Joa Braïthe Mangombi

Nadine N'dilimabaka
email: nadinendilimabaka@yahoo.fr

Joa Braïthe Mangombi

Jean-Bernard Lekana-Douki

Octavie Banga

Sydney Maghendji-Nzondo

Mathieu Bourgarel

Eric Leroy

Florence Fenollar

Oleg Mediannikov

First investigation of pathogenic bacteria, protozoa and viruses in rodents and shrews in context of forest-savannah-urban areas interface in the city of Franceville (Gabon)

Keywords: Orientia spp., Occidentia spp., Leptospira spp., Streptobacillus moniliformis, Coxiella burnetii, and Yersinia pestis; parasites

First investigation of pathogenic bacteria, protozoa and viruses in rodents and shrews in context of forest-savannah-urban areas interface in the city of Franceville (Gabon)

Introduction

For several decades, rodents have been recognized as reservoirs or hosts carrying zoonotic pathogens [START_REF] Luis | A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special?[END_REF][START_REF] Meerburg | Rodent-borne diseases and their risks for public health[END_REF][START_REF] Mills | The role of rodents in emerging human disease: examples from the hantaviruses and arenaviruses[END_REF][START_REF] Gratz | Rodents as carriers of disease[END_REF] that can have very dramatic impacts on the economy and public health [START_REF] Meerburg | Rodent-borne diseases and their risks for public health[END_REF]. These zoonoses include the plague [START_REF] Keeling | Metapopulation dynamics of bubonic plague[END_REF][START_REF] Perry | Yersinia pestis etiologic agent of plague[END_REF], Lassa hemorrhagic fever (LHF) [START_REF] Olayemi | New Hosts of The Lassa Virus[END_REF], and hemorrhagic fever with renal syndrome [START_REF] Jiang | Hemorrhagic fever with renal syndrome: Pathogenesis and clinical picture[END_REF]. Even today, zoonotic diseases involving rodents may cause hundreds or even thousands of deaths worldwide [START_REF] Rabaan | The rise of pneumonic plague in Madagascar: Current plague outbreak breaks usual seasonal mould[END_REF][START_REF]Lassa Fever-Nigeria Disease outbreak news[END_REF][START_REF] Mofolorunsho | Outbreak of lassa fever in nigeria: Measures for prevention and control[END_REF]. Many of these rodent-borne diseases are often misdiagnosed. For example, leptospirosis cases can easily be misdiagnosed as dengue or malaria infection because of the similarity of the initial symptoms [START_REF] Salmo ´n-Mulanovich | Community perceptions of health and rodent-borne diseases along the Inter-Oceanic Highway in Madre de Dios, Peru[END_REF]. Such misdiagnosis is especially frequent in countries of sub-Saharan Africa, where access to the necessary diagnostic tools is limited [START_REF] Diagne | Serological Survey of Zoonotic Viruses in Invasive and Native Commensal Rodents in Senegal, West Africa[END_REF][START_REF] Dobigny | Urban Market Gardening and Rodent-Borne Pathogenic Leptospira in Arid Zones: A Case Study in Niamey, Niger[END_REF].

Countries of sub-Saharan Africa are experiencing a remarkable expansion of their urban agglomerations [START_REF] Aliyu | Urbanization, cities, and health: The challenges to Nigeria-A review[END_REF][START_REF] Neiderud | How urbanization affects the epidemiology of emerging infectious diseases[END_REF][START_REF] Hove | The urban crisis in Sub-Saharan Africa: A threat to human security and sustainable development[END_REF][START_REF] Alirol | Urbanisation and infectious diseases in a globalised world[END_REF]. This growth of cities is so dramatic that it may exceed the absorption and management capacity of municipal environmental services, leading to the development of large informal areas characterized by particularly degraded socioenvironmental conditions (high human density, waste accumulation, precarious dwellings, etc.) [START_REF] Alirol | Urbanisation and infectious diseases in a globalised world[END_REF][START_REF] Cohen | Urbanization in developing countries: Current trends, future projections, and key challenges for sustainability[END_REF][START_REF] Gratz | Urbanization, arthropod and rodent pests and human health[END_REF]. Such living conditions are reported risk factors favorable to rodent infestations [START_REF] Bonner | Poor housing quality increases risk of rodent infestation and lassa fever in refugee camps of sierra leone[END_REF], leading to the assertion that the level of infestation of cities by rodents is correlated with the rapid growth of these cities [START_REF] Feng | The secret life of the city rat: A review of the ecology of urban Norway and black rats (Rattus norvegicus and Rattus rattus)[END_REF]. Finally, when the density of a rodent population increases, contacts (direct or indirect) between rodents and humans will become more common, and the likelihood of disease transmission will increase [START_REF] Gratz | Urbanization, arthropod and rodent pests and human health[END_REF][START_REF] Ahmed | Does urbanization make emergence of zoonosis more likely? Evidence, myths and gaps[END_REF][START_REF] Tong | Infectious Diseases, Urbanization and Climate Change: Challenges in Future China[END_REF].

In developing countries in sub-Saharan Africa, the contribution of rodents to human disease is very poorly understood [START_REF] Diagne | Serological Survey of Zoonotic Viruses in Invasive and Native Commensal Rodents in Senegal, West Africa[END_REF][START_REF] Dobigny | Urban Market Gardening and Rodent-Borne Pathogenic Leptospira in Arid Zones: A Case Study in Niamey, Niger[END_REF]. The relevant studies that have been reported to date have often focused on specific major pathogen agents such as Lassa virus, which causes thousands of cases and deaths each year in West Africa [START_REF] Olayemi | Arenavirus Diversity and Phylogeography of Mastomys natalensis Rodents, Nigeria[END_REF], or plague, which is widely studied in Madagascar, where new cases are still recorded every year [START_REF] Andrianaivoarimanana | Trends of human plague, madagascar, 1998-2016[END_REF]. However, West Africa is an exception to this situation. Indeed, numerous studies have been conducted in Senegalese rodents addressing topics ranging from rodent ecology to invasive rodents as well as the bacterial, parasitic and virus communities carried by these rodents [START_REF] Diagne | Serological Survey of Zoonotic Viruses in Invasive and Native Commensal Rodents in Senegal, West Africa[END_REF][START_REF] Diagne | Ecological and sanitary impacts of bacterial communities associated to biological invasions in African commensal rodent communities[END_REF][START_REF] Diagne | Parasites and invasions: changes in gastrointestinal helminth assemblages in invasive and native rodents in Senegal[END_REF][START_REF] Dalecky | Range expansion of the invasive house mouse Mus musculus domesticus in Senegal, West Africa: a synthesis of trapping data over three decades, 1983-2014[END_REF][START_REF] Brouat | Host habitat patchiness and the distance decay of similarity among gastrointestinal nematode communities in two species of Mastomys (southeastern Senegal)[END_REF][START_REF] Brouat | Population genetic structure of two ecologically distinct multimammate rats: the commensal Mastomys natalensis and the wild Mastomys erythroleucus in southeastern Senegal[END_REF][START_REF] Brouat | Host ecology and variation in helminth community structure in Mastomys rodents from Senegal[END_REF][START_REF] Duplantier | 24 Rodents as definitive hosts of Schistosoma, with special reference to S. mansoni transmission[END_REF][START_REF] Duplantier | Rodents as reservoir hosts in the transmission of Schistosoma mansoni in Richard-Toll, Senegal, west Africa[END_REF][START_REF] Duplantier | Swimming ability in six West-African rodent species under laboratory conditions[END_REF]. Similarly, studies on the ecology of rodents and rodent-borne diseases in urban areas are emerging in Niger [START_REF] Dobigny | Urban Market Gardening and Rodent-Borne Pathogenic Leptospira in Arid Zones: A Case Study in Niamey, Niger[END_REF][START_REF] Dobigny | Molecular survey of rodentborne Trypanosoma in Niger with special emphasis on T. lewisi imported by invasive black rats[END_REF][START_REF] Garba | Spatial Segregation between Invasive and Native Commensal Rodents in an Urban Environment: A Case Study in Niamey, Niger[END_REF][START_REF] Tatard | Rodent-borne Trypanosoma from cities and villages of Niger and Nigeria: A special role for the invasive genus Rattus?[END_REF], Mali [START_REF] Schwan | Fleas and trypanosomes of peridomestic small mammals in sub-Saharan Mali[END_REF] and Benin [START_REF] Houemenou | Leptospira spp. Prevalence in Small Mammal Populations in Cotonou, Benin[END_REF][START_REF] Houemenou | Ecologie, diversite ´spe ´cifique et abondance des petits mammifères de la ville de Cotonou au Be ´nin (Afrique de l ' Ouest)[END_REF][START_REF] Dobigny | Spatio-temporal survey of small mammal-borne Trypanosoma lewisi in Cotonou, Benin, and the potential risk of human infection[END_REF][START_REF] Hima | Native and invasive small mammals in urban habitats along the commercial axis connecting Benin and Niger, West Africa[END_REF][START_REF] Houe ´me ´nou | Pathogenic Leptospira in Commensal Small Mammals from the Extensively Urbanized Coastal Benin[END_REF].

