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Therya

On-line version ISSN 2007-3364

Therya vol.10 n.2 La Paz May./Aug. 2019

https://doi.org/10.12933/therya-19-722 

Special contributions

Detection of Bartonella and Rickettsia in small mammals and their ectoparasites in México

Sokani Sánchez-Montes1 

Martín Yair Cabrera-Garrido2 

César A. Ríos-Muñoz1  3 

Ali Zeltzin Lira-Olguín2 

Roxana Acosta-Gutiérrez2 

Mario Mata-Galindo1 

Kevin Hernández-Vilchis1 

D. Melissa Navarrete-Sotelo1 

Pablo Colunga-Salas1  2 

Livia León-Paniagua2 

Ingeborg Becker1  * 

1 Centro de Medicina Tropical, Unidad de Medicina Experimental, Facultad de Medicina. Universidad Nacional Autónoma de México. Dr. Balmis 148, CP. 06726, Ciudad de México. México. Email: sok10108@gmail.com (SSM), rmunoz98@gmail.com (CARM), mata316@ciencias.unam.mx (MMG), chopa95@ciencias.unam.mx (KHV), dmel1231@gmail.com (MNS), colungasalas@gmail.com (PCS), becker@servidor.unam.mx (IB).

2 Museo de Zoología “Alfonso L. Herrera”, Departamento de Biología Evolutiva, Facultad de Ciencias, Universidad Nacional Autónoma de México. Avenida Universidad 3000, CP. 04510, Ciudad de México México. Email: bmw_mark@comunidad.unam.mx (MYCG), alizeltzin@ciencias.unam.mx (AZLO), roxana_a2003@yahoo.com.mx (RAG), colungasalas@gmail.com (PCS), llp@ciencias.unam.mx (LLP).

3 Laboratorio de Arqueozoología, Subdirección de Laboratorios y Apoyo Académico, Instituto Nacional de Antropología e Historia. Moneda 16, CP. 06060, Ciudad de México. México. Email: rmunoz98@gmail.com (CARM).


Abstract:

Fleas and sucking lice are important vectors of multiple pathogens causing major epidemics worldwide. However these insects are vectors of a wide range of largely understudied and unattended pathogens, especially several species of bacteria’s of the genera Bartonella and Rickettsia. For this reason the aim of the present work was to identify the presence and diversity of Bartonella and Rickettsia species in endemic murine typhus foci in Hidalgo, México. A cross-sectional study was carried out to collect small mammals and their associated ectoparasites during October, 2014. Samples of liver and ear of hosts, and ectoparasites were fixed in absolute ethanol and examined to identify the presence of Bartonella and Rickettsia DNA by the amplification of specific fragments of the gltA and ompB genes using conventional PCR. The recovered sequences were compared with those deposited in GenBank, and phylogenetic analyzes were carried out to identify the position of the pathogens detected with respect to the valid species previously reported worldwide. A total of 47 fleas and 172 sucking lice, belonging to five families (Ceratophyllidae, Leptopsyllidae, Ctenophtalmidae, Hoplopleuridae, Polyplacidae) and related to six species were collected from 40 rodents of four species and one shrew. Only four hosts (two P. beatae, and two R. norvergicus) were positive to Bartonella elizabethae, Bartonella vinsonii and Rickettsia typhi. In the case of ectoparasites, 23 specimens of two flea species (Peromyscopsylla hesperomys and Plusaetis mathesoni) tested positive for B. vinsonii. No evidence of Bartonella or Rickettsia was detected in any lice. Our findings represent the first record of Bartonella elizabethae a confirmed zoonotic pathogen causing endocarditis in México and several new associations of Bartonella with Mexican flea species, which highlight the importance of the establishment of active entomological surveillance in wildlife.

Keywords: Bartonella elizabethae; emerging diseases; Rickettsia typhi; small mammals; vectors

Resumen:

