meu mestrado. A você, tenho só a agradecer, pois a sua ajuda foi importante desde o início. Agradeço a todos os colegas do Laboratório que estão ou estiveram aqui no mesmo período que eu, pela troca de conhecimento, respeito e convivência. À FCAV e aos professores do Programa de Pós-Graduação em Medicina Veterinária agradeço pela contribuição na minha formação. Aos meus grandes amigos André Menezes, Alexsander Moraes, Cleber Eduardo, Érica Verneque, Larissa Pitelli, Leonardo Olbrick, Marcos Valério, Nícolas Roncato, Rafael Zanata e Thaíze Oliveira pela confiança que sempre depositei em vocês e a fidelidade que juntos construímos. Obrigado meus amigos, cada um de vocês são especiais para mim. Ao meu irmão Thiago de Souza Santana e à minha cunhada Tatiane Laurentino Silva Santana por me apoiarem e serem presentes em minha vida. Saibam que a garra de vocês me inspira a lutar todos os dias. E, por último, não menos importante, agradeço aos meus pais, que são a minha base e fonte de amor. Acreditar que tenho vocês ao meu lado para me apoiarem em todos os meus sonhos, só me dá forças para seguir adiante. Obrigado por todo gesto de amor e carinho, como também compreensão que só vocês, pais, tem por nós, filhos. Amo vocês de todo meu coração. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. OBRIGADO! 41 CAPÍTULO 2 – Molecular detection of vector-borne agents in wild boars (Sus scrofa) and associated ticks from Brazil, with evidence of putative new genotypes of Ehrlichia, Anaplasma and hemoplasmas Matheus de Souza Santana1, Estevam Guilherme Lux Hoppe1, Paulo Eduardo Carraro1, Ana Cláudia Calchi1, Laryssa Borges de Oliveira1, Renan Bressianini do Amaral1, Anna Claudia Baumel Mongruel1, Dália Monique Ribeiro Machado1, Karina Paes Burger1, Darci Moraes Barros-Batestti1, Rosangela Zacarias Machado1, Marcos Rogério André1* 1Department of Pathology, Reproduction, and One Health - Faculty of Agricultural and Veterinary Sciences/University State Paulista (FCAV/UNESP), Jaboticabal, SP, Brazil * Corresponding author: Prof. Dr. Marcos Rogério André, Laboratory of Immunoparasitology, Departament of Patology, Reproduction, and One Health, Faculty of Agricultural and Veterinary Sciences Júlio de Mesquita Filho (UNESP), Jaboticabal Campus, Access Road Prof. Paulo Donato Castellane, s/n, Rural Zone, CEP: 14884-900, Jaboticabal, São Paulo, Brazil. Phone: +55 (16) 3209-7302 Fax: +55 (16) 3202- 4275. e-mail: mr.andre@unesp.br Abstract The present study aimed to investigate, by molecular techniques, the occurrence of Anaplasmataceae, Bartonellaceae, Rickettsiaceae, Mycoplasmataceae, mailto:marcos_andre@fcav.unesp.br 42 Coxiellaceae e Babesiidae/Theileriidae agents in blood samples of free-living wild boars (Sus scrofa) and associated ticks in southeastern Brazil. For this purpose, 67 blood samples and 265 ticks (264 Amblyomma sculptum and one A. ovale) were analyzed. In the screening for Anaplasmataceae agents by a PCR assay based on the 16S rRNA gene, 5.97% blood samples and 50.54% ticks were positive. In the PCR assay for Ehrlichia spp. based on the dsb gene, 9.24% of ticks were positive. Despite the low occurrence, a possible new 16S rRNA genotype of Anaplasma sp. was detected in a wild boar’s blood sample. According to phylogenetic analyses based on the groEL, gltA, sodB genes and ITS (23S-5S rRNA) intergenic region, it was found that A. sculptum and A. ovale ticks