In Gabon, located at the equator in the western portion of the Central Africa, there are many circulating pathogens. These pathogens include the etiologic agents responsible for viral hemorrhagic fever, examples Chikungunya fever, Dengue fever and Ebola hemorrhagic fever, reviewed by Bourgarel et al, [START_REF] Bourgarel | Emerging viral threats in Gabon: Health capacities and response to the risk of emerging zoonotic diseases in Central Africa-Emerging zoonotic viral threats in Gabon[END_REF]. Several diseases of parasitic origin are also reported in the region, such as toxoplasmosis [START_REF] Mercier | Additional Haplogroups of Toxoplasma gondii out of Africa: Population Structure and Mouse-Virulence of Strains from Gabon[END_REF] and malaria, which is the most common parasitosis in tropical Africa, [START_REF] Richard-Lenoble | Malaria in Gabon I. Study of 500 children with fever in Libreville[END_REF][START_REF] Richard-Lenoble | Malaria in Gabon 2. Evaluation of the qualitative and quantitative prevalence of parasites in the total school and preschool population of the country[END_REF][START_REF] Borrmann | Reassessment of the resistance of Plasmodium falciparum to chloroquine in Gabon: implications for the validity of tests in vitro vs. in vivo[END_REF][START_REF] Bouyou-Akotet | Evidence of decline of malaria in the general hospital of Libreville, Gabon from 2000 to 2008[END_REF] and shows no improvement compared to other countries in the region according to the most recent data [START_REF] Assele | No evidence of decline in malaria burden from 2006 to 2013 in a rural Province of Gabon: implications for public health policy[END_REF]. Many such pathogens may be carried by rodents, but in Gabon, very few studies have focused on inventories or the identification of potentially zoonotic infectious agents in these animals. The existing studies have focused on one virus at a time [START_REF] Mombo | Detection of novel astroviruses among rodents of Gabon, Central Africa[END_REF][START_REF] Dilimabaka | Evidence of Lymphocytic Choriomeningitis Virus (LCMV) in Domestic Mice in Gabon: Risk of Emergence of LCMV Encephalitis in Central Africa[END_REF] and one plasmodium parasite [START_REF] Boundenga | Rodent malaria in Gabon: Diversity and host range[END_REF] in rodents. They do not provide sufficient data to reveal the diversity and abundance of infectious agents carried by rodents in Gabon.

Franceville is the third largest city in Gabon. It is located in the southeast of the country and is characterized by a spatial structure in which constructed, forest and savannah areas come into contact, referred to as a forest mosaic and savannah [START_REF] Laporte | ´ographie des Relations Ville-Forêt en Afrique Centrale: Approche Re ´gionale[END_REF]. This heterogeneity of habitats makes Franceville city an excellent model for the study of zoonoses since the human population is in close contact with both domestic and wild animals in this area.

In this study, we sought to identify a wide range of potential zoonotic bacteria, protozoans and viruses hosted by rodents in the city of Franceville. The aims were to (i) identify and characterize these pathogens and (ii) compare their distribution according to the different types of habitats encountered within the city. This is the first time such a study has been conducted on rodents in Gabon.

Materials and methods

Franceville, study area

Franceville is a Gabonese city located 500 km southeast of the capital, Libreville. Its population increased dramatically between 1993 and 2013, from 31,193 to 110,568 inhabitants [START_REF]website of the municipality of Franceville[END_REF][START_REF]Direction de la statistique[END_REF]. It continues to grow at a moderate rate; the current population is approximately 129,000 [START_REF]Population Data[END_REF].

Franceville is an atypical city characterized by the presence of buildings and vegetation referred to as mosaic forest and savannah.

Rodents were captured during four trapping sessions in houses and small savannah and forest islands in 2014. Trapping took place in houses in six (6) districts of the city of Franceville (Fig 1 ), including four peripheral districts (Mangoungou, Mbaya Sable and Ye ´ne ´) and two central districts (Ombe ´le ´and Potos). It should also be noted that these districts are located along the main access routes to the city (roads and railways). These districts display many of the dominant characteristics of the city as a whole in terms of the level of connectivity and the aggregation of buildings as well as the presence or absence of vegetation. Mbaya and Ye ´ne ´are the two main entry points of the city, by road and railway, respectively. Sable and Mangoungou are more isolated districts. Mbaya is mainly industrial. Potos is the central trade district, including large storehouses and the main open market [START_REF] Mangombi | Urban population genetics of the invasive black rats in Franceville, Gabon[END_REF].

Rodent and organ sampling

Rodents were sampled according to a standardized live-trapping protocol as previously described [START_REF] Mangombi | Urban population genetics of the invasive black rats in Franceville, Gabon[END_REF]. Live-trapped rodents were brought back to our laboratory, euthanized, weighed, sexed, measured and autopsied. During autopsy, various organs and tissues, including the kidney, liver, brain, lungs, and spleen, were collected. All of these samples were stored at -80˚C. In this study, all bacteria and protozoa were screened on the liver, except Leptospira on the kidney and Toxoplasma on the brain. While, all viruses were screened in the spleen.

Ethics statements

Trapping campaigns were performed with prior (oral) agreement from local authorities (city Mayor, district chief and family heads). All sampling procedures were approved by the Ethics Committee named "Comite ´Nationale d'Ethique pour la Recherche" under the number: Prot n˚0020/2013/SG/CNE. Live-trapped rodents were brought back to our laboratory, euthanized with a halothane solution and autopsied in accordance with guidelines of the American Society of Mammalogists [START_REF] Sikes | Mammalogists T animal care and use commitee of the AS of. Guidelines of the American Society of Mammalogists for the use of wild mammals in research[END_REF]. None of the rodent species captured in the present study had a protected status (CITES lists and IUCN).