Las pulgas y los piojos son vectores de patógenos causantes de epidemias de importancia histórica. Sin embargo, estos insectos son vectores de una amplia gama de patógenos poco estudiados y no atendidos, especialmente varias especies de bacterias de los géneros Bartonella y Rickettsia. Por este motivo, el objetivo del presente trabajo fue identificar la presencia y diversidad de las especies de Bartonella y Rickettsia en un foco de tifus murino en el estado de Hidalgo, México. Se realizó un estudio transversal para recolectar hospederos y sus ectoparásitos durante octubre de 2014. Las muestras de hígado y oreja de los hospederos y los ectoparásitos se fijaron en etanol absoluto y se examinaron para identificar la presencia de ADN de Bartonella y Rickettsia mediante la extracción de DNA y amplificación de fragmentos específicos de los genes gltA y ompB. Las secuencias obtenidas fueron comparadas con aquellas depositadas en GenBank y se realizaron análisis filogenéticos para identificar la posición de los patógenos detectados respecto a las especies válidas previamente reportadas a nivel mundial. Se recolectaron un total de 47 pulgas y 172 piojos chupadores, pertenecientes a seis especies de cinco familias (Ceratophyllidae, Leptopsyllidae, Ctenophtalmidae, Hoplopleuridae, Polyplacidae) asociados con 40 roedores de cuatro especies y una musaraña. Sólo cuatro hospederos (dos P. beatae, y dos R. norvergicus) resultaron positivos para Bartonella elizabethae, Bartonella vinsonii y Rickettsia typhi. En el caso de los ectoparásitos, 23 ejemplares de dos especies de pulgas (Peromyscopsylla hesperomys y Plusaetis mathesoni) fueron positivos para B. vinsonii. No se detectó evidencia de ninguno de los dos patógenos en los piojos analizados. Nuestros hallazgos representan el primer registro de Bartonella elizabethae, un patógeno zoonótico confirmado que causa endocarditis en México y varias asociaciones nuevas de Bartonella con especies de pulgas mexicanas, lo cual resalta la necesidad de implementar vigilancia entomológica activa para el monitoreo de estos patógenos en animales silvestres.

Introduction

Fleas and sucking lice are important vectors of multiple pathogens causing major epidemics worldwide, such as plague (Yersinia pestis) and epidemic typhus (Rickettsia prowazekii). Despite the historical importance of both diseases, this group of ectoparasites has been little studied with respect to other vectors such as mosquitoes or ticks (Gillespie et al. 2009; Bitam et al. 2010; Eisen and Gage 2012). However, these groups of insects are hosts for a wide range of largely understudied pathogens, especially several species of bacteria of the genera Bartonella and Rickettsia (Bitam et al. 2010). The genus Bartonella includes at least 33 species of Gram-negative, intracellular and slow-growing coccobacilli with complex life cycles including multiple vertebrate hosts and vectors, such as B. elizabethae and B. vinsonii arupensis, declared pathogens causing endocarditis in humans and dogs (Breitschwerdt and Kordick 2000; Tsai et al. 2011; Kosoy et al. 2012; Regier et al. 2016). On the other hand, Rickettsia encompasses 26 species of obligate intracellular bacteria which are transmitted by different groups of hematophagous arthropods such as ticks, lice and fleas (Fournier and Raoult 2009; Merhej et al. 2014). Rickettsia species are classified into four groups, two of which are pathogens for man: members of the Spotted Fever group [SGF] (R. conorii, R. massiliae, R. rickettsii and R. parkeri) and Typhus group [TG] (R. prowazekii and R. typhi), this latter group is transmitted exclusively by lice and fleas, which cause epidemic and murine typhus (Fournier et al. 2003; Fournier and Raoult 2009).

In recent decades with the advent of molecular biology techniques, the number of species or strains of both bacteria genera has increased exponentially (Merhej et al. 2014; Regier et al. 2016). Particularly, fleas and sucking lice associated with rodents are the groups in which more studies have focused for the detection of pathogens, with the identification of 16 validated species of Bartonella, nine of Rickettsia and more than 17 new linages near to several validated taxa (but which require isolation for formal identification) for both genera, associated with 45 flea species and seven sucking lice which are also associated with 42 species of rodents in 24 countries around the world (Table 1).

Table 1 Bartonella and Rickettsia species detected in fleas and sucking lice associated with rodents worldwide 

