New records of bacteria in different species of fleas from France and Spain

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: COMPARATIVE IMMUNOLOGY, MICROBIOLOGY AND INFECTIOUS DISEASES on June 2021, available at: https://doi.org/10.1016/j.cimid.2021.101648 .” 1 New records of bacteria in different species of fleas from France and Spain. 1 2 Antonio Zurita1, Linda Benkacimi2,3, Khalid El Karkouri2,3, Cristina Cutillas1, Philippe 3 Parola2,3, Maureen Laroche2,3* 4 1Department of Microbiology and Parasitology. Faculty of Pharmacy. University of 5 Seville. Professor García González 2, 41012 Seville, Spain. 6 2 IHU-Méditerranée Infection, Marseille, France. 7 3 Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, Marseille, France. 8 9 * Corresponding author: Dr. Maureen Laroche. VITROME, IHU Méditerranée 10 Infection. 19-21 Boulevard Jean Moulin 13005 Marseille, France Phone: + 33 (0) 4 13 11 73 24 01. Fax: + 33 (0) 4 13 73 24 02. E-mail address: [email protected] 12 13 14 15 16 17 18 19 20 © 2021 published by Elsevier. This manuscript is made available under the CC BY NC user license https://creativecommons.org/licenses/by-nc/4.0/ Version of Record: https://www.sciencedirect.com/science/article/pii/S0147957121000400 Manuscript_ea8593487337033a652e6a9159b3613b 2 Abstract 21 In this study, we assessed the presence of vector-borne microorganisms in different 22 species of fleas collected from different hosts in diverse areas of South-Western Europe 23 by molecular methods. A total of 319 fleas belonging to eight different species was 24 tested for the presence of eight microorganisms. Wolbachia spp. endosymbionts were 25 detected in Ctenocephalides felis, Pulex irritans, Archaeopsylla erinacei and 26 Ctenophthalmus baeticus boisseauorum specimens. Rickettsia felis, an emerging 27 pathogen, was detected in C. felis, A. erinacei and Ct. b. boisseauorum. Rickettsia typhi, 28 the agent of murine typhus was detected for the first time in A. erinacei and 29 Mycobacterium spp. were detected for the first time in fleas (C. felis, P. irritans and A. 30 erinacei). Lastly, five different species of Bartonella were detected in fleas’ DNA in 31 this study, including a possible new bacterium belonging to this genus. With this study, 32 we updated the knowledge of the flea-borne bacteria present in the South-West of 33 Europe reinforcing the idea about the necessity to expand and increase the current 34 knowledge on flea-borne pathogens. 35 36 Keywords: Siphonaptera; flea-borne diseases; Bartonella spp.; Rickettsia felis; 37 Rickettsia typhi. 38 39 40 41 1. Introduction 42 3 Vector-borne diseases cause significant morbidity and mortality throughout the world. 43 The majority of recent vector-borne zoonoses research focuses on tick- and mosquito- 44 borne illnesses, such as Lyme disease and arboviral diseases, respectively [1]. 45 Nevertheless, the public health importance of flea-borne infections has been reported in 46 several studies in recent years [2]. 47 Several studies published in the last twenty years have warned about the possibility of 48 emergence or reemergence of certain vector-borne diseases in several geographic areas, 49 including flea-borne diseases [3, 4, 5, 6, 7, 8, 9]. In this sense, health care professionals 50 and health authorities should be aware of the flea-borne pathogens circulating in their 51 area in order to improve diagnosis and vector surveillance, and if necessary be able to 52 manage a potential outbreak [2]. 53 The role of fleas in the transmission of several pathogenic agents such as bacteria, 54 viruses and helminths is well known [2, 10]. Therefore, in the last years, numerous 55 studies have assessed the presence of pathogenic microorganisms in fleas using 56 molecular methods [3] or even proteomics methods more recently [11]. Nevertheless, 57 the role of fleas as competent vectors of several zoonotic pathogens such as rickettsiae 58 or Bartonella species has been poorly studied. Moreover, epidemiological data is still 59 missing for several flea species which may play an important role in the transmission of 60 zoonotic vector borne pathogens. 