collected from wild boars carry Ehrlichia genotypes phylogenetically associated with E. ewingii, E. ruminantium, and new Ehrlichia genotypes previously detected in horses, peccaries, and ticks collected from jaguars. In the screening for hemoplasmas by a qPCR based on the 16S rRNA gene, 88.06% of blood samples and 8.69% of ticks were positive. Mycoplasma suis, M. parvum and a possible new hemoplasma genotype were detected in wild boars in southeastern Brazil. In the screening for Bartonella spp. using a nuoG-based qPCR assay, 3.8% of tick samples were positive. Phylogenetic inferences positioned four nuoG and one r gltA Bartonella sequences into the same clade as Bartonella machadoae. No blood or tick samples from wild boars showed to be positive in the qPCR for Coxiella burnetii based on the IS1111 gene. On the other hand, only 1.6% of ticks was positive in the nested PCR assay for piroplasmids based on the 18S rRNA gene. A 18S rRNA sequence detected in a pool of A. sculptum nymphs was phylogenetically close to Cytauxzoon felis sequences previously detected in cats from the United States. Rickettsia sp. 43 closely related to R. bellii was detected in a pool of A. sculptum nymphs. This is the first report of hemoplasmas, B. machadoae and Cytauxzoon spp. in A. sculptum. Wild boars and associated ticks do not seem to participate in the epidemiological cycle of C. burnetii in the region studied. This invasive mammal species may act as a potential disperser of ticks infected with Ehrlichia spp., Bartonella spp., hemotropic mycoplasmas, and Cytauxzoon, and may bring important epidemiological implications in the transmission of bartonelosis, ehrlichiosis, hemoplasmosis, and cytauxzoonosis to humans and animals, more specifically to horses, rodents, pigs, and cats. KeyWords: Anaplasmataceae, Amblyomma spp., Bartonella sp., Coxiella burnetii, Cytauxzoon sp., hemoplasmas, Rickettsia sp. 1. Introduction Wild boars (Sus scrofa) belong to the Suidae family and are native to Eurasia. In Brazil, they were introduced for consumption in the states of Rio Grande do Sul and São Paulo in the 1960’s. After dispersing across the country, they are currently found in 15 states (Brasil, 2017). Due to the great expansion of this invasive species in the Brazilian territory, the normative instruction 03/2013 was created, which allows the legal control of these animals and their hybrids for population control. Considered an invasive alien species, they are responsible for causing socio-economic and environmental problems, such as destruction of crops and springs (Hardin et al., 1960; Lee and Lee, 2019). According to the World Health Organization, 60% of the described pathogens that affect humans are zoonotic, which are responsible for 75% of the emerging diseases; out of these, 22.8% are transmitted by arthropod vectors 44 (Taylor et al., 2001; Jones et al, 2008; Zanella, 2016; Machado, 2017). Understanding the dynamics of transmission and maintenance of vector-borne diseases will allow the adoption of prevention measures. Regarding vector-borne pathogens in wild boars, those belonging to the families Anaplasmataceae, Rickettsiaceae, Bartonellaceae, Coxiellaceae, Mycoplasmataceae, Babesiidae and Theileriidae are of major importance. The presence of DNA of Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), the causative agent of human and animal granulocytic anaplasmosis, has