Bacteria species Flea Host Country References
B. birtlesii Ctenophtalmus andorrensis catalanensis Apodemus sylvaticus Spain Cevidanes et al. 2017
Leptopsylla taschenbergi amitina A. sylvaticus Spain Cevidanes et al. 2017
B. coopersplainsensis Stephanocircus pectinipes Rattus fuscipes Australia Kaewmongkol et al. 2011
B. doshiae Xenopsylla cheopis Rattus sp. Afghanistan Marie et al. 2006
B. elizabethae Leptopsylla segnis Mus spretus Algeria Bitam et al. 2012
Synosternus cleopatrae Gerbillus pyramidum Israel Morick et al. 2010
Synopsyllus fonquerniei Rattus rattus Madagascar Brook et al. 2017
X. cheopis Rattus norvergicus Algeria Bitam et al. 2012
USA Frye et al. 2015
R. rattus Algeria Bitam et al. 2012
Rattus tanezumi Indonesia Winoto et al. 2005
Rattus sp. Afghanistan Marie et al. 2006
Nigeria Kamani et al. 2013
B. grahamii Ctenophthalmus agyrtes ND Lithuania Lipatova et al. 2015
Ct. andorrensis catalanensis A. sylvaticus Spain Cevidanes et al. 2017
Ctenophthalmus nobilis Myodes glareolus England Bown et al. 2004
Megabothris turbidus ND Lithuania Lipatova et al. 2015
Megabothris walkeri ND Lithuania Lipatova et al. 2015
Sy. cleopatrae ND Israel Rzotkiewicz et al. 2015
Xenopsylla ramesis ND Israel Rzotkiewicz et al. 2015
B. henselae X. ramesis ND Israel Rzotkiewicz et al. 2015
Meriones tristrami Israel Morick et al. 2010
B. koehlerae Xenopsylla gerbilli Meriones lybicus Afghanistan Marie et al. 2006
B. phoceensis X. cheopis R. tanezumi Indonesia Winoto et al. 2005
B. queenslandensis X. cheopis Rattus sp. Thailand Klangthong et al. 2015
B. quintana X. gerbilli Meriones lybicus Afghanistan Marie et al. 2006
B. rattaustraliani Stephanocircus dasyure R. fuscipes Australia Kaewmongkol et al. 2011
B. rattimassiliensis X. cheopis R. tanezumi Indonesia Winoto et al. 2005
B. rochalimae X. cheopis R. norvergicus USA Frye et al. 2015
B. taylorii Ct. agyrtes ND Lithuania Lipatova et al. 2015
Ct. andorrensis catalanensis A. sylvaticus,
C. russula, M. spretus
Spain Cevidanes et al. 2017
Ct. nobilis M. glareolus England Bown et al. 2004
Ctenophthalmus uncinatus ND Lithuania Lipatova et al. 2015
Hystrichopsylla talpae ND Lithuania Lipatova et al. 2015
L. taschenbergi amitina A. sylvaticus Spain Cevidanes et al. 2017
M. turbidus ND Lithuania Lipatova et al. 2015
M. walkeri ND Lithuania Lipatova et al. 2015
X. gerbilli M. lybicus Afghanistan Marie et al. 2006
B. tribocorum Ctenophtalmus sp. ND Nigeria Kamani et al. 2013
X. cheopis R. norvergicus USA Reeves et al. 2007a; Frye et al. 2015
R. rattus Algeria Bitam et al. 2012
R. tanezumi flavipectus China Li et al. 2007
Rattus sp. Thailand Klangthong et al. 2015
B. vinsonii Polygenis bohlsi bohlsi Thrichomys fosteri Brazil de Sousa et al. 2018
Polygenis gwyni Sigmodon hispidus USA Abbot et al. 2007
B. vinsonii arupensis Malareus sinomus Peromyscus eremicus México Zapata-Valdés et al. 2018
Orchopeas leucopus P. eremicus
Peromyscus leucopus, Peromyscus maniculatus Fernández-González et al. 2016
Pleochaetis exilis Onycomys torridus Zapata-Valdés et al. 2018
B. vinsonii vinsonii Ctenophthalmus pseudagyrtes Microtus sp. USA Reeves et al. 2007a
Meringis parkeri Onychomys arenicola, Onychomys leucogaster México Fernández-González et al. 2016
Orchopeas sexdentatus Neotoma albigula México Fernández-González et al. 2016
Pleochaetis exilis N. albigula, O. arenicola, O. leucogaster, P. maniculatus México Fernández-González et al. 2016
B. washoensis Orchopeas hirsuta Cynomys sp. USA Stevenson et al. 2003; Reeves et al. 2007b
Cynomys ludovicianus México Zapata-Valdés et al. 2018
Orchopeas howardi Sciurus carolinensis USA Durden et al. 2004
Oropsylla montana Otospermophilus beecheyi USA Osikowicz et al. 2016
Pulex sp. C. ludovicianus México Fernández-González et al. 2016
Thrassis fotus Cynomys sp. USA Reeves et al. 2007b
Bartonella near birtlesii O. howardi S. carolinensis USA Reeves et al. 2005b
Bartonella near clarridgeiae Ctenophthalmus lushuiensis Eothenomys sp. China Li et al. 2007
L. segnis R. rattus Egypt Loftis et al. 2006
P. gwyni S. hispidus USA Abbot et al. 2007
Bartonella near doshiae Ct. andorrensis catalanensis A. sylvaticus Spain Cevidanes et al. 2017
L. taschenbergi amitina A. sylvaticus Spain Cevidanes et al. 2017
Bartonella near elizabethae Ct. andorrensis catalanensis A. sylvaticus Spain Cevidanes et al. 2017
Leptopsylla algira ND Israel Rzotkiewicz et al. 2015
Mus musculus Israel Morick et al. 2010
L. taschenbergi amitina A. sylvaticus Spain Cevidanes et al. 2017
Ornithophaga sp. M. spretus Portugal De Sousa et al. 2006
Stenoponia tripectinata M. spretus Portugal De Sousa et al. 2006
R. rattus Portugal De Sousa et al. 2006
Sy. cleopatrae ND Israel Rzotkiewicz et al. 2015
G. pyramidum Israel Morick et al. 2010
X. cheopis Rattus sp. Thailand Klangthong et al. 2015
X. ramesis ND Israel Rzotkiewicz et al. 2015
Bartonella near grahamii Meringis altipecten O. arenicola, O. leucogaster, Dipodomys merriami México Fernández-González et al. 2016
Meringis arachis O. arenicola, O. leucogaster, D. merriami México Fernández-González et al. 2016
M. parkeri O. arenicola, O. leucogaster, D. merriami México Fernández-González et al. 2016
Nosopsyllus fasciatus Rattus surifer Thai-Myanmar Border Parola et al. 2003
P. exilis O. arenicola, O. leucogaster México Fernández-González et al. 2016
Sy. cleopatrae Meriones sacramenti Israel Morick et al. 2010
X. ramesis ND Israel Rzotkiewicz et al. 2015