Specific identification of rodent species

Specific species identification was carried out according to the identification keys provided by Jean-Marc DUPLANTIER and Violaine NICOLAS following their studies on rodents in Gabon [START_REF] Nicolas | Assessment of three mitochondrial genes (16S, Cytb, CO1) for identifying species in the Praomyini tribe (Rodentia: Muridae)[END_REF][START_REF] Nicolas | Phylogeographic structure and regional history of Lemniscomys striatus (Rodentia: Muridae) in tropical Africa[END_REF][START_REF] Nicolas | Seasonal variations in population and community structure of small rodents in a tropical forest of Gabon[END_REF][START_REF] Nicolas | Population structure and reproduction of Heimyscus fumosus in south-western Gabon[END_REF][START_REF] Nicolas | Geographical distribution and morphometry of Heimyscus fumosus[END_REF][START_REF] Nicolas | Variabilite structurale des peuplements forestiers de rongeurs (Muridae) et musaraignes (Soricidae) dans les Monts Doudou[END_REF][START_REF] Duplantier | Les rongeurs myomorphes forestiers du Nord-est du Gabon: structure du peuplement, de ´mographie, domaines vitaux[END_REF][START_REF] Duplantier | Critères d'identification des principales espèces[END_REF][START_REF] Duplanrier | Les rongeurs myomorphes forestiers du nord-est du Gabon: peuplements, utilisation de l'espace et des ressources alimentaires ro ˆle dans la dispersion et la germination des graines[END_REF].

Rattus rattus was the only rat species identified in our study. Morphologically, it is easily distinguishable from Rattus norvegicus. In addition, the Rattus sample collected in this study was the subject of a population genetic study validating the identification of R. rattus [START_REF] Mangombi | Urban population genetics of the invasive black rats in Franceville, Gabon[END_REF]. Similarly, the Mus musculus domesticus specimens included in this study were genetically identified previously [START_REF] Dilimabaka | Evidence of Lymphocytic Choriomeningitis Virus (LCMV) in Domestic Mice in Gabon: Risk of Emergence of LCMV Encephalitis in Central Africa[END_REF]. Lemniscomys striatus and Lophuromys sikapusi were identified to the species level by using morphological identification keys. Praomys and Mus (Nannomys) were identified to the subgenus level. Nevertheless, the determination of host species was performed only in pathogen-positive samples. A fragment of the 16S ribosomal RNA gene (16S rRNA) was amplified as previously described [START_REF] Nicolas | Assessment of three mitochondrial genes (16S, Cytb, CO1) for identifying species in the Praomyini tribe (Rodentia: Muridae)[END_REF] and sequenced under BigDyeTM terminator cycling conditions. All sequences from this study have been deposited in GenBank under accession numbers MT256376 to MT256385 for rodent host species and MT677677 to MT677695 for shrews host species.

DNA and RNA extraction from organs and tissues

Small pieces of the liver, kidney, spleen and brain of rodents were collected and placed individually in Eppendorf tubes. Total DNA or RNA was extracted with a BioRobot EZ1 system (Qiagen, Courtaboeuf, France) using a commercial EZ1 DNA/RNA Tissue Kit (Qiagen) (Qiagen, Courtaboeuf, France) following the manufacturer's instructions. DNA and RNA were eluted in 100 μl of TE buffer. DNA was stored at 4˚C until being used for PCR amplification, while RNA was stored at -20˚C.

Molecular detection of virus, bacterial and protozoan DNA in rodents and shrews

Virus molecular detection. The following virus families or species were screened by onestep RT-PCR in RNA extracts from rodent spleens: Hantavirus spp., Mammarenavirus spp., Flavivirus, Paramyxovirus, Lymphocytic choriomeningitis mammarenavirus (LCMV) and Zika virus (ZIKV). Methodological details and primer sequences are provided in Table 1.

Bacteria and protozoan molecular detection. The real-time PCR (qPCR) was performed to screen all rodent samples using previously reported primers and probes for Bartonella spp., Anaplasmataceae, Coxiella burnetii, Borrelia spp., Rickettsiaceae, Mycoplasma spp., Orientia spp., Occidentia spp., Yersinia pestis, Leptospira spp., Streptobacillus moniliformis., Piroplasmida., Toxoplasma gondii., and Kinetoplastida (including the Trypanosoma and Leishmania genera). The sequences of the primers and probes are shown in Table 2. For all systems, any sample with a cycle threshold (Ct) value of less than 40 Ct was considered positive. Conventional PCR analysis was performed for all qPCR-positive samples using the primers and conditions described in Table 2. The amplification reaction was conducted in a final volume of 25 μl containing 12.5 μl of AmpliTaq Gold master mix, 0.75 μl of each primer [20 μM], 5 μl of DNA template, and 6 μl of water. The thermal cycling profile consisted of one incubation step at 95˚C for 15 min, 45 cycles of 30 s at 95˚C, 30 s to 1 min at the annealing temperature (Table 2) and 1 min at 72˚C, and a final extension step of 5 min at 72˚C. Successful amplification was confirmed by electrophoresis in a 1.5% agarose gel, and the amplicons were completely sequenced on both strands. Quantitative real-time PCR was performed on the CFX96 Real-Time system (Bio-Rad) with the Roche LightCycler 480 Probes Master Mix PCR kit (Roche Applied Science, Mannheim, Germany). For each assay, DNA extracts of the targeted bacteria or parasites were used as positive controls and distilled water as negative control (S1 Table ). For the viral families Bunyaviridae and Arenaviridae, the positive controls used were plasmids, designed during the PREDICT project. For Flaviviridae, we used the Yellow fever virus RNA (vaccinal strain 17D) and RNA transcripts from mumps, measles, and respiratory syncytial viruses, for Paramyxoviridae. Conventional PCR was performed in an automated DNA thermal cycler (GeneAmp PCR Systems Applied Biosystems, Courtaboeuf, France). Sequencing analyses were performed on the ABI Prism 3130XL Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, France) using the DNA sequencing BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA, Perkin-Elmer) according to the manufacturer's instructions. The BigDye products were purified on Sefadex G-50 Superfine gel filtration resin (Cytiva, Formerly GE Healthcare Life Science, Sweden).

The sequences were compared to sequences available in the GenBank database using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Multispacer sequence typing (MST) genotyping of Coxiella burnetii. The multispacer sequence typing (MST) method was used for Coxiella burnetii genotyping. For this purpose, five (5) different spacers, which were previously described [START_REF] Glazunova | Coxiella burnetii Genotyping[END_REF], were selected and amplified (Cox 2, 5, [START_REF] Alirol | Urbanisation and infectious diseases in a globalised world[END_REF][START_REF] Feng | The secret life of the city rat: A review of the ecology of urban Norway and black rats (Rattus norvegicus and Rattus rattus)[END_REF][START_REF] Garba | Spatial Segregation between Invasive and Native Commensal Rodents in an Urban Environment: A Case Study in Niamey, Niger[END_REF]. Conventional PCR was performed as described below with a hybridization temperature of 59˚C. Then, the web-based MST database (https://ifr48.timone.univ-mrs. fr/mst/coxiella_burnetii/groups.html) was used for MST identification.

Phylogenetic and statistical analyses

Phylogenetic analysis. The obtained sequences were analyzed using ChromasPro version 1.3 (Technelysium Pty, Ltd., Tewantin, Queensland, Australia) for assembly and were aligned with other sequences of targeted bacteria or parasite species from GenBank using CLUS-TALW, implemented in BioEdit v7.2 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Phylogenetic trees were constructed with MEGA software v.7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF]. The maximum likelihood method based on the Hasegawa-Kishino-Yano model (HKY) was used to infer the phylogenetic analysis with 500 bootstrap replicates.

Statistical analysis. Statistical analysis was performed with R software V3.2.5 [START_REF] Development | R: A language and environment for statistical computing[END_REF] using chi-square/Fisher's exact tests for data comparisons between the prevalence of infected rodents for all parasites according to habitat type. A p-value� 0.05 was considered to be significant.