already been reported in wild boars sampled in Europe (Strasek Smrdel et al., 2009; Kiss et al., 2014; Silaghi; Pfister; Overzier, 2014; Nahayo et al., 2014; Reiterová et al., 2016; Kazimírová et al., 2018; Hrazdilová et al., 2020). In the United States, Beard et al. (2011) detected Bartonella henselae, Bartonella koehlerae, and Bartonella vinsonii subsp. berkhoffii (Rhizobiales: Bartonellaceae) in feral pigs evaluated. Hoelzle et al. (2010) detected Mycoplasma suis (Mycoplasmatales: Mycoplasmataceae) in wild boars sampled in Germany. Regarding the detection of Coxiellaceae agents (Order Leigionellales), Lim et al. (2020) reported the presence Coxiella sp. DNA in Haemaphysalis hystricis ticks collected from wild boars in Malaysia. Rickettsia sp. has been molecularly detected in ticks collected from wild boars from France (Defaye et al., 2021), Italy (Sgroi et al., 2020), China (Wang et al., (2020), and Spain (Castillo-Contreras et al., 2021). Piroplasmids (Piroplasmida) were detected in wild boars sampled from the Czech Republic (Hrazdilová et al., 2020) and Japan (Morikawa et al., 2021). On the other hand, studies reporting the occurrence of arthropod vector- borne agents in wild boars in Brazil are scarce. For instance, the presence of anti- 45 Rickettsia spp. antibodies in wild boars, hunting dogs, and hunters in the southern and central-western Brazil (Kmetiuk et al. (2019). Hemoplasmas were molecularly detected in wild boars from southeastern (Dias et al., 2019) and central-western Brazil (Fernandes et al., 2021). The present study aimed to investigate the molecular occurrence of arthropod vector-borne agents (Anaplasmataceae, Rickettsiaceae, Bartonellaceae, Mycoplasmataceae, Coxiellaceae, and Babesiidae/Theileriidae) in free-living boars and associated ticks sampled in southeastern Brazil. 2. Material and Methods 2.1 Sampling Between the years 2018 and 2020, wild boar captures were conducted with the assistance of legalized hunters registered by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA). After the wild boars were slaughtered, blood samples were obtained by cardiac puncture and ticks were collected after external inspection of the animals' bodies. Blood samples were collected from wild boars by intracardiac route and ticks (when possible) in the municipalities of Barretos, Colina, Guaraci, Jaboticabal, Monte Alto, Monte Azul Paulista, Morro Agudo, Olímpia and Torrinha (Figure 1). 46 Figure 1. Regions where blood samples and tick were collected from free-ranging wild boars in São Paulo state, southeastern Brazil. 2.2 Taxonomic identification of collected ticks Taxonomic identification of the collected ticks was performed using a stereomicroscope (Olympus Corporation, Tokyo, Japan) and following previously described taxonomic keys for adult ticks (Barros-Battesti et al., 2006; Martins et al., 2016) and Amblyomma nymphs (Martins et al., 2010). 