Bartonella near henselae Or. howardi Glaucomys volans USA Reeves et al. 2007a
Sy. cleopatrae Gerbillus andersoni allenbyi Israel Morick et al. 2010
Bartonella near phoceensis X. cheopis R. norvergicus, R. rattus Egypt Loftis et al. 2006
Bartonella near quintana Or. howardi S. carolinensis USA Durden et al. 2004
Bartonella near rochalimae L. taschenbergi amitina A. sylvaticus Spain Cevidanes et al. 2017
X. cheopis R. norvegicus Algeria Bitam et al. 2012
X. ramesis ND Israel Rzotkiewicz et al. 2015
Bartonella near taylorii Ct. lushuiensis Eothenomys sp. China Li et al. 2007
Bartonella near tribocorum X. cheopis R. rattus Benin Leulmi et al. 2014
Bartonella near vinsonii arupensis Sy. cleopatrae ND Israel Rzotkiewicz et al. 2015
Bartonella sp. Echinophaga gallinacea Dipodomys spectabilis México Fernández-González et al. 2016
Ct. andorrensis catalanensis C. russula Spain Cevidanes et al. 2017
M. arachis D. spectabilis México Fernández-González et al. 2016
M. altecpin D. spectabilis, O. arenicola México Fernández-González et al. 2016
Or. hirsuta Cynomys sp. USA Reeves et al. 2007b
Sy. cleopatrae ND Israel Rzotkiewicz et al. 2015
Thrassis aridis D. spectabilis México Fernández-González et al. 2016
X. cheopis R. norvegicus Algeria Bitam et al. 2012
R. rattus Algeria, Israel Morick et al. 2010; Bitam et al. 2012
R. conorii Stivalius aporus Mus caroli Taiwan Kuo et al. 2016
R. felis Acropsylla episema Apodemus agrarius Taiwan Kuo et al. 2016
Anomiopsyllus nudata N. albigula USA Stevenson et al. 2005
Ctenocephalides felis Peromyscus yucatanicus México Peniche Lara et al. 2015
R. norvegicus Cyprus Psaroulaki et al. 2006
R. rattus Cyprus Psaroulaki et al. 2006
Ct. agyrtes Apodemus flavicollis Lithuania Radzijevskaja et al. 2018
Ctenophthalmus calceatus calceatus Lophuromys aquilus Tanzania Leulmi et al. 2014
Ctenophtalmus sp. R. norvegicus Portugal De Sousa et al. 2006
H. talpae Micromys minutus Lithuania Radzijevskaja et al. 2018
L. segnis Mus sp. Algeria Bitam et al. 2009
Polygenis odiosus Ototylomys phyllotis México Peniche Lara et al. 2015
S. aporus M. caroli Taiwan Kuo et al. 2016
X. cheopis R. norvegicus Cyprus Christou et al. 2010
R. rattus Cyprus, Madagascar Christou et al. 2010; Rakotonanahary et al. 2017
Rattus sp. Afghanistan, Algeria Marie et al. 2006; Bitam et al. 2009
R. helvetica Ct. agyrtes A. flavicollis Lithuania Radzijevskaja et al. 2018
M. turbidus A. flavicollis
M. minutus
M. walkeri A. flavicollis
R. japonica S. aporus M. caroli Taiwan Kuo et al. 2016
R. monacensis Ct. agyrtes A. flavicollis Lithuania Radzijevskaja et al. 2018
R. raoultii ND A. flavicollis, Myodes glareolus Germany Obiegala et al. 2016
R. typhi Ctenophthalmus congeneroides A. agrarius South Korea Kim et al. 2010
L. segnis R. norvegicus Cyprus Christou et al. 2010
R. rattus Cyprus, Egypt, Portugal De Sousa et al. 2006, Loftis et al. 2006; Christou et al. 2010
Rhadinopsylla insolita A. agrarius South Korea Kim et al.2010
Xenopsylla brasiliensis Mastomys natalensis Tanzania Leulmi et al. 2014
R. rattus Tanzania Leulmi et al. 2014
Rattus sp. Democratic Republic of the Congo Leulmi et al. 2014
X. cheopis R. norvegicus Cyprus, Egypt Loftis et al. 2006; Christou et al. 2010
R. rattus Benin, Cyprus, Egypt, Madagascar Loftis et al. 2006; Christou et al. 2010; Leulmi et al. 2014, Rakotonanahary et al. 2017
Rattus sp. Argelia Bitam et al. 2009
Rickettsia prowazekii Or. howardii G. volans USA Sonenshine et al. 1978
Candidatus Rickettsia Asemboensis E. gallinacea R. rattus Egypt Loftis et al. 2006
S. cleopatrae ND Israel Rzotkiewicz et al. 2015
X. ramesis Gerbillus dasyurus, Meriones tristrami, M. musculus Israel Rzotkiewicz et al. 2015
Rickettsia felis-like X. ramesis ND Israel Rzotkiewicz et al. 2015
Rickettsia near monacensis Oropsylla hirsuta Cynomys sp. USA Reeves et al. 2007b
Rickettsia sp. Oh16 Or. howardi S. carolinensis USA Reeves et al. 2005
Rickettsia sp. TwKM01 S. aporus A. agrarius Taiwan Kuo et al. 2016
Rickettsia endosymbiont of Eucoryphus brunneri Ct. agyrtes A. flavicollis Lithuania Radzijevskaja et al. 2018
B. henselae Neohaematopinus sciuri S. carolinensis USA Durden et al. 2004
B. phoceensis Hoplopleura pacifica R. norvegicus Egypt Reeves et al. 2006
Polyplax spinulosa R. norvegicus Taiwan Tsai et al. 2010
Polyplax sp. R. rattus Madagascar Brook et al. 2017
Rattus sp. Thailand Klangthong et al. 2015
B. rattimassiliensis Hoplopleura pacifica R. norvegicus Egypt Reeves et al. 2006
Polyplax spinulosa R. norvegicus Egypt, Taiwan Reeves et. al. 2006; Tsai et al. 2010
Polyplax sp. R. rattus Madagascar Brook et al. 2017
Rattus sp. Thailand Klangthong et al. 2015
B. tribocorum Polyplax spinulosa R. norvegicus Taiwan Tsai et al. 2010
B. vinsonii Hoplopleura hirsuta S. hispidus México Sánchez-Montes et al. 2016b
B. washoensis Neohaematopinus sciuri S. carolinensis USA Durden et al. 2004
Bartonella near tribocorum Polyplax spinulosa R. norvegicus Egypt Reeves et al. 2006
Bartonella near washoensis Hoplopleura sciuricola S. carolinensis USA Durden et al. 2004
Bartonella sp. Polyplax sp. Thrichomys apereoides Brazil Fontalvo et al. 2017
R. prowazekii Neohaematopinus sciuropteri G. volans USA Sonenshine et al. 1978
Polyplax spinulosa* R. norvegicus México Mooser et al. 1931
R. typhi Enderleinellus marmotae Marmota monax USA Reeves et al. 2005
Hoplopleura pacifica R. norvegicus Egypt Reeves et al. 2006

