THERYA, 2019, Vol. 10 (2): 69-79 DOI: 10.12933/therya-19-722 ISSN 2007-3364

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-Olguin2; Roxana Acosta-Gutiérrez2, Mario Mata-Galindo1, Kevin Hernández-Vilchis1, D. Melissa Navarrete-Sotelo1, Pablo Colunga-Salas1, 2, Livia León-Paniagua2 and 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).

* Corresponding author.

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.

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.

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

© 2019 Asociación Mexicana de Mastozoología, www.mastozoologiamexicana.org

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).

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].

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).

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.

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.

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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Associated editor: Jesús Fernández

Submitted: November 27, 2018; Reviewed: March 18, 2019;

Accepted: April 24, 2019; Published on line: April 30, 2019.

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

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

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

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.

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.