2.3 Molecular assays 2.3.1 DNA extraction and endogenous reaction control DNA was extracted from blood samples and tick specimens collected from wild boars using Instagene Matrix kits (Bio-Rad, Hercules, California, USA) and 47 Biopur Extraction Mini Spin Plus (Mobius Life Science, Pinhais, Paraná, BR), respectively, following the manufacturers' instructions. While tick nymphs had their DNA extracted in pools of up to five individuals, adults were individually submitted to DNA extraction, totaling 184 samples of ticks. Subsequently, DNA blood samples were submitted to a conventional PCR (PCR) targeting the endogenous mammalian gapdh (glyceraldehyde-3-phosphate dehydrogenase) gene (Birkenheuer et al., 2003), in order to ascertain the presence of amplifiable DNA. Tick DNA samples were submitted to a PCR assay based on the 16S rRNA gene (Black and Piesman, 1994). DNA samples positive in the abovementioned PCR assays were submitted to PCR assays for Anaplasma spp., Ehrlichia spp., Bartonella spp., hemotropic Mycoplasma spp., Coxiella spp., Babesia/Theileria spp., and Rickettsia spp. (ticks only) (Figure 2). 48 Figure 2. Molecular and phylogenetic analyses used in the present study to detected and characterize Anaplasmataceae agents, Bartonella spp., hemoplasmas, Coxiella burnetii, Rickettsia spp. (only ticks) and piroplasmids in wild boars’ blood and tick samples. 49 2.3.2 PCR assays for Anaplasmataceae agents Blood and tick samples were screened by PCR assays targeting fragments of the 16S rRNA gene for Anaplasmataceae agents (Inokuma et al., 2001) and Anaplasma spp. (Massung et al., 1998), as well as for the dsb gene of Ehrlichia spp. (Doyle et al., 2005). Additionally, DNA samples were submitted to a multiplex quantitative real-time PCR (qPCR) assay for Ehrlichia spp. and Anaplasma spp. based on the groEL gene (Benevenute et al., 2017). Positive samples for the abovementioned PCR assays performed were molecularly characterized using additional PCR assays based on the groEL (Müller et al., 2018) and gltA (Gofton et al., 2016) genes, and 23S-5S intergenic region (Rejmanek et al., 2012) of Anaplasma spp. and Ehrlichia spp., and to a PCR assay for Ehrlichia spp. based on the sodB gene (O'Nion et al., 2015) (Table 1). Table 1. Description of primers and probes used in quantitative real-time PCR assays (qPCR) for Ehrlichia spp., Anaplasma spp., Bartonella spp., hemoplasmas and Coxiella burnetii. Agent (Target Gene) PCR Primer sequences TaqMan Hydrolysis probe Fragment size Reference Ehrlichia spp. (groEL)* qPCR F-Ehr (5’- GCGAGCATAATTAC TCAGAG-3)’ R-Ehr – (5’ CAGTATGGAGCAT GTAGTAG-3’) TET- 5’- CATTGGCTCTTGCTATTGC TAAT3’[BHQ2a-Q]3’ 83 bp Benevenute et al., 2017 Anaplasma spp. (groEL)* qPCR F-Anap (5’- TTATCGTTACATTG AGAAGC-3’) R-Anap (5’- GATATAAAGTTAT TAAAAGTATAAAGC-3’) Cy-5- 5’- CCACCTTATCATTACACTG AGACG3’[BHQ2a-Q]3’ 83 bp Benevenute et al., 2017 Bartonella spp. (nuoG) qPCR F-Bart (5’- CAATCTTCTTTTGCTT CACC-3’) e R-Bart (5’- TCAGGGCTTTATGTGA ATAC-3’) TexasRed-5’- TTYGTCATTTGAACA CG-3’[BHQ2a-Q]3’ 83 bp André et al., 2015 Hemotropic Mycoplasma spp. (16S rRNA) qPCR Fsuis-F (5′-CCC TGA TTG TAC TAA TTG AAT AAG-3’) Rsuis-R (5′-GCG AAC ACT TGT MGBsuis2: 5 ′ FAM- TGR ATA CAC AYT TCA G -MGBNFQ 3 ′ 157 bp Guimarães et al., 2011 50 TAA GCA AG-3’) Coxiella burnetti (IS1111) qPCR Cox-F (5’GTCTTAAGGTGGGC TGCGTG-3’) Cox-R (5’- CCCCGAATCTCATT GATCAGC-3’) Cox-TM = FAM- AGCGAACCATTGGT ATCGGACGTT- TAMRA-TATGG 295 bp Klee et al., 2006 *qPCR multiplex 2.3.3 PCR assays for hemoplasmas A molecular screening for hemoplasmas was performed by using a qPCR based on the 16S rRNA gene (Guimarães et al., 2011) (Table 2). DNA samples positive in the abovementioned qPCR with an estimated quantification higher than 104 copy numbers of a 16S rRNA gene fragment per microliter (Gatto et al., 2019) were additionally characterized by a PCR based on the 23S rRNA gene (Mongruel et al., 2020) (Table 1). 