In México, nine taxa of fleas (Ctenocephalides felis, Maleareus sinomus, Meringis parkeri, Orchopeas hirsuta, O. leucopus, O. sexdentatus, Pleochaetis exilis, Pulex sp., and Polygenis odiosus) and two species of sucking lice (Hoplopleura hirsuta and Polyplax spinulosa) tested positive for at least one of four validated species of Bartonella (B. vinsonii and B. washoensis) and Rickettsia (R. felis and R. prowazekii). Additionally new lineages of Bartonella have been registered in six more flea species (Echinophaga gallinacea, Meringis altipecten, M. arachis, M. parkeri, Pleochaetis exilis, Thrassis aridis, Table 1). These records came from isolated studies carried out in wildlife from the southeast and northern parts, lacking data regarding central México where there is a report of human cases of murine typhus (Centro Nacional de Vigilancia Epidemiológica y Control de Enfermedades 2018; Sánchez-Montes et al. 2019). Additionally, for México, 172 species of fleas and 44 species of sucking lice, have been recorded, then, the inventory of species of both bacteria genera is still far from complete (Sánchez-Montes et al. 2013; Acosta-Gutiérrez 2014).

Due to the great diversity of potential vectors and the historical presence of human cases of murine typhus in the centre of the country; the purpose of this study was to identify the presence and diversity of Bartonella and Rickettsia species in a focus of murine typhus in Hidalgo, México.