General linear mixed models (GLMMs) run using the lme4 package [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF] were also employed to examine the potential determinants of parasite richness (number of parasite species in a host), as reported in a previous similar study [START_REF] Dahmana | Rodents as Hosts of Pathogens and Related Zoonotic Disease Risk[END_REF]. We assumed a Poisson distribution for the parasite richness data. The sampling site was considered a random factor, and other

Results

Rodents sampled for this study

Bacterial, protozoan and viral nucleic acids detected in rodents and shrews

All rodents were found negative for all viral pathogens screened in the spleen by conventional PCR and qPCR, including Hantavirus spp., Mammarenavirus spp., Flavivirus, Paramyxovirus, Lymphocytic choriomeningitis mammarenavirus (LCMV) and Zika virus (ZIKV). Similarly, all rodents were found negative by qPCR on tissues for several bacteria and protozoa, specifically Borrelia sp, Leishmania sp, Mycoplasma sp, Orientia sp, Occidentia sp, Streptobacillus moniliformis, Rickettsia sp and Yersinia pestis. In contrast, 49/198 (24,7%) rodents were positive for 8 of the 16 pathogens (bacteria and protozoans) tested via qPCR. In total, 7 genera of pathogenic microorganisms were identified, including bacterial Anaplasma spp. ( 3). All microorganisms were detected in the liver samples except for Leptospira spp. and Toxoplasma gondii, which were only detected in the kidney and brain, respectively.

Multiple infections (i.e., many infectious agents in the same organ of an individual rodent) were found in 11 rodents (5.5%), including 10 double infections (5%) and one triple infection (0.5%). Rattus rattus and Le. striatus presented the highest carriage rate for all of the identified pathogens, including 5 out of 7 infectious agents, while M. m. domesticus appeared to harbor the fewest pathogens (1/7). Other species carried between 3 and 4 pathogens (Table 3).

Phylogenetic analysis for the taxonomic description of detected pathogens

Bartonella. The sequencing of 733 bp of the ITS gene from the DNA extracts of 13 qPCRpositive individuals revealed five sequences of Bartonella ranging from 690 to 722 bp (Gen-Bank: MN968369 to MN968373). BLAST analysis of three sequences obtained from Lophuromys sp. hosts showed that the most closely related species was Bartonella queenslandensis (GenBank: EU111769.1), which presented the highest score and a percentage of identity of 84-86% (611/721, 546/634, 550/635). This percentage of identity below 95% and the fact that all these three sequences were grouped in the same cluster (with 99% of identity between each other) in the phylogenetic tree suggested that the obtained Bartonella pathogen represented a new species, an undescribed species. We provisionally named this probable new genotype Candidatus Bartonella gabonensis. The fourth sequence obtained from a Cricetomys sp. rodent matched the B. massiliensis OS23 and OS09 strains (HM636450 and HM636449) with 96.7% (699/723) and 96.1% (700/728) identity, respectively. The last sequence, obtained from R. rattus, matched Bartonella sp. 'Tel Aviv' of the Bartonella elizabethae complex (GenBank: CP031843.2) with 100% (690/690) identity. It is referred to here as B. elizabethae (Fig 2). Anaplasma. Among 16 qPCR-positive individuals, six sequences ranging from 623 to 683 bp (GenBank: MT269268 to MT269273) were obtained after the sequencing of the Anaplasma 23S rRNA gene [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France. Parasites and Vectors[END_REF][START_REF] Benevenute | Assessment of a quantitative 5 0 nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil[END_REF]. BLAST analysis of these sequences showed identities with A. phagocytophilum (KM021418) ranging from 91% to 92% (578/633, 606/659, 607/659, 606/660, 607/ 659, 607/659). The percentage of identity below 95% suggests that the obtained pathogen is a new or undescribed species, with similarity to Anaplasma phagocytophilum. However, the dissimilarity between the Anaplasma sequences, as shown in our data (Fig 3), could also suggest that the amplified genetic material would come from organisms of a different genus. Finally, not being able to conclude on the basis of our results, we refer to it here as Uncultured Anaplasma sp.

Coxiella burnetti. The genotyping of Coxiella burnetii from 10 qPCR-positive individuals via MST analysis showed the following profile allele codes: 3-2-6-5-4, corresponding to Cox 2-Cox5-Cox 18-Cox22-Cox37, respectively. This profile identified MST group 20. This genotype has been found in Europe and the United States [START_REF] Glazunova | Coxiella burnetii Genotyping[END_REF] and is associated with human and animal disease. The same genotype, MST20 was also found on domestic animal ticks in Ethiopia in Africa [START_REF] Kumsa | Occurrence and genotyping of coxiella burnetii in ixodid ticks in oromia, Ethiopia[END_REF]. In Franceville, C. burnetii MST group 20 was found in all the samples tested from the five rodent species mentioned Praomys sp, R. rattus, Mus Nannomys sp, Le. striatus and Lo. sikapusi. Cricetomys sp; one, from Praomys sp (1/198), showed 100% (522/522) identity to both T. brucei brucei (XR_002989635) and T. brucei gambiense (FN554966.1); and two others from R. rattus and Mus Nannomys (AB190228) (2/198) were identified as T. otospermophili, with 97% (453/ 467) identity (Fig 4).

Theileria. The two samples that were positive according to the pan-Piroplasma 5.8S qPCR analysis and were sequenced (GenBank: MT269266 and MT269267) were shown 98% (869/ 883, 868/881) identity to Theileria sp strain HaD-2019a (MK484070.1) found in Senegalese rodents (Fig 5). These two infected rodents were Le. striatus and Praomys sp.

Toxoplasma gondii. Of the tested brain samples, only one (0.5%, N = 198) was positive in the qPCR screening of T. gondii according to the ITS1 gene, with a Ct value of 35.1. However, it could not be identified; the sample came from an Le. striatus rodent.

Habitats and pathogens in rodents

We categorized the sampling areas into three groups as follows: central districts (Ombe ´le

´, Potos), peripheral districts (Mbaya, Ye ´ne ´, Sable, and Mangoungou) and non urban areas of vegetation (savannah-forest) (Fig 6). The prevalence of infection by the pathogens in each group was 32.3% (11/32), 21.6% (24/111) and 52.8% (28/53) for the central districts, peripheral districts and vegetated areas, respectively.

In terms of overall prevalence, a significant difference was found between the average prevalence in the infected rodents in the three habitat types (X-squared = 16.659, df = 2, P < 0.0002413). The residual value of the X-squared test showed that the difference was attributed to the vegetated areas. The rodents from vegetated areas showed the highest infection prevalence in Franceville. Similarly, at the pathogen group scale (only for pathogens found in more than 5 rodents), the rodents from vegetated areas showed the highest prevalence of infection by Bartonella sp (P <0.001) and C. burneti (P <0.004) pathogens (Fig 6). However, a different result was obtained in rodents from central districts, which showed a significantly higher rate of infection by Trypanosoma pathogens than the rodents coming from the 2 other habitats (P< 0.002).

Host factors and pathogens in rodents

GLMM analysis revealed a strong association effect between the parasite richness and body mass of host rodents (t-value = 0.791, P< 0.0449) (Table 4). Parasitic richness was positively correlated with the weight of the rodents. Conversely, no association could be identified between parasitic richness and the other factors tested among the rodents in Franceville.

Discussion

Rodents are hosts of numerous zoonotic diseases caused by bacteria, protozoans, or viruses all around the world [START_REF] Han | Rodent reservoirs of future zoonotic diseases[END_REF]. Luis et al., 2013 [1] noted the importance of paying serious attention to rodents because they are the most diverse mammals and carry many pathogens responsible for emerging viral zoonoses. In Gabon, over the last five years, some studies have focused specifically on rodent viruses [START_REF] Mombo | Detection of novel astroviruses among rodents of Gabon, Central Africa[END_REF][START_REF] Dilimabaka | Evidence of Lymphocytic Choriomeningitis Virus (LCMV) in Domestic Mice in Gabon: Risk of Emergence of LCMV Encephalitis in Central Africa[END_REF] and plasmodium parasites [START_REF] Boundenga | Rodent malaria in Gabon: Diversity and host range[END_REF]. Herein, we broaden the spectrum of these studies by investigating a wide range of bacteria, protozoa and viruses in the rodents of Franceville in Gabon.