2.3.4. Cloning The hemoplasma 23S rRNA amplicons for which the obtained sequences presented electropherograms containing multiple peaks were submitted to cloning, using pGEM-T Easy (Promega® Madison, WI, USA), according to the manufacturer's recommendations. Two clones were selected from each positive sample according to the blue/white colony system. Colonies were subjected to plasmid DNA extraction using the alkaline lysis method (Sambrook & Russell, 2006 et al., 2006). Subsequently, a PCR assay was performed using the primers M13 F (5′-CGCCAGGGTTTTCCCAGTCACGAC3′) and M13 R (5′- GTCATAGCTGTTTCCTGTGTGA-3′) (Lau et al., 2010), which flank the multiple cloning site of the pGEM-T Easy plasmid, in order to include the analyzed gene 51 fragments. The obtained amplicons were purified and sent for sequencing as described later. 2.3.5 PCR assays for Bartonella spp. A nuoG gene-based qPCR for Bartonella spp. was performed following a previously described protocol (André et al., 2015a) (Table 2). Samples positive in the qPCR were molecularly characterized by conventional PCR assays based on the nuoG (400 bp) (Colborn et al., 2010), gltA (750 bp) (Norman et al., 1995), rpoB (458 bp) (Renesto et al., 2001) and ribC (540 bp) (Johnson et al., 2003) genes. 2.3.6 PCR assays for Coxiella burnetii A qPCR assay based on the repetitive element IS1111 for Coxiella burnetti was performed following a previously described protocol (Klee et al., 2006) (Table 2). 2.3.7 PCR assays for Rickettsia spp. The molecular screening of ticks for Rickettsia spp. was performed using a PCR based on the gltA gene (Labruna et al., 2004a). Positive samples were submitted to PCR assays based on the ompA (Regnery, Spruill & Plikaytis, 2006), ompB (Choi et al., 2005), and 17KDa (Labruna et al., 2004b) genes (Table 1). 2.3.8. PCR assays for piroplasmids A molecular screening for piroplasmids was performed by a nested PCR (nPCR) assay based on the 18S rRNA gene (Jefferies et al., 2008) (Table 1). 52 2.3.9. PCR assay reaction conditions All qPCR assays were performed with a total final volume of 10 μL, containing a mixture of 1 μL (approximately 50 ng) of the sample DNA, 0.2 μM of each primer oligonucleotide and hydrolysis probe, PCR buffer (IQ Multiplex Power Mix, BioRad®, Hercules, California, United States) and sterile ultrapure water (Nuclease-Free Water, Promega®, Madison, Wisconsin, United States). The amplification reactions were conducted in a CFX96 Thermal Cycler (BioRad®, Hercules, California, United States). The quantification of the target DNA copy number/μL was performed using plasmids (IDT psmart, Integrated DNA Technologies®) containing the target sequences. Serial dilutions were made in order to build standards with different concentrations of plasmid DNA containing the target sequence (2.0 x 107 copies/μL to 2.0 x 100 copies/μL), in order to obtain the efficiency and correlation coefficient of the reactions. The plasmid copy number was determined according to the formula (Xg/μL DNA/ [plasmid size (bp) x 660]) x 6.022x1023 x plasmid copies/μL. Assays were performed including duplicates of each DNA sample. All duplicates showing Cq difference values greater than 0.5 were retested in triplicate. Amplification efficiency (E) was calculated from the slope of the standard curve in each run using the following formula (E = 10 -1/slope) (Bustin et al., 2009). The reactions