61 Other pathogens have been detected in fleas such as Coxiella burnetii [12], Leishmania 62 infantum [13], Francisella tularensis [14] or Borrelia burgdorferi [15]. As for 63 Wolbachia spp., they are considered a common endosymbiont of several arthropod 64 species including fleas [16]. 65 4 The aim of this work was to assess the presence and prevalence of several 66 microorganisms (Bartonella spp., Rickettsia spp., C. burnetii, Borrelia spp., Leishmania 67 spp. and Mycobacterium spp., Wolbachia spp.) in different flea species collected from 68 domestic, peridomestic and wild animals from different geographical areas of Spain and 69 France. 70 2. Material and Methods 71 2.1 Ethical statement 72 Catching and handling procedures of animals in this study were carried out according to 73 the guidelines provided by the Institutional Animal Care of the University of Seville 74 based on RD 53/2013 of January 1 st , 2013 (BOE number 34 of February 8 th , 2013) of 75 the Spanish government. Rodents were trapped using Sherman traps (H. B. Sherman 76 Inc., Tallahassee, Florida) and released into their natural habitat after flea collecting 77 procedures. Dogs belonged to private owners and public dog kennels and the consent of 78 owners and dog kennels chairman was obtained for the inclusion of the dogs in the 79 study. Rodents and dogs were exhaustively and manually examined for fleas through an 80 inspection of head, neck, body, sides, tail, and ventral regions of each animal. 81 Hedgehogs killed on roads at night were collected by hand early morning. Collected 82 hedgehogs were taken to the laboratory and visually inspected for ectoparasites on a 83 white sheet. No specific approval of any Institutional Animal Care and Use Committee 84 were needed in this study since no anesthesia substances were applied for animal 85 handling procedures. 86 2.2 Sample collection and morphological identification 87 Fleas were collected from March 2011 to April 2018 from dogs (Canis lupus 88 familiaris), hedgehogs (Erinaceus europaeus) and rodents (Rattus sp., Mus sp., 89 5 Apodemus sp. and Arvicola terrestris) from different localities of the South of Europe 90 (Spain and France). Details about geographical areas, number and sex of samples, 91 preservation methods, date of collection and hosts species tested in this study are 92 showed in Table 1. Dogs were randomly selected by breed, sex and age in the study 93 area. All dogs handled in this work were domestic dogs. Owners reported flea 94 infestation in their pets to their veterinarians. Pets were subsequently included in the 95 study after obtaining owners´ permission to examined their dogs for fleas. Rodents were 96 trapped using live traps, whereas, hedgehogs killed on roads at night were located and 97 collected by hand early morning. Collected hedgehogs were taken to the laboratory and 98 then placed on a white sheet of paper in order to be visually examined for ectoparasites. 99 Dogs, hedgehogs and rodents were exhaustively examined for fleas through an 100 inspection of head, neck, body, sides, tail, and ventral regions of each animal. All these 101 hosts were examined by combing for a standardized period of time of 15 minutes. On 102 the other hand, some specimens were collected from a neglected horse stable with a flea 103 infestation. This place had been previously populated by horses, dogs and probably 104 rodents; however, specimens were directly caught from the ground. All flea specimens 105 were preserved in 70% and 96% ethanol at room temperature until morphological 106 identification. Fleas were collected and morphologically identified using different 107 taxonomic keys. Genus and species identification were carried out according to different 108 authors and taxonomic keys [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29]. For 109 morphological analysis, all specimens were examined and photographed using a Nikon 110 eclipse Ci microscope (Minato City, Tokyo, Japan) equipped with a camera lucid 111 system and a photomicroscope. 