We did not identify the presence of viruses in these rodents. Several hypotheses can be put forth to explain this result. We suggest that a low viral load could explain the failure to detect the targeted viral fragments. In such instances, the use of high-throughput sequencing, particularly next generation sequencing methods [START_REF] Gu | Clinical Metagenomic Next-Generation Sequencing for Pathogen Detection[END_REF], could be more effective and efficient, as reported by Diagne et al., 2017 [27]. Another hypothesis is that the failure to detect pathogens may be due to the absence in our sample of rodent species that are reservoirs for the targeted pathogens. For example, Mastomys natalensis and Mastomys erythroleucus, which are LHF reservoirs whose distribution area includes Gabon, were missing from our sample. Nevertheless, R. rattus and M. m. domesticus are reservoirs of many pathogens, including Hantavirus and Lymphocytic choromeningite virus (LCMV), respectively, but we did not succeed in identifying these viruses. Nevertheless, this hypothesis is supported by the fact that these two species are invasive rodents that may lose associated viruses in new environments, referred to as the "enemy release" effect [START_REF] Colautti | Is invasion success explained by the enemy release hypothesis?[END_REF]. The third hypothesis that may explain the lack of virus detection is that Franceville rodents are secondary hosts for the screened viruses [START_REF] N'dilimabaka | Absence of arenavirus RNA among animal's samples from potential reservoirs in Gabonof[END_REF]. The studied rodents may, however, harbor many other viruses that may or may not be related to the viral species targeted in our study. Negative results in such cases can be explained by insufficiently broad primer specificity.

To obtain support for this hypothesis, serological analyses would be appropriate for determining whether the pathogen continues to circulate when nucleic acids are absent. Indeed, serological diagnoses of circulating viral infectious agents have already been carried out and shown to be effective in other studies conducted in Africa [START_REF] Diagne | Serological Survey of Zoonotic Viruses in Invasive and Native Commensal Rodents in Senegal, West Africa[END_REF]100,101].

Our study included the first detection and description of several bacterial and protozoan species in rodent populations in Gabon. Our results revealed an overall prevalence of 25.7% (51/198) for the 7 identified microparasites. This diversity of pathogens is not surprising. Due to the location of Gabon in the Congo basin, it is expected to be a biodiversity hotspot including various pathogens [START_REF] Bourgarel | Emerging viral threats in Gabon: Health capacities and response to the risk of emerging zoonotic diseases in Central Africa-Emerging zoonotic viral threats in Gabon[END_REF]. In Senegal, a well-diversified bacterial community of 12 zoonotic OTUs (Operational Taxonomic Unit) was identified despite the Sahelian context and the use of different detection methods. The difference in the composition of these microparasitic communities shows the need to expand investigations to improve our knowledge of them, especially in Africa, where most studies focus on a single zoonotic agent [START_REF] Dobigny | Spatio-temporal survey of small mammal-borne Trypanosoma lewisi in Cotonou, Benin, and the potential risk of human infection[END_REF]102], except when investigating the helminth community [START_REF] Diagne | Parasites and invasions: changes in gastrointestinal helminth assemblages in invasive and native rodents in Senegal[END_REF][START_REF] Brouat | Host habitat patchiness and the distance decay of similarity among gastrointestinal nematode communities in two species of Mastomys (southeastern Senegal)[END_REF].

Studies on infectious agents hosted by rodents are very limited in many developing countries in sub-Saharan Africa [102], particularly in Central Africa. Existing studies have previously revealed the presence in African rodents of the bacteria and protozoans that we detected in our study. These organisms include Bartonella spp. [103-105], Coxiella burnetii [102, 106], Anaplasma sp. [107], Theileria sp. [START_REF] Dahmana | Rodents as Hosts of Pathogens and Related Zoonotic Disease Risk[END_REF], Trypanosoma sp. [START_REF] Dobigny | Spatio-temporal survey of small mammal-borne Trypanosoma lewisi in Cotonou, Benin, and the potential risk of human infection[END_REF], Toxoplasma gondii [108] and Leptospira sp. [START_REF] Dobigny | Urban Market Gardening and Rodent-Borne Pathogenic Leptospira in Arid Zones: A Case Study in Niamey, Niger[END_REF][START_REF] Houemenou | Leptospira spp. Prevalence in Small Mammal Populations in Cotonou, Benin[END_REF]. The presence of these bacterial genera shows the need to explore and implement surveillance in host rodents in Gabon. These bacterial genera include species that can act as zoonotic pathogens capable of inducing severe diseases that are often misdiagnosed in Africa [START_REF] Sokhna | Point-of-Care Laboratory of Pathogen Diagnosis in Rural Senegal[END_REF]109,110].

Indeed, the detected species of pathogens included C. burnetii, which constitutes a monospecific genus and is the causative agent of Q fever, which is a disease found worldwide. Zoonotic Q fever can be acute or chronic and exhibits a wide spectrum of clinical manifestations. The natural cycle of C. burnetii is not reported to include humans, who are considered incidental hosts [111]. Coxiella burnetii is associated with sylvatic or domestic transmission cycles, with rodents being suspected to link the two transmission cycles [102]. Consequently, human infections with C. burnetii are systematically associated with infected livestock and ticks. Nevertheless, Q fever outbreaks associated with contact with infected rodents were recently reported in Zambia [102]. We identified the C. burnetii pathogenic genotype MST 20, which was detected for the first time in central Africa but has previously been reported in an East African country, Ethiopia [START_REF] Kumsa | Occurrence and genotyping of coxiella burnetii in ixodid ticks in oromia, Ethiopia[END_REF], in the United States and Europe [START_REF] Glazunova | Coxiella burnetii Genotyping[END_REF]. Other known genotypes of C. burnetii in Africa are: MST 6,[START_REF] Cohen | Urbanization in developing countries: Current trends, future projections, and key challenges for sustainability[END_REF]35 and 36 [109], discovered on a wide range of hosts, including ruminants and human febrile patients.

Leptospira borgpetersenii, L. kirschneri, L. interrogans and L. weilii are highly pathogenic leptospires, which are agents of leptospirosis, an emerging zoonotic disease that affects both animals and humans worldwide [112]. Based on phylogenetic analyses of DNA-DNA hybridization (DDH) and 16S rRNA data, the Leptospira genus has been divided into three distinct clades. The pathogenic leptospire clade includes 10 pathogens that can infect and cause disease in humans and animals [113]. In another clade, the intermediate clade, there are five leptospires that have been isolated from humans and animals that may cause various mild clinical manifestations of leptospirosis. In the third clade, the so-called saprophytes, there are seven leptospires that are unable to cause disease [112,114]. Pathogenic Leptospira spp. colonize the proximal renal tubules of reservoir hosts and are excreted through urine into the external environment, where they can survive in water for several months [115].