performed followed the standards established by the MIQE Guidelines (Bustin et al., 2009). All conventional and nested PCR assays were performed using 5 μL (approximately 70-120 ng) of the DNA samples in a mixture containing 1.25 U Platinum Taq DNA Polymerase (Invitrogen®, Carlsbad, California, United 53 States), PCR Buffer (PCR buffer 10 X - 100nM Tris-HCl, pH 9.0, 500 mM KCl), 0.2 mM deoxynucleotides (dATP, dTTP, dCTP and dGTP) (Invitrogen®, Carlsbad, California, United States), 1.5 mM Magnesium Chloride (Invitrogen®, Carlsbad, California, United States), 0.5 μM of each primer oligonucleotide (Invitrogen®, Carlsbad California, United States) and sterile ultrapure water (Invitrogen®, Carlsbad, California, United States) q. s.p. 25μL. In the nested PCR assays, 1 μL of the amplified product from the first PCR reaction was used as a template. DNA samples of Ehrlichia canis (Jaboticabal strain) (Nakaghi et al., 2010) and A. phagocytophilum (kindly provided by Prof. Dr. John Stephen Dumler, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA) were used as positive controls in the PCR assays for Anaplasmataceae agents. DNA samples of Babesia vogeli (de Sousa et al., 2018) and Mycoplasma suis (Sonálio et al., 2020) were used as positive controls in PCR assays for piroplasmids and hemotrophic mycoplasmas, respectively. Sterile ultrapure water (Nuclease-Free Water, Promega Corporation, Madison, USA) was used as a negative control in all PCR assays. Rickettsia vini DNA, kindly provided by Prof. Dr. Marcelo Labruna (University of São Paulo), was used as a positive control in the PCR assays for Rickettsia spp. DNA sample from the Jaboticabal strain of Bartonella henselae isolated from cat blood samples (Furquim et al., 2021) was used as a positive control in the qPCR/PCR assays for Bartonella spp. Amplified products were separated by electrophoresis in 1% agarose gels stained with ethidium bromide (Life Technologies™, Carlsbad, California, USA) under 100 V/150 mA electric current for 50 minutes. The agarose gels were 54 subjected to ultraviolet light (ChemiDoc MP Imaging System, Bio Rad, Hercules, California, USA) and photographed using Image Lab Software version 4.1. 55 Table 2. Description of primers used in conventional (PCR) and nested (nPCR) PCR assays used in this study. Agents (Target gene) PCR technique Primer sequences Frag ment size Reference Hemotropic Mycoplasma spp. (23S rRNA) PCR 23S_HAEMO_F (5'- TGAGGGAAAGAGCCCAGAC-3') 23S_HAEMO_R (5’-GGACAGAATTTACCTGACAAGG- 3') 800 bp Mongruel et al, 2020 Ticks (16 rRNA) PCR Cox16SF1 (5’-CGTAGGAATCTACCTTRTAGWGG-3’) Cox16SR1 (5’-ACTYYCCAACAGCTAGTTCTCA-3’) Cox16SF2 (5’-TGAGAACTAGCTGTTGGRRAGT-3’) Cox16SR2 (5’-GCCTACCCGCTTCTGGTACAATT-3’) 1321- 1416 bp Duron et al, 2014 Piroplasmada (18S rRNA) nPCR BTF1 (5’- GGCTCATTACAACAGTTATAGCCCAAAGACTTTGAT TTCTCTC-3’) BTR1 (5’- CCGTGCTAATTGTAGGGCTAATACGGACTACGACG GTATCTGATCG-3’) BTF2 (5’- GGCTCATTACAACAGTTATAGCCCAAAGACTTTGAT TTCTCTC-3’) BTR2 (5’- CCGTGCTAATTGTAGGGCTAATACGGACTACGACG GTATCTGATCG-3’) 930 bp 800 bp Jefferies et al., 2008 Ehrlichia spp. (dsb) PCR DSB-330 (F) (5′- GATGATGTCTGAAGATATGAAACAAAT-3 ′) DSB-728 (R) (5′- CTGCTCGTCTATTTTACTTCTTAAAGT-3’) 409 bp Doyle et al., 2005 Anaplasma spp. (16S rRNA) nPCR ge3a (5′-CACATGCAAGTCGAACGGATTATTC-3’) ge10r 5’-TTCCGTTAAGAAGGATCTAATCTCC-3’) ge9f (5′-AACGGATTATTCTTTATAGCTTGCT-3’) ge2 (5′-CTGGCAGCAGTAA-3’) 932 bp 546 bp Massung