112 2.3 Molecular detection of pathogens 113 6 Prior to DNA amplification, the abdomen of each specimen was cut off and transferred 114 to a 1.5 mL tube containing 180 μL of G2 lysis buffer (Qiagen, Hilden, Germany) and 115 20 μL of proteinase K (Qiagen), and incubated at 56º C overnight. DNA extraction was 116 individually performed with an EZ1 DNA Tissue Kit (Qiagen) according to the 117 manufacturer’s recommendations. Flea DNAs were then eluted in 100 μL of Tris EDTA 118 buffer using the DNA extracting EZ1 Advanced XL Robot (Qiagen). The DNA was 119 either immediately used or stored at −20° C until molecular analysis. The DNA 120 extracting EZI Advanced XL Robot was disinfected after each batch of extraction as per 121 the manufacturer’s recommendations, to avoid cross-contamination. 122 Quantitative PCR was individually performed according to the manufacturer's protocol 123 using a PCR detection system: a CFX Connect™ Real-Time (Bio-Rad) with the Light 124 Cycler 480 Probes Master Mix (Roche Diagnostics, Meylan, France). The qPCR 125 reaction contained 10 μl of Light Cycler 480 Probes Master Mix (Roche Diagnostics, 126 Meylan, France) , 3 μl sterile distilled water, 0.5 μl of each of the primers, 0.5 μl of 127 Uracil-DNA-glycosylase (UDG), 0.5 μl of probe and 5 μl of the DNA extract. All 128 samples were screened using primers and probes targeting specific sequences of the 129 following microorganisms: Rickettsia spp., R. felis, R. typhi, Anaplasmataceae, 130 Wolbachia spp., Borrelia spp., Bartonella spp., B. henselae, B. quintana, C. burnetii, 131 Leishmania spp. and Mycobacterium spp. The primers and probes references and the 132 targeted sequences used to detect each pathogen by real-time quantitative PCR are 133 summarized in table 2. DNA from our laboratory cultures of R. montanensis, R. felis, R. 134 typhi, Anaplasma phagocytophilum, W. pipientis, Borrelia crocidurae, B. quintana, B. 135 henselae, C. burnetii and Mycobacterium ulcerans were used as PCR positive controls. 136 For C. burnetii two genes were tested in qPCR in order to avoid false positives, thus 137 only the samples positive for both genes were considered positive. For all samples 138 7 tested by qPCR, only those with a cycle threshold (Ct) value lower than 35 were 139 considered positive. The Bartonella spp.-positive samples that were negative for B. 140 henselae- and B. quintana-specific qPCRs were subsequently subjected to standard PCR 141 prior to sequencing in order to obtain identification to the species level. The same 142 protocol was carried out for the other bacteria genera; each time we were not able to 143 obtain a specific identification by qPCR, we used sequencing methods. Details about 144 the primers used for standard PCR in this study are also listed in table 2. A Bio-Rad 145 Thermocycler (Bio-Rad Laboratories, Hercules, California, USA) was used to amplify 146 the target sequences prior to sequencing. The cycling protocol and PCR mix were 147 described by Diarra et al. [47]. Amplificated products were visualized by 148 electrophoresis through a 1.5 % agarose-tris-borate-EDTA gel containing SYBR Safe. 149 PCR products were sequenced in both directions using a Big Dye Terminator kit and an 150 ABI PRISM 3130 Genetic Analyzer (Applied BioSystems, Courtabeauf, France). The 151 sequences were analysed using the ABIPRISM DNA Sequencing Analysis software 152 version 3.0 (Applied BioSystems) and compared with sequences available in the 153 GenBank database using the BLAST algorithm (http: //blast.ncbi.nlm. 154 nih.gov/Blast.cgi). 155 2.4 Phylogenetic analysis 156 The obtained partial 16S-23S Intergenic Spacer (ITS) sequences of Bartonella spp. 157 were firstly subjected to BLAST analysis using the NCBI GenBank database. 158 Phylogenetic analyses were then carried out using included reference sequences of 159 different Bartonella species which are commonly detected in fleas. Phylogenetic trees 160 were inferred using nucleotide data and performed using two methods: Maximum 161 Likelihood (ML) trees were generated using the PHYML package from [48] whereas 162 Bayesian inferences (B) were generated using MrBayes-3.2.6 [49] JMODELTEST [50] 163 8 program was used to determinate the best-fit substitution model for the bacteria data. 164 Models of evolution were chosen for subsequent analyses according to the Akaike 165 Information Criterion [51, 52]. Support for the topology was examined using 166 