healthy individuals usually recover without complication when treated with doxycycline; however, immunocompromised individuals may have a more severe illness with poor outcomes including multiorgan system failure and death (Chapman et al., 2006). Ehrlichiosis, most commonly caused by Ehrlichia chaffeensis and E. ewingii in humans, was historically reported primarily from the South, Southeast and mid-Atlantic regions of the US, reflecting the distribution
∗ Corresponding author at: Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA. Tel.: +1 507 538 8128; fax: +1 507 284 4272. E-mail address: [email protected] (B.S. Pritt). 1 Current address: University of Michigan Health System, Ann Arbor, MI, USA. 2 Current address: Georgia Southern University, Statesboro, GA, USA. http://dx.doi.org/10.1016/j.ttbdis.2014.11.006 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
of the tick vector, Amblyomma americanum (Demma et al., 2005; Childs and Paddock, 2003). In 2009, 4 cases of human ehrlichiosis caused by an unnamed Ehrlichia species were diagnosed in Minnesota and Wisconsin (Pritt et al., 2011). DNA from this organism was also detected in at least 17 of 697 Ixodes scapularis ticks collected in Wisconsin and Minnesota (Pritt et al., 2011). Genetically, this Ehrlichia sp. is most closely related to E. muris, which is present in Ixodes ticks in the Asia subcontinent and Japan (Eremeeva et al., 2007; Spitalska et al., 2008; Rar et al., 2005). The taxonomic status of this organism has not yet been determined and it hereto be referred to as the “Ehrlichia murislike” or EML agent. Vector surveillance conducted in proximity to the human cases has detected the EML agent in Ixodes scapularis ticks (tested individually or in pools of five), suggesting a potential role for this tick in the life cycle of this organism (Pritt et al., 2011). The natural animal reservoir of the EML agent is unknown. However, recent work has shown that lethal and persistent infections can be successfully established in laboratory murine models (Saito et al., 2014; Karpathy et al., 2010). Therefore, the purpose of this study was to investigate the occurrence of the EML agent in wild rodents and deer from areas where human and tick infections were previously demonstrated.
156
C.G. Castillo et al. / Ticks and Tick-borne Diseases 6 (2015) 155–157
Table 1 Primers and probes used in the groEL heat-shock protein operon PCR assay used for testing of animal and tick specimens. These represent a slightly modified version of the originally published assay (Bell and Patel, 2005). Name
Sequence (5 to 3 )
EHR1 primer EHR2 primer EHR3 probea EHR3a probea EHR4 probeb
TAC TCA GAG TGC TTC TCA ATG T GCA TAC CAT CAG TTT TTT CAA C ATT TCA GCT AAT GGA GAT AAG AAT ATA CAT TGT CTG CGA ATG GAG ACA AGA ACA TAG GA GTA AGA TTG CAC AGT GTG TTC AAG AAG TCG GTA
a b
Labeled with fluorescein on 3 end. Labeled with LC640 on 5 end and a phosphate on 3 end.
Materials and methods Blood collection Blood was sampled from small mammals in Fall Creek, Eau Claire County, Wisconsin (June 2010) and Camp Ripley, Morrison County, Minnesota (August 2010) following approved Institutional Animal Care and Use Committee protocols from the University of Wisconsin and the U.S. Army. Small mammals were trapped using Sherman traps (Tallahassee, FL) baited with bread and peanut butter, anesthetized using 5% isoflurane, and blood was obtained using retroorbital sampling. Blood (20–60 L) was spotted onto WhatmanTM FTATM DNA filter cards (GE Healthcare Life Sciences, Pittsburg, PA) and stored at room temperature prior to DNA extraction. The Whatman FTA technology allows for cells lysis and protein denaturation upon application of the blood to the card, allowing DNA to remain intact in the card fiber matrix for long term storage at room temperature (GE Healthcare Life Sciences, 2014). After the sample was obtained, small mammals were released at the site of capture. Blood was also collected onto Whatman FTA cards from available wounds on white-tailed deer (Odocoileus virginianus) killed during Minnesota archery season (September 14–December 31, 2010) at Camp Ripley, MN. DNA processing and extraction Two spots containing approximately 10–20 L of blood each were obtained from each filter card using a standard ¼-inch singlehole punch and washed in sterile water. The sample was then eluted into 500 L of sterile water by heating at 95 ◦ C for 30 min followed by vigorously vortexing. The DNA was extracted from 200 L of each sample using the Total DNA kit on the MagNA Pure LC 2.0® System (Roche Applied Science® , Indianapolis, IN, Total DNA kit, 100 L final volume). Extracted DNA was stored at 4 ◦ C prior to the testing. The hole punch was cleaned between filter cards with 10% bleach followed by 70% ethanol. DNA amplification Extracted DNA from each blood spot was individually tested for the EML agent, E. chaffeensis, E. ewingii and Anaplasma phagocytophilum using a multiplex PCR assay targeting a conserved region of the groEL heat-shock protein operon (Bell and Patel, 2005). Modifications were made to the mastermix components and thermocycling profile to standardize with other clinical assays in the diagnostic laboratory, with minor changes to the FRET probe sequences (Table 1) to produce well-defined melting peaks. The reaction mix was prepared using the LC FastStart® DNA Master Hybridization Probes Kit (Roche Applied Science) with the following final concentrations of reagents: 3 mM MgCl2 , 0.5 M each of primers ESP-F and ESP-R, 0.2 mM each of probes APHPF and ECHPF, and 0.4 mM of probe ESPHPR. Amplification parameters included 40 ◦ C × 10 min at 20 ◦ C/s, denaturation at
Fig. 1. Genetic relationships of Ehrlichia sp. Wisconsin and selected members of the Family anaplasmataceae. Sequence comparison was conducted with MEGA version 5 (www.megasoftware.net). The phylogenetic optimal tree was inferred by using the Neighbor-Joining method, and distances were evaluated by implementing the Kimura 2-parameter model of substitution (sum of branch length = 0.83048643). In total, 315 nt sites GroEL gene fragment were evaluated; primer sequences and sites containing gaps and deletions were excluded from the analysis. Statistical reliability of the tree is based on 1000 bootstrap replicates; only bootstrap values >90 are shown above the branches. The corresponding sequences of reference species and isolates were obtained from the National Center for Biotechnology Information GenBank database; their accession numbers are shown next to the sequence names. Corresponding groEL sequences from human blood (Wisconsin, HM543746) and rodent blood (Hull54, KF986479) are identical and shown on the same branch.