For some time now, the incidence of tick-borne diseases in humans and animals has been increasing due to several factors that together favor the chance of contact among wild animals, their ectoparasites, domestic animals and humans [107]. The detected bacteria of the genera Anaplasma and Bartonella as well as Theileria sp. protozoa are responsible for tick-borne diseases. Theileriosis and anaplasmosis are mostly diseases of domestic ruminants that are responsible for economic losses to varying degrees depending on the species of the pathogen and the region of the epidemic [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France. Parasites and Vectors[END_REF]116]. However, an increasing number of human cases of anaplasmosis (human granulocytic anaplasmosis, HGA) with A. phagocytophilum have been reported, including some fatal cases [117]. It is this species in particular that is mostly found in rodents [118][119][120]. Although rodents are suspected to be potential reservoirs of this bacterium, their role in the epidemiologic cycles affecting domestic and wild animals as well as humans has not been demonstrated [119]. In Africa, rodents infected with Ehrlichia sp. have been detected [START_REF] Benevenute | Assessment of a quantitative 5 0 nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil[END_REF]107,121]. The most recent study revealed the probable presence of a new species of Anaplasmatacae infecting rodents in Senegal: Candidatus Ehrlichia senegalensis [START_REF] Dahmana | Rodents as Hosts of Pathogens and Related Zoonotic Disease Risk[END_REF]. Among the 16 animals that were qPCR positive for Anaplasmatacae, six sequences were identified by 23S rRNA gene amplification, two of which came from R. rattus and four from Le. striatus. The 6 sequences were almost identical (98.72% to 99.99%). Based on the phylogenetic tree topology and the percentage of identity (91%) after BLAST analysis, our results suggest that the sequences obtained by 23S rRNA gene amplification represent an organism not yet described, close to A. phagocytophylum and could be a new species. We refer to it here as Uncultured Anaplasma spp. The pathogenicity of this new genotype in humans and animals is unknown.

For piroplasms, two samples were positive by qPCR, which were successfully amplified from the rodents Le. striatus and Praomys sp. The obtained sequences were closely related to an isolated sequence from the rodent Arvicanthis niloticus in Senegal designated Candidatus Theileria senegalensis, with 98% identity [START_REF] Dahmana | Rodents as Hosts of Pathogens and Related Zoonotic Disease Risk[END_REF]. Here, we refer to it as Theileria sp. This is presumably the same species of bacteria isolated from different rodents, suggesting that they are not reservoirs of this bacterium; however, it could be that the source of infection is either a tick or a ruminant. To date, no human case of theileriosis has been reported; in contrast Babesia, particularly Babesia microti, has been shown to be responsible for human babesiosis, with rodents serving as hots [START_REF] Meerburg | Rodent-borne diseases and their risks for public health[END_REF]122]. Babesia and Theileria are closely related genera within the Piroplasmida order.

Conversely, in the Bartonella genus, there are many species responsible for human diseases, such as B. henselae, causing cat scratch disease, B. quintana, causing trench fever, and B. bacilliformis, causing Carrio ´n disease [123]. Indeed, among the 53 Bartonella species currently described [124], more than twenty are associated with rodents. These are the species that usually cause human infections [103]. We found three different genotypes of Bartonella, which were identified as Bartonella elizabethae from R. rattus; these genotypes were closely related to Bartonella massiliensis recently described [125] from Cricetomys sp. as well as a new genotype proposed as Candidatus Bartonella gabonensis from Lo. sikapusi. Thus, we identified 13 (6,6%) positive individuals with the qPCR system targeting 16S-23S rRNA, including one from R. rattus, two from Cricetomys sp and ten from Lophuromys sikapusi. Among the three genotypes that we have highlighted, the pathogenicity of two, B. massiliensis and Candidatus B. gabonensis, is not yet known. B. elizabethae is known to cause febrile human diseases [126], usually resulting from close contact between humans and rodents. Here, B. elizabethae was described in R. rattus captured inside a house, which implies close contact with the human inhabitant of this house and therefore an increased risk of infection with Bartonella, as reported [127].

Trypanosoma species are flagellated protozoan parasites including species that are highly pathogenic to humans, such as T. cruzi, responsible for American Chagas disease, and T. brucei, which is the causal agent of human African trypanosomiasis (HAT), also known as African sleeping sickness. Both are transmitted to humans by biting insects (Triatominae and tsetse flies, respectively) [128]. In rodents, many studies have revealed the presence of T. lewisi, for which rats were initially considered the only specific hosts [129]. However, T. lewisi has also been found in other rodents [START_REF] Tatard | Rodent-borne Trypanosoma from cities and villages of Niger and Nigeria: A special role for the invasive genus Rattus?[END_REF] and even in insectivores [130]. This parasite is transmissible to humans, although instances of lethal human infection have been reported in both Asia and Africa [131,132]. In the present study, we did not find T. lewisi. However, among the 14 individuals that tested positive for Trypanosoma spp. by qPCR, we successful amplification was achieved from 6. Two of these sequences were identical to a T. congolense riverine/forest-type from Le. striatus and Cricetomys sp.; one was identical to both T. brucei brucei and T. brucei gambiense from Praomys sp.; and the last two were identical to T. otospermophili from R. rattus and Mus (Nannomys) sp. Trypanosoma congolense riverine/forest-type trypanosomes are the most economically important trypanosomes causing African animal trypanosomiasis (AAT) and losses in domestic animals (cattle, goats, sheep, horses, pigs and dogs) in sub-Saharan Africa [133,134]. Trypanosoma congolense has been classified as savannah, riverine-forest and Kilifi types, which are morphologically identical but genetically heterogeneous types that vary in their virulence, pathogenicity, vectors and geographical distribution [135]. Nevertheless, studies have frequently identified coinfections of different T. congolense types in livestock and tsetse flies [136]. On the other hand, Trypanosoma otospermophili is a species hosted by rodents that is very poorly studied [137]. Trypanosoma brucei brucei is the only subspecies among the three that is not infectious to humans. T. brucei gambiense and T. brucei rhodesiense cause a chronic form and an acute form of sleeping sickness, respectively [128]. The identification of T.brucei gambiense reveals the risk of transmission of this pathogen from rodents to humans in Franceville. Nevertheless, a specific analysis (another gene or more variable regions) would be necessary to determine precisely which species is described here, T. brucei brucei or T. brucei gambiense.

Toxoplasma gondii is an intracellular protozoan responsible for toxoplasmosis, which is an anthropozoonosis that is widely distributed around the world. Domestic or wild felids are the definitive hosts of this parasite, and a wide range of terrestrial or marine mammals and birds, including rodents, are intermediate hosts [138]. We found one individual positive for Toxoplasma from Le. striatus by qPCR, but amplification to describe the genotype was unsuccessful. Indeed, T. gondii is a monospecific genus; however, it presents several strains whose virulence profiles are variable according to the host species [108]. This result reflects the lack of specific PCR amplification tools, which are currently being developed following the recommendations of various collaborators.

Furthermore, our results show that the prevalence of pathogens is higher in native rodents, notably species such as Lemniscomys sp. (55%), Lophuromys sp. (62%) and Praomys sp. that are associated with vegetation, compared to that found in the commensal invasive species R. rattus (23%) and M. m. domesticus (3.4%) (Table 3). This situation is similar to what was recently highlighted in Senegal [START_REF] Dahmana | Rodents as Hosts of Pathogens and Related Zoonotic Disease Risk[END_REF]. It would be difficult to speculate here about the involvement of these microparasite communities in the invasion of the black rat or even the domestic mouse, as described, for example, in Senegal [START_REF] Diagne | Ecological and sanitary impacts of bacterial communities associated to biological invasions in African commensal rodent communities[END_REF]. We do not have enough data to make such inferences.

Otherwise, the pathogen species detected and described according to phylogenetic trees (Figs 2345) are pathogens associated with rural areas, peridomestic areas or even plants. With the exception of Leptospira species, B. elizabaethae and T. gondii, the other pathogens identified here, including the Trypanosoma species, are not infectious to humans but cause disease in domestic animals [139]. Therefore, these results highlight the relationship between vegetation and pathogens, more specifically, the implication of interactions between wildlife and domestic fauna in the circulation of infectious agents and their transmission to humans. Our results also highlight multihost pathogens, particularly T. otospermophili ( Conversely, pathogenic agents of monospecific hosts have also been detected, as indicated by the phylogenetic tree of Bartonella species. The probable presence of a new species, referred to as Candidatus Bartonella gabonensis, was observed in the rodent species Lo. sikapusi. In addition, our results provide evidence of the circulation of new bacterial genotypes: Candidatus Bartonella gabonensis and Uncultured Anaplasma spp.