Material and Methods

During August to September 2014, we sampled in two private ranches from Mineral del Monte and Tulancingo de Bravo (Figure 1), in the state of Hidalgo, México, close to sites where human murine typhus cases have been reported (CENAPRECE 2016). This study was approved by the Ethics and Research Committee of the Medical Faculty of the Universidad Nacional Autónoma de México [FMED/CI/JMO/004/2012].

Figure 1 Sampling sites along the state of Hidalgo, México. Green: State of Hidalgo; Brown: Huasca de Ocampo; Yellow: Mineral del Monte. 

In order to identify the presence of several flea-borne and louse-borne pathogens (Rickettsia and Bartonella) in small mammals and their associated ectoparasites, we trapped small mammals using Sherman traps following Romero-Almaraz et al. (2007), under permission FAUT-0170 from the Secretaría del Medio Ambiente y Recursos Naturales. All mammals were sacrificed in accordance with the Guidelines of the American Society of Mammalogists for the Use of Wild Mammals in Research (Sikes et al. 2016). We performed the necropsy of each animal, extracting a portion of liver and ear which were fixed in 96 % ethanol until its processing in the laboratory. Additionally, fleas and lice were recovered from host’s bodies by manual inspection and fixed in absolute ethanol. Hosts and fleas were identified and deposited at the Mammal Collection and the Flea Collection of the Museo de Zoología “Alfonso L. Herrera” Facultad de Ciencias (MZFC) and Colección del Centro de Medicina Tropical, Facultad de Medicina (CMTFM), both belonging to Universidad Nacional Autónoma de México.

For morphological determination, fleas and lice were mounted on slides using the modified techniques of Kim et al. (1986) and Wirth and Marston (1968). Species were identified using specialized taxonomic keys such as Kim et al. (1986) for lice and Acosta and Morrone (2003), Hastriter (2004), Hopkins and Rothschild (1971), Morrone et al. (2000), and Traub (1950) for fleas.

From collected ectoparasites and hosts tissues, we extracted DNA with the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany). As an endogenous internal control and for molecular identification of the ectoparasites, we amplified a fragment of 400 bp of Cytochrome Oxidase Subunit I (COI) gene. For pathogens detection, we amplified a fragment of gltA and ompB genes specific for each group using primers and temperature conditions previously reported (Table 2).

Table 2 Oligonucleotide primers used in this study. 

Gen Primers Sequence (5´-3´) Length (bp) Reference
Fleas and lice
COI (Cytochrome oxidase subunit I) L6625 CCGGATCCTTYTGRTTYTTYGGNCAYCC 400 Hafner et al. 1994
H7005 CCGGATCCACNACRTARTANGTRTCRTG
Rickettsia sp.
gltA (Citrate synthase) RpCS.415 GCTATTATGCTTGCGGCTGT 806 de Souza et al. (2006)
RpCS.1220 TGCATTTCTTTCCATTGTGC
ompB (Outer membrane protein B) 120-M59 CCGCAGGGTTGGTAACTGC 862 Roux and Raoult, 2000
120-807 CCTTTTAGATTACCGCCTAA
Bartonella sp.
gltA (Citrate synthase) BhCS781.p GGGGACCAGCTCATGGTGG 379 Norman et al. 1995
BhCS1137.n AATGCAAAAAGAACAGTAAACA

The reaction mixture consisted of 12.5 μL of GoTaq® Green Master Mix, 2X of Promega Corporation (Madison, WI, USA), the pair of primers (100 ng each), 6.5 μL nuclease-free water and 30 ng DNA in a final volume of 25 μL (Sánchez-Montes et al. 2016a, b).

PCR products were resolved in 2 % agarose gels using TAE buffer at 85 V during 45 minutes and visualized using an ODYSSEY CLx Imaging System (LICOR Biosciences). Purified amplification products were submitted for sequencing at Macrogen Inc., Korea.