bootstrapping (heuristic option) [53] over 1000 replications to assess the relative 167 reliability of clades. For ML inference, best-fit nucleotide substitution model included 168 general time reversible model with gamma-distributed rate variation GTR + G. The 169 commands used in MrBayes-3.2.6 for BI were nst = 6 with gamma rates. For Bayesian 170 inference, the standard deviation of split frequencies was used to assess if the number of 171 generations completed was sufficient; the chain was sampled every 500 generations and 172 each dataset was run for 10 million generations. Adequacy of sampling and run 173 convergence was assessed using the effective sample size diagnostic in TRACER 174 program version 1.6 [54]. Trees from the first million generations were discarded based 175 on an assessment of convergence. Burn-in was determined empirically by examination 176 of the log likelihood values of the chains. The Bayesian Posterior Probabilities (BPP) 177 was percentage converted. 178 The ftsZ partial sequences from our Bartonella spp. and those obtained from GenBank 179 database belonging to different Bartonella species were aligned and subjected to 180 phylogenetic analysis in order to carry out a specific identification. Phylogenetic trees 181 were inferred using Neighbor-joining (NJ) and Maximum Likelihood (ML) under 182 Kimura-2-parameter +G and GTR+G+I models, respectively. The trees were plotted 183 using the MEGA version X [55] software. Support for the topology was examined using 184 bootstrapping [53] over 1000 and 200 replications for NJ and ML respectively. 185 3. Results 186 3.1 Morphological identification 187 15 sequence Mycobacterium qPCR-positive samples by standard PCR in order to obtain an 336 identification to the species level. This fact could be explained by the higher sensitivity 337 of qPCR compared to standard PCR [87]. Therefore, although we could not determine 338 which species of Mycobacterium we dealt with, this study constitutes the first report of 339 the detection of Mycobacterium spp. in fleas. In this sense, this bacterium genus should 340 be included in flea epidemiological studies in order to increase the knowledge about 341 Mycobacterium species potentially carried by fleas. Nevertheless, experimental models 342 would still be needed to evaluate if fleas could really play a role in Mycobacterium spp. 343 transmission. 344 Nowadays, 11 species of Bartonella are known to cause human diseases [88]. These 345 infections can result in significant complications such as endocarditis, bacillary 346 angiomatosis, peliosis hepatis, chronic bacteremia, chronic lymphadenopathy, or 347 neurological disorders [88]. Particularly, B. henselae and B. clarridgeiae are considered 348 the main causative agents of cat scratch disease, while B. quintana, B. elizabethae, B. 349 henselae, or B. koehlerae have been related to human and animal endocarditis in several 350 countries [89, 90]. A large list of mammals is considered as reservoirs of Bartonella 351 spp., out of which several species are kept as pets [91]. Arthropods are known to 352 transmit Bartonella spp. either through saliva (fleas, sand flies) or through their feces 353 (fleas and lice) [92]. 354 Within these pathogenic Bartonella species, at least 8 (B. henselae, B. clarridgeiae, B. 355 quintana, B. koehlerae, B. tribocorum, B. taylorii, B. grahamii and B. elizabethae) have 356 been detected in different flea species worldwide by molecular methods [3]. Five 357 different species of Bartonella infesting fleas were detected in this study: B. quintana, 358 B. henselae, B. taylorii, B. elizabethae and B. doshiae. This genus showed a prevalence 359 of 8.7 % in P. irritans isolated from Seville. Our results are in agreement with Márquez 360 16 et al. [93] who observed a prevalence of 31.8 % of this pathogenic bacterium in P. 361 irritans isolated from red foxes from Andalusian region (Huelva and Jaén). On the other 362 hand, three different species of Bartonella (B. quintana, B. henselae and B. elizabethae) 363 were detected in S. t. tripectinata isolated from Corsica (France), whereas B. elizabethae 364 and B. henselae were detected in N. fasciatus collected from the same island. This 365 completes recent results from the Canary Islands reporting B. elizabethae as the sole 366 Bartonella species found in S. t. tripectinata fleas [94]. As far as we know, this work is 367 the first investigation on the prevalence of Bartonella species in fleas from this 368 geographical area. Furthermore, L. taschenbergi was shown to harbor B. doshiae and B. 369 elizabethae, the latter was also detected in Ct. b. boisseauorum. This works provides an 370 updated list of flea species which could potentially play a role in the transmission of this 371 pathogen in Europe. 