Our results concerning the pathogens C. burnetii and Anaplasma spp. could also correspond to the spill back hypothesis [141] because these pathogens associated with forest and savannah rodents are found in the invasive rodent R. rattus. Additional data would be required to confirm this assumption.

None of the factors analyzed here as potential determinants of parasitic richness in Franceville rodents can be questioned except for animal weight, where we found that the heaviest rodents were the most infected (Table 4), as previously reported [START_REF] Diagne | Ecological and sanitary impacts of bacterial communities associated to biological invasions in African commensal rodent communities[END_REF][START_REF] Dahmana | Rodents as Hosts of Pathogens and Related Zoonotic Disease Risk[END_REF]142]. Larger individuals may have a larger home range, which increases their frequency of contact with parasites [143]. In addition, since body mass can be considered an indicator of host age, the generally positive correlation between infection and body mass may reflect the longer duration of exposure in older rodents [START_REF] Diagne | Ecological and sanitary impacts of bacterial communities associated to biological invasions in African commensal rodent communities[END_REF].

This study is the first epidemiological investigation of infectious agents carried by rodents in Franceville and thus contributes to the identification and taxonomic description of infectious agents circulating in Gabon. It highlights the presence of 7 kinds of infectious agents, including several pathogenic agents, particularly Coxiella burnetii, Leptospira spp., Bartonella elizabethae and Toxoplasma gondii in Gabonese rodents native to the forest and the savannah rodents Lophuromys sp, Le. striatus and Praomys sp. as well as the invasive rodent R. rattus. These results show that many infectious agents that are pathogenic to humans are in circulation and reveal the need for systematic detection methods for these infectious agents in humans. Indeed, in Africa, many febrile diseases of unknown etiologies can be attributed to these agents. Our results also reveal the need for further studies to establish the zoonotic risks associated with these new potential species of circulating pathogens, particularly Uncultured Anaplasma spp, Candidatus Bartonella gabonense and Theileria sp., to determine whether these agents (new and already known) could be responsible for human cases of febrile diseases of unknown etiology in Gabon.

Fig 1 .

 1 Fig 1. Map of Franceville. Study area and location of the six districts sampled for rodents. https://doi.org/10.1371/journal.pone.0248244.g001

Table 2 .

 2 Method of investigation of targeted bacteria and protozoa.

Table 3 .

 3 Bacteria and protozoa identified in Franceville rodents.

  identified and described in this study and the rodents associated with them. One hundred and ninety-eight (198) rodents, collected in N1, N2 and N3 were analyzed by qPCR. Ŧ indicates invasive rodents. � Mang represents the Mangoungou district. https://doi.org/10.1371/journal.pone.0248244.t003

Fig 2 .

 2 Fig 2. Taxonomic tree and description of the identified Bartonella. Phylogenetic tree of Bartonella spp. identified in rodents in Franceville. The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. The analysis involved 36 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 189 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. Sequences obtained in this study are indicated in bold. The hosts are indicated after the underscore. https://doi.org/10.1371/journal.pone.0248244.g002

  Leptospira. Seven samples were positive in qPCR screening of the 16S rRNA gene of Leptospira sp. The sequencing of a portion of the LipL 32 gene from the DNA extracts of 7 qPCRpositive individuals revealed two Leptospira sequences (MT274303 and MT274304). Sequence MT274303 was 99.1% (442/446; 444/448) similar to both Leptospira borgpetersenii serovar Hardjo (CP033440.1) and Leptospira weilii (AY461930.1), identified from Lophuromys sp. Sequence MT274304 was 100% (474/474) similar to both Leptospira kirshneri (JN683896.1) and Leptospira interrogans (KC800991.1), identified in the Crocidura goliath shrew (MT256384.1). Trypanosoma. Five sequences of Trypanosoma ranging from 464 to 571 bp were obtained (GenBank: MT271793 to MT271797) after the sequencing of 550bp of the 28 S gene from 14 qPCR-positive individuals identified when screening for Trypanosoma spp. (10 positive individuals) and the Kinetoplastidae class (4 positive individuals). Two presented as T. congolense riverine/forest-type (U22319) (2/198) with 99% (570/571) of identity from Le. striatus and

Fig 3 .

 3 Fig 3. Taxonomic tree and description of the identified Candidatus Anaplasma gabonense. Phylogenetic tree of Anaplasma species identified in rodents of Franceville. The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. The analysis involved 24 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 420 positions in the final dataset. Sequences obtained in this study are indicated in bold. Evolutionary analyses were conducted in MEGA7. The hosts are indicated after the underscore. Ls, Lemniscomys striatus; Rr, Rattus rattus. https://doi.org/10.1371/journal.pone.0248244.g003

Fig 4 .

 4 Fig 4. Taxonomic tree and description of the identified Trypanosoma sp. Maximum likelihood tree of Trypanosoma species identified in rodents in Franceville. Sequences obtained in this study are indicated in bold. The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. The analysis involved 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 415 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. The hosts are indicated after the underscore. Ls, Lemniscomys striatus; C. sp., Cricetomys sp.; MN, Mus Nannomys sp. https://doi.org/10.1371/journal.pone.0248244.g004

Fig 5 .Fig 6 .

 56 Fig 5. Taxonomic tree and description of the identified Theileria sp. Phylogenetic tree of Theileria species identified in rodents in Franceville. Sequences obtained in this study are indicated in bold. The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. The analysis involved 36 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 749 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. Ls, Lemniscomys striatus. https://doi.org/10.1371/journal.pone.0248244.g005

Fig 4 )

 4 and Candidatus Anaplasma gabonensis (Fig 3), which infect both R. rattus and Mus (Nannomys) sp. or both R. rattus Praomys sp. and Le. striatus, respectively [140].

Table 1 . Method of investigation of targeted viruses. Virus familly Target virus (group) Technique Target gene Primer names Sequences (5'-3') Amplification Amplicon References