Sequences were analysed and edited using Bioedit version 5.0.9 Sequencing Alignment Editor Copyright © program and deposited in GenBank under accession numbers (MG952757 to MG952772). In order to identify the species of Bartonella and Rickettsia, we used the similarity criteria of the gltA and ompB genes proposed by La Scola (2003), Fournier and Roult (2009) and Fournier et al. (2003). Global alignments were done using Clustal W (Thompson et al. 1994) and the best substitution model was selected based on the lowest BIC (Bayesian Information Criterion) score for each gene using MEGA 6.0 (Tamura et al. 2011; Sánchez-Montes et al. 2016c). Additionally phylogenetic reconstruction was done using Maximum Likelihood also in MEGA 6.0 and branch support was evaluated over 10,000 bootstrap replications.

Results

We collected 40 rodents from four species (Mus musculus, Peromyscus beatae, Rattus norvergicus, and Reithrodontomys sumichrasti), and one shrew (Sorex ventralis), which are deposited in the MZFC under the following catalogue numbers LRR001 to LRR040. We detected the presence of Bartonella DNA in four samples of liver of two P. beatae (2/26 = 7.69 %) and two R. norvergicus (2/4 =50 %). Sequences recovered from P. beatae exhibited a similarity of 98 % with B. vinsonii vinsonii (a member of the Bartonella vinsonii complex) and those from R. norvergicus corresponded in a 100 %, respectively with B. elizabethae (Figure 2). In the case of Rickettsia detection, a single specimen of R. norvergicus (1/4 = 25 %) tested positive in samples from liver and ear; we recovered sequences of gltA and ompB genes which exhibited a similarity of 99 % and 100 % with R. typhi (Accesion number AE017197) deposited in GenBank (Figure 3). A single R. norvergicus specimen presents co-infection between B. elizabethae and R. typhi.

Figure 2 Maximum likelihood (ML) phylogenetic tree generated with gltA gene (300 bp) from several members of the genus Bartonella. The nucleotide substitution model was the Tamura three parameter model (T92) with discrete Gamma distribution (+G). Bootstrap values higher than 50 are indicated at the nodes. Sequences recovered in the study are marked with blue rhombuses and red triangles. 

Figure 3 Maximum likelihood (ML) phylogenetic tree generated with gltA and ompB genes concatenated (1547 bp) from several members of the genus Rickettsia. The nucleotide substitution model was the Tamura three parameter model (T92) with discrete Gamma distribution (+G). Bootstrap values higher than 50 are indicated at the nodes. Sequences recovered in the study are marked with red triangles. 

Hosts were infested by 47 fleas (18 females, 29 males), and 172 sucking lice (60 females, 39 males, 73 nymphs), distributed in six taxa, five species belonging to five families and six genera (Table 3). No fleas or lice were recovered from M. musculus and S. ventralis. After morphological identification was done, we amplified a fragment of 400 bp of Cytochrome oxidase subunit I (COI) in all ectoparasites recovered, in order to corroborate the identification of all samples, especially of those damaged specimens and nymphal stages. DNA sequences of the COI for four of the six species analysed were deposited in GenBank with the following accession numbers: C. tecpin (MG952757), P. hesperomys adelpha (MG952758); P. mathesoni (MG952759), P. spinulosa (MG952772) and H. reithrodontomydis (KT151126). No complete sequences were obtained for J. b. breviloba. We detected the presence of the same Bartonella lineage previously refereed in P. beatae, in two flea species (six P. hesperomys adelpha and 17 P. mathesoni) recovered from the two hosts which tested positive and from three others that were negative (Table 3). Sequences from fleas and hosts shape a single cluster within our phylogenetic analysis (Fig. 1). None of the flea or sucking lice species analysed was positive for Rickettsia DNA.

Table 3 Ecological parameters of Bartonella and Rickettsia species detected in fleas, sucking lice and small mammals in Hidalgo, México. 

Host Ectoparasite
Family Species n HI % BAD Family Species HP EA % A II EI % BAD
Ranch 1 Tulancingo de Bravo
Cricetidae Peromyscus beatae 20 2 10 Bartonella vinsonii Ceratophyllidae Jellisonia breviloba breviloba 2 3 10 0 2 0 0 ND
Plusaetis mathesoni 10 27 5 1 3 17 57 Bartonella vinsonii
Ctenophtalmidae Ctenophtalmus tecpin 2 3 10 0 2 0 0 ND
Leptopsyllidae Peromyscopsylla hesperomys adelpha 4 7 20 0 2 6 86 Bartonella vinsonii
Reithrodontomys sumichrasti 2 0 0 ND Hoplopleuridae Hoplopleura reithrodontomydis 1 4 50 2 4 0 0 ND
Soricidae Sorex ventralis 1 0 0 ND NR NR 0 NR (-) (-) (-) NR NR ND
Ranch 2 Mineral del Monte
Cricetidae Peromyscus beatae 6 0 0 ND Ceratophyllidae Plusaetis mathesoni 1 3 17 1 3 0 0 ND
Muridae Mus musculus 8 0 0 ND NR NR 0 NR (-) (-) (-) NR NR ND
Rattus norvergicus 4 2 50 Bartonella elizabethae Polyplacidae Polyplax spinulosa 4 172 100 43 43 0 0 ND
1 25 Rickettsia typhi

n: Host collected; HI: Number of hosts infected; %: Prevalence; BAD: Bacterial agents detected; HP: Host parasitized; EA: Ectoparasites collected; A: Mean abundance; II: Intensity of infestation; EI: Ectoparasites infected; NR: Not recovered; ND: Not detected.