372 Lice are considered the main vector in the transmission of B. quintana [95]; but, Kernif 373 et al. [96] reported the fact that cat fleas (C. felis) can transmit this bacterium by 374 releasing viable microorganisms into their faeces. Thus, they suggested that fleas might 375 play a role as vectors of trench fever or other infections caused by B. quintana. In our 376 study, it should be noted the presence of B. quintana, the causative agent of trench 377 fever, was detected in S. t. tripectinata fleas. 378 Based on epidemiological data provided in this work it seems plausible that different 379 Bartonella species could be circulating among diverse rodent genus (Rattus sp., 380 Arvicola sp., Apodemus sp and Mus sp.) and infecting different flea species in Corsica 381 Island and North of Spain. Although the vectorial competence of these flea species 382 needs to be confirmed with experimental studies, the zoonotic character of these 383 infections must alert public health authorities and the veterinary community about the 384 risk of possible diseases associated with these flea species and their rodent hosts. 385 17 As for B. taylorii, it was detected in three different species of rodent fleas: Ct. b. 386 boisseauorum, Ct. a. allani and L. taschenbergi, with the prevalence (6.6 %). This 387 bacterium can cause infection in animals, but the real pathogenic potential of this 388 Bartonella species is yet unknown [97]. Nevertheless, the fact that rodent fleas could be 389 substantial vectors for the transmission of B. taylorii among rodents has been reported 390 in several former studies [97, 98, 99, 100]. Therefore, in the last years, B. taylorii has 391 been detected in more and more flea species encouraging veterinary and medical 392 community to assess the possible pathogenic potential of this Bartonella species in 393 humans and animals and to investigate which flea species could be implicated in the 394 transmission of this bacterium. Lastly, complementary molecular and phylogenetic 395 analysis based on ftsZ and gltA genes allowed us to detect B. doshiae in one specimen of 396 L. taschenbergi collected from the French Pyrenees. This bacterium was detected in 397 fleas for the first time using rodent fleas collected from Afghanistan [100]. Our results 398 constitutes the first evidence of the presence of this bacterium in fleas from Western 399 Europe. It is noteworthy, that Vayssier-Taussat et al. [101] found this species in the 400 blood of French human patients reporting tick bites. These patients exhibited non- 401 specific symptoms, suggesting a zoonotic potential of this bacterium. 402 Phylogenetic analysis based on ftsZ and gltA genes did not allow to characterize the 403 second Bartonella species detected in L. taschenbergi. Thus, it would be necessary to 404 conduct complementary studies involving the isolation of the possible novel strain of 405 Bartonella spp. in order to confirm a possible new species. 406 5. Conclusions 407 With this study we updated the knowledge of the flea-borne pathogens present in some 408 regions of south of France and Spain. Additional studies are needed to expand the 409 18 current knowledge on flea-borne pathogens and characterize new host-vector 410 relationships. 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Nosopsyllus fasciatus. 