 1

	[70]							[53]				[71]			[72]			[73]				[73]				[73]			[74]		[75]
	640 bp			460 bp							236 bp			388 bp			497 bp				250 bp				300 bp			160 bp		280 bp	210 bp
	95˚C for 2min, then 40 cycles of	95˚C-30 S, 55˚C-30 S and 72˚C	-1 min. Extension 72˚C -5min.	Same for the both round			95˚C -20 sec, then 45cycles of	95˚C-3 sec 57˚C-30 sec			95˚C 2 m, then 45 cycles of 95˚C	-15 S, 52˚C-30 S and 72˚C -1m.	72˚C -1 m	95˚C-5 m, then 45 cycles of	96˚C-30 S, 60˚C-35 S and 72˚C	-50 S. finish with 72˚C-5min.	60˚C-1 min for denaturing, 44 to	50˚C-30 min (for RT), 94˚C-2	min, and then 40 cycles of 94˚C-	15 s, 48 to 50˚C-30 s, 72˚C-30	s, and final extension 72˚C-7 min	For semi-nested: 94˚C-2 min,	and then 40 cycles of 94˚C-15 s,	48 to 50˚C-30 s, 72˚C-30 s, and	final extension 72˚C-7 min			95˚C -20 sec, then 45cycles of	95˚C-3 sec 57˚C-30 sec	45˚C-20 min, 95˚C-2 min,	then45 cycles of 95˚C-25 sec,	51˚C-30 sec, 68˚C-30 sec. End	68˚C-5 min Semi-nested: 94˚C-2	min,then45 cycles of 94˚C-25	sec, 5˚C-30 sec, 72˚C-30 sec.	End 72˚C-5 min
	Arenaviridae Mammarenavirus � Nested PCR S ARS16V GGCATWGANCCAAACTGATT	ARS1 CGCACCGGGGATCCTAGGC	ARS3V CATGACKMTGAATTYTGTGACA	ARS7C ATRTGYCKRTGWGTTGG	(modified)	Arenaviridae LCMV qRT-PCR GPC LCMVS GGGATCCTAGGCTTTTTGGAT	LCMVAS GCACAATAATGACAATGTTGAT	LCMVP-CCTCAAACATTGTCACAATCTGACCCAT	FAM	Hantaviridae Hantavirus � Conventionnal S UHantaF2 GGVCARACWGCHGAYTGG	PCR UHantaR2 TCITCWGCYTTCATDSTDGA	L HAN-L-F2 TGCWGATGCHACIAARTGGTC	HAN-L-R2 GCRTCRTCWGARTGRTGDGCAA	Paramyxoviridae Paramyxovirus One step RT-PCR L RMH-F1 TCITTCTTTAGAACITTYGGNCAYCC	(Respirovirus, / semi-nested PCR RMH-F2 Morbillivirus, GCCATATTTTGTGGAATAATHATHAAYGG RMH-R Henipavirus) CTCATTTTGTAIGTCATYTTNGCRAA	Paramyxovirus One step RT-PCR L AR-F1 GGTTATCCTCATTTITTYGARTGGATHCA	(Avulavirus, / semi-nested PCR AR-F2 Rubulavirus) ACACTCTATGTIGGIGAICCNTTYAAYCC AR-R GCAATTGCTTGATTITCICCYTGNAC	Pneumoviridae Paramyxovirus One step RT-PCR L PNE-F1 GTGTAGGTAGIATGTTYGCNATGCARCC	(Pneumovirinae) / semi-nested PCR PNE-F2 ACTGATCTIAGYAARTTYAAYCARGC	PNE-R GTCCCACAAITTTTGRCACCANCCYTC	Flaviviridae Zika virus One step RT-NS5 ZIKV 1086 CCGCTGCCCAACACAAG	qPCR ZIKV CCACTAACGTTCTTTTGCAGACAT	1162c	ZIKV AGCCTACCTTGACAAGCAGTCAGACACTCAA	1107-FAM	Flaviviridae Flavivirus One step RT-PCR NS5 PF1S TGYRTBTAYAACATGATGGG	/ semi-nested PCR PF3S ATHTGGTWYATGTGGYTDGG	PF2Rbis GTGTCCCAiCCNGCNGTRTC	Oligonucleotide sequences of the primers and probes used for virus detection in rodent spleens in this study.	� Analyses were performed with cDNA using Superscript III following the manufacturer's instructions.	https://doi.org/10.1371/journal.pone.0248244.t001

  Oligonucleotide sequences of primers and probes used for real-time PCR and conventional PCR to screen bacteria and protozoans in this study.https://doi.org/10.1371/journal.pone.0248244.t002factors, including host factors (species, sex, weight, body mass), status (native vs. invasive), trap location (inside vs. outside the door), habitat type (central districts, peripheral districts, vegetal areas) and seasons (dry season and rainy season), were considered fixed effects (S2 and S3 Tables). The significance of the interactions of different effects was estimated by using the Akaike information criterion (AIC) for model selection. AIC changes were evaluated when model parameters were modified (added or removed). Full-model averages, available in the MuMIn package[START_REF] Barton | Multi-model inference[END_REF], were used for AIC estimation. The best model showed a null ΔAICC.

		[88]
		~550 bp
		58
	6-FAM-GTTGAAGAACGCAGCAAAGGCGAT	ACCAAGGAGTCAAACAGACG	ACGCCACATATCCCTAAG
		F2 28S	R1 28S
		Broad-range	conventional PCR
		28S

  A total of 198 small mammals were captured including 49 in Mbaya,[START_REF] Cohen | Urbanization in developing countries: Current trends, future projections, and key challenges for sustainability[END_REF] in Mangoungou, 19 in Ombe ´le ´, 15 in Potos, 25 in Sable, 18 in Ye ´ne ´and 53 in vegetative areas (S2 and S3 Tables). The captured animals included two (2) invasive species of rodents, Rattus rattus (N = 54) and Mus musculus domesticus (N = 29), five (5) native rodent taxa, Lophuromys sp. (N = 27), Lemnisc-According to the three types of established habitats, small mammals were distributed as follows 29 rodents (3 Lemniscomys striatus, 2 M. m. domesticus, 1 Praomys sp., 3 Mus (Nannomys) sp. and 20 R. rattus) and 5 shrews in central districts; 102 rodents (3 Cricetomys sp, 8 Le. striatus, 7 Lophuromys sp, 27 M. m. domesticus, 17 Mus (Nannomys) sp., 6 Praomys sp., 34 R. rattus) and 9 shrews in peripheral districts and 48 rodents (16 Le. striatus, 20 Lo. sikapusis, 2 Mus (Nannomys) sp., 10 Praomys sp.) and 5 shrews in forest-savannah areas (Table3).

omys striatus (N = 27), Praomys sp. (N = 17), Mus (Nannomys) sp. (N = 22) and Cricetomys sp. (N = 3), and shrews (N = 19).

  8.1%; 16/198), Bartonella spp. (6.6%; 13/198), Coxiella burnetii (5.1%; 10/198), and Leptospira spp. (3.5%; 7/198). The protozoans that we identified included Trypanosoma sp (7%; 14/198), Piroplasma sp (1%; 2/ 198) and Toxoplasma gondii (0.5%; 1/198) (Table

Table 4 . Factors and parasitic richness association. Fixed effects: Estimate SE t -value 2.5% 97.5% p-value

 4

	(Intercept)	0.321	0.146	2.192	0.034	0.608	0.030
	Sex a	0.007	0.081	0.083	-0.152	0.166	0.934
	Trap location b	-0.128	0.119	-1.070	-0.362	0.106	0.286
	Season c	-0.158	0.087	-1.821	-0.327	0.012	0.070
	Weight	0.001	0.000	2.019	0.000	0.002	0.045
	Species status d	0.092	0.117	0.791	-0.136	0.321	0.430
	Habitat types e	0.082	0.142	0.576	-0.197	0.360	0.586
	Evaluation of the Poisson generalized linear mixed models fitted to estimate the host factors and parasitic richness
	association.
	The reference categories corresponding to:
	a male,
	b indoor,
	c rainy season,

d native and e vegetal areas. https://doi.org/10.1371/journal.pone.0248244.t004

PLOS ONE | https://doi.org/10.1371/journal.pone.0248244March 8, 2021

PLOS ONE | https://doi.org/10.1371/journal.pone.0248244 March 8, 2021 / 28

Acknowledgments

We thank the Association pour la recherche en infectiologie (APRI) for his support in finalizing this study. We thank the authorities of the city of Franceville as well as the leaders of the districts where rodent sampling took place. We also express our deep gratitude to those individuals who have kindly given us access to their homes. We also thank Mr AWOUNDJA Lauriano for assistance with the trapping of rodents and the interpretation of local languages and Randy Lyn ESSONO for his help in mapping. Finally, this acknowledgements section would be incomplete without conveying our gratitude to Dr Ce ´lia BOUMBANDA KOYO, Handi DAHMANA and Marielle BEDOTTO-BUFFET for their help with molecular biology analyses in the laboratory and Dr Serge DIBAKOU for his support in statistical analysis.

JBM 1: AUF-Agence universitaire de la Francophonie. https://www.auf.org/afriquecentrale-grands-lacs/nouvelles/actualites/listebeneficiaires-college-doctoral-regionalmathematiques-informatique-biosciencesgeosciencesstudy design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the paper and its Supporting Information files.

Supporting information

S1