95 ◦ C × 10 min followed by 45 cycles of: 95 ◦ C × 10 s at 20 ◦ C/s slope, 55 ◦ C × 15 s at 20 ◦ C/s slope, and 72 ◦ C × 15 s at 20 ◦ C/s slope. Melting curve analysis was performed as follows: 95 ◦ C × 0 s at 20 ◦ C/s slope, 40 ◦ C × 60 s at 20 ◦ C/s slope, and 85 ◦ C × 0 s at 0.2 ◦ C/s slope with continuous fluorescence acquisition. Amplicons with a melting temperature consistent with Ehrlichia sp. Wisconsin (51.5–53.5 ◦ C) were sequenced using a 3730 DNA Analyzer (Applied Biosystems® , Life TechnologiesTM , Grand Island, NY) and analyzed using Sequencher® DNA sequence analysis software, version 4.2 (Gene Codes Corporation, Ann Arbor, MI). Further confirmation was performed upon detection and sequencing of the variable fragment of Anaplasmataceae 16S ribosomal RNA gene as described previously (Eremeeva et al., 2007). Results Blood specimens were obtained and tested by PCR and sequencing from 59 Peromyscus leucopus, one P. maniculatus, one Microtus pennsylvanicus, and one unidentified rodent at the Wisconsin location, from which EML agent DNA was detected in one P. leucopus sample (1.6% of small mammals tested). Specimens were also obtained and tested from 80 P. leucopus, one P. maniculatus, one Myodes gapperi, one Blarina brevicauda and one Tamias striatus at the Minnesota location. DNA of one P. leucopus blood sample (1.2% of small mammals tested) was positive for the EML agent. Ehrlichia sp. sequences detected in rodent blood (NCBI accession #KF986479) were identical to homologous sequences of 16S rRNA gene previously detected in humans (NCBI accession #HM543746) and ticks from the same or adjacent areas (Fig. 1). A. phagocytophilum was also detected in blood from five P. leucopus and one T. striatus in the Minnesota rodents; no co-infections with A. phagocytophilum and the EML agent were detected. Ehrlichia muris-like agent DNA was not detected in 181 O. virginianus blood specimens. Conclusions The EML agent is a newly detected human pathogen that appears to be geographically restricted to Wisconsin and Minnesota based on human testing performed to date (Pritt et al., 2011). Prior to the initial description of the EML agent, ehrlichioses
C.G. Castillo et al. / Ticks and Tick-borne Diseases 6 (2015) 155–157
were recognized as human diseases mostly limited to the Southeast and central regions of the US, transmitted solely by the A. americanum. This study confirms the presence of the EML agent DNA in P. leucopus mice in Minnesota and Wisconsin, suggesting a potential role for this rodent in the natural life cycle of this organism. It is likely that the prevalence and distribution of the EML agent is larger and broader than reported here since only a small number of rodents and white-tailed deer were tested. In addition, it is unknown if detection of the EML agent in the blood of P. leucopus rodents represents productive infection or transient introduction by feeding nymphs. To better assess the spatial and temporal distribution of this pathogen in small mammals and deer to further understand the transmission cycle and maintenance of this pathogen, we recommend additional testing using PCR, we recommend additional testing using PCR, organism-specific serologic studies, culture and/or passage in animals. Testing whole blood or the buffy coat fraction of blood rather than samples collected on FTA paper may increase the sensitivity of detection. In summary, we provide the first evidence for EML agent DNA in two wild P. leucopus from Wisconsin and Minnesota, areas in which ehrlichiosis was previously thought to be rarely found. Health Care providers in these states should consider a diagnosis of ehrlichiosis in the appropriate clinical context and seek confirmatory PCR testing when available. Continued monitoring for the EML agent among geographically diverse collections of ticks, rodents and humans in the United States will help to further characterize the epidemiology and ecology of this pathogen. Acknowledgements The authors would like to thank Members of the Camp Ripley Environmental Office and the Central Lakes College (Natural Resources Club) for their assistance in collecting the deer blood samples at Camp Ripley, MN, and Ellison Stead for her laboratory assistance in mouse species identification.
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