728 Figure 2. Phylogenetic tree of Bartonella spp. based on 16S-23S Intergenic Spacer 729 Region (ITS region) inferred using the Bayesian (B) and Maximum-Likelihood (ML) 730 methods and Bayesian topology. The percentage of replicate trees in which the 731 associated taxa clustered together in the bootstrap test (1000 replicates) is shown on the 732 branches (B/ML). Bootstrap and BPP values lower than 60 % are not shown. Bayesian 733 posterior probabilities (BPP) are percentage-converted. (*) A total of four ITS 734 sequences of Bartonella spp., which did not, showed nucleotide differences among 735 them. In bold those sequences, which showed low percentage of homology with B. 736 rochalimae in BLAST analysis. 737 Figure 3. Phylogenetic tree of Bartonella spp. based on ftsZ partial gene inferred using 738 Maximum-Likelihood (ML) and Neighbor-Joining (NJ) methods and ML topology. The 739 percentage of replicate trees in which the associated taxa clustered together in the 740 bootstrap test (1000 replicates) is shown on the branches (ML/NJ). Bootstrap values 741 32 Table 1. Geographical origin, number and sex, preservation method, date of collection and hosts of eight different species of fleas used in 742 this study. 743 Species Geographical area Number of samples (male/female) Preservation method Date of collection Hosts C. felis Seville (Spain) Villamanique de la Condesa, Seville (Spain) Dos Hermanas, Seville (Spain) Sanlúcar de Barrameda, Cádiz (Spain) 7 (1/6) 13 (3/10) 12 (1/11) 21 (1/20) 96 % Ethanol 96 % Ethanol 96 % Ethanol 70 % Ethanol May 2016 August 2014 January-June 2016 March-September 2011 Neglected horse stable with a flea infestation 1 owned dog (Canis lupus familiaris) 2 owned dogs (Canis lupus familiaris) 3 owned dogs (Canis lupus familiaris) P. irritans Seville (Spain) 23 (10/13) 96 % Ethanol May 2016 Neglected horse stable with a flea infestation N. fasciatus Corse (France) 23 (5/18) 70 % Ethanol January-June 2016 6 rodents (Rattus sp.) S. t. tripectinata Corse (France) 22 (1/21) 70 % Ethanol January-June 2016 5 rodents (Rattus sp., Mus sp., Apodemus sp.) A. erinacei Almonte, Huelva (Spain) 16 (5/2) 96 % Ethanol April 2018 3 hedgehogs (Erinaceus europaeus) Dos Hermanas, Seville (Spain) 2 (0/2) March 2017 L. taschenbergi Conat, Pyrenee (France) 16 (6/10) 70 % Ethanol June-September 2016 12 rodents (Apodemus sp.) Py, Pyrenee (France) 44 (20/24) Ct. b. boisseauorum Asturias (Spain) 60 (60/0) 70 % Ethanol June 2016 13 rodents (Arvicola terrestris) Ct. a. allani Asturias (Spain) 60 (60/0) 70 % Ethanol June 2016 16 rodents (Arvicola terrestris) 744 745 746 747 748 33 Table 2. Primers and probes references used for real-time quantitative and standard PCR in this study. 749 qPCR Microorganism Targeted sequence Reference Rickettsia spp. gltA [30] R. felis Phosphatase gene [31] R. typhi Hypothetical protein [32] Bartonella spp. ITS2 [33] B. henselae Pap31 [34] B. quintana yopP [35] Borrelia spp. ITS4 [36] 16S [37] Anaplasmataceae spp. 23S [38] Wolbachia spp. 23S [39] Leishmania spp. Kinetoplastic DNA [40] C. burnetii IS30A [41] IS1111A [42] Mycobacterium spp. ITS [43] Standard PCR Bartonella spp. ITS [44] Bartonella spp. ftsZ [45] Bartonella spp. gltA [46] Mycobacterium spp. rpoB [47] 750 751 752 34 Table 3. Percentage of microorganism-positive fleas detected by PCR (qPCR and standard PCR). 753 Anaplasmataceae Rickettsia sp. R. typhi C. burnetti Mycobacterium spp. Borrelia spp. Bartonella spp. Leishmania spp. C. felis 32/53 ( Wolbachia spp.) (60.4 %) 15/53 (R. felis) (28.3 %) - - 1/53 (2.0 %) - - - P. irritans 22/23 ( Wolbachia spp.) (95.7 %) - - - 1/23 (4.3 %) - 2/23 (Bartonella spp.) (8.7 %) - S. t. tripectinata - - - - - - 2/22(B. henselae) (9.1 %); 3/22 (B. quintana) (13.6 %) 3/22 (B. elizabethae) (13.6 %) - N. fasciatus - - - - - - 3/23 (B. henselae) (13.0 %); 1/23(B. elizabethae) (4.3 %) 3/23 (Bartonella spp.) (13.0 %) - A. erinacei 10/18(Wolbachia spp.) (55.5 %) 6/18 (R. felis) (33.3 %) 1/18 (5.5 %) - 1/18 (5.5 %) - - - L. taschenbergi - - - - - - 4/60 (B. taylorii) (6.6 %); 4/60 (B. elizabethae) (6.6 %) 2/60 (Bartonella spp.) (3.3 %) 1/60 (B. doshiae) (1.6 %) - Ct. b. boisseauorum 10/60(Wolbachia spp.) (16.7 %) 1/60 (R. felis) (1.6 %) - - - - 4/60 (B. taylorii) (6.6%); 1/60 (B. elizabethae) (1.6%) - Ct. a. allani - - - - - - 4/60 (B. taylorii) (6.6 %); 1/60 (Bartonella spp.) (1.6%) - C. felis P. irritans Ct. apertus allani A. erinacei St. t. tripectinata L. taschenbergi Ct. b. boisseauorum N. fasciatus