Discussion

We report for the first time the presence of two species of Bartonella and one of Rickettsia in the state of Hidalgo, México. The first Bartonella species is a member of the B. vinsonii complex, closely related with previous sequences detected in Cricetid rodents and fleas of the northern México (Rubio et al. 2014; Fernández-González et al. 2016). Also, this is the first study to report the presence of a Bartonella in the fleas P. hesperomys adelpha and P. mathesoni and in the host P. beatae (Table 1). Our phylogenetic analysis grouped sequences of B. vinsonii from P. hesperomys adelpha, P. mathesoni and P. beatae in a single cluster, then, our inference is that both flea species could be the potential vectors of these. Additionally, positive P. hesperomys adelpha were recovered from negative hosts, suggesting that these fleas may disseminate the pathogen in non-infected individuals among the rodent population bacteria (Kosoy et al. 1997; Morick et al. 2010). However, it is necessary to carry out tests to verify their vectorial capacity. Both species of fleas have a restricted distribution in México, which extend along the northeastern and central parts of the country, parasitizing several cricetid species such as Peromyscus levipes, P. maniculatus, Reithrodontomys megalotis (P. mathesoni) and P. difficilis (P. hesperomys adelpha), so it is not unexpected that this strain of bacteria is widely distributed in the country (Ponce-Ulloa and Llorente-Bousquets 1993; Hoffman et al. 1989; Whitaker and Morales-Malacara 2005; Acosta and Fernández 2015).

We also report for the first time the presence of B. elizabethae in México, a zoonotic bacterial that may causes endocarditis and neuroretinitis in humans. This agent was reported for the USA in the 1990’s, however, is has become an emerging problem in several countries of Southeast Asia, Portugal and France (Regier et al. 2016; Tay et al. 2016). Bartonella elizabethae is mainly transmitted by the rat flea Xenosylla cheopis (Table 1); however, in our study we did not recovered any fleas from the four R. norvergicus analysed. The higher prevalence of B. elizabethae in collected murid rodents suggests the presence of this flea or other competent vector in the area (Bitam et al. 2012). Additionally, we compiled evidence for the first time of the presence of R. typhi in rodents of the state of Hidalgo. This Rickettsia produces febrile cases with a wide range of severity that can lead to systemic failure in less than 5% percent of cases (Zavala-Castro et al. 2009). In the state of Hidalgo, three cases of murine typhus had been reported between 2005 to 2010, nevertheless, in 2015 there was an outbreak with 12 cases (Centro Nacional de Vigilancia Epidemiológica y Control de Enfermedades 2018).

Only one rat reported coinfection by B. elizabethae and R. typhi, a phenomenon that has been previously reported, probably because both pathogens are transmitted by the same flea species (Table 1). This reinforces the hypothesis of the presence of this vector in the study area (Marie et al. 2006; Bitam et al. 2012; Frye et al. 2015). The presence of positive Norway rats for these two zoonotic pathogens is a risk to human health, because this rodent species invade suburban and urban areas, live and thrive in human settlements and could carry fleas that can feed on human hosts and produce urban outbreaks. Our findings represent the first record of several confirmed zoonotic pathogens that can cause murine typhus and endocarditis in México, which highlight the importance of the establishment of active entomological surveillance in wildlife.

Acknowledgements

We thank to A. Villalpando, O. Escorza and G. Cruz for their help in the logistics and direction of sampling. Additionally to Y. N. Lozano Sardaneta for editing our images. We are indebted to J. C. Sánchez-Montes of the Department for Teaching and Research Branch of the General Directory for Preventive Medicine in Secretaria de Comunicaciones y Transportes, who kindly reviewed our manuscript and provided a number of valuable comments. This work was supported by grants CONACyT 221405 and PAPIIT IN211418. There are no financial or commercial conflicts of interest. Daniel Sokani Sánchez Montes was supported by a fellowship from CONACyT and was a Ph.D. student of Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, UNAM.

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1Associated editor: Jesús Fernández

Received: November 27, 2018; Revised: March 18, 2019; Accepted: April 24, 2019

* Corresponding author. Ingeborg Becker becker@servidor.unam.mx

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