et al., 1998 Anaplasmataceae (16S rRNA) PCR EHR16SD (5’GGTCCYACAGAAGTCC3’) EHRSR (5’TAGCACTCATCGTTTACAGC3’) 345 bp Inokuma et al., 2001 Ehrlichia spp. (sodB) PCR sodbEhr1600-F (5′- ATGTTTACTTTACCTGAACTTCCATATC-3′) sodbEhrl600-R (5′- ATCTTTGAGCTGCAAAATCCCAATT-3′) 600 bp O’nion et al., 2015 Anaplasma spp. (ITS - 23S-5S) PCR ITS2F (5′-AGGATCTGACTCTAGTAACGAG-3 ′) ITS2R (5′-CTCCCATGTCTTAAGACAAAG-3 ′) 300 bp Rejmanek et al., 2012 Bartonella spp. (nuoG) PCR F-nuoG (5’-GGCGTGATTGTTCTCGTTA-3’) R-nuoG (5’- CACGACCACGGCTATCAAT-3’) 400 bp Colborn et al., 2010 Bartonella spp. (gltA) PCR CS443f (5′-GCTATGTCTGCATTCTATCA-3′ ) CS1210r (5′-GATCYTCAATCATTTCTTTCCA-3′) 750 bp Norman et al., 1995 56 2.4. Amplicon Purification and Sequencing The products of the PCR assays were purified using the ExoSAP-IT PCR Product Cleanup Reagent enzyme (Applied Biosystems TM) and sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific™, Waltham, MA, USA) and the ABI PRISM 310DNA Analyzer (Applied Biosystems™, Foster City, CA, USA) (Sanger, 1997). 2.5 Phylogenetic Analyses The obtained sequences were submitted to a screening test in Phred- Phrap version 23 software (Ewing & Green, 1998; Ewing et al., 1998) in order to evaluate the quality of the electropherograms and to obtain consensus sequences from the alignment of sense and antisense sequences. Subsequently, the BLASTn analysis tool (Altschul, 1990) was used to analyze the Bartonella spp. (rpoB) nPCR 400F (5’-CGCATTGGCTTACTTCGTAT-3’) 2300R (5’-GTAGACTGATTAGAACGCTG G-3’) 2300R (5’-GTAGACTGATTAGAACGCTGG-3’) 1596F (5’-CGCATTATGGTCGTATTTGTCC-3’) 800 bp Renesto et al., 2001 Bartonella spp. (ribC) PCR BARTON-1 (5’-TAACCGATATTGGTTGTGTTGAAG-3’) BARTON-2 (5’- TAAAGCTAGAAAGTCTGGCAACATAACG-3’) 540 bp Johnson et al., 2003 Rickettsia spp. (gltA) PCR CS-239 (5-GCTCTTCTCATCCTATGGCTATTAT-3) CS-1069 (5-CAGGGTCTTCGTGCATTTCTT-356) 834 bp Labruna et al., 2004a Rickettsia spp. (17KDa) PCR 17KD1 (5’-ACTTGGTTCTCAATTCGGTCAC-3’) 17kD2 (5’-GACACTTGCACCGATTTGTCC-3’) 440 bp Labruna et al., 2004b Rickettsia spp. (ompA) PCR Rr190.70p (5′–ATGGCGAATATTTCTCCAAAA-3′) Rr190.602n (5′–AGTGCAGCATTCGCTCCCCCT-3′) 530 bp Regnery, Spruill & Plikaytis et al., 1991 Rickettsia spp. (ompB) PCR 120-M59 (5’- CCGCAGGGTTGGTAACTGC-3’) 120–807 (5’-CCTTTTAGATTACCGCCTAA-3) 862 bp Roux e Raoult, 2000 57 nucleotide sequences by comparing them with sequences previously deposited in the GenBank database (Benson et al., 2017). The obtained consensus sequences were aligned together with other sequences of the same gene previously deposited in GenBank using MAFFT software (Katoh and Standley, 2002). Phylogenetic inferences based on the Maximum Likelihood method were performed using IQTree analysis software (Trifinopoulos et al., 2016). The bootstrap was accessed with 1,000 replicates and the best evolutionary model was selected by the software according to the BIC (Bayesian Information Criterion) criterion, based on the characteristics of each sequence group. Editing and rooting (via external groups) of the phylogenetic trees were performed using Treegraph 2.0.56-381 beta software (Stover & Müller, 2010). 3. Results 3.1. Identification of ticks Out of 97 boars sampled, ticks were collected, by convenience, from 34 (35.05%) animals. Out of 265 ticks collected, 264 (99.51%) were identified as A. sculptum (100 nymphs and 164 adults [116 males and 48 females]) and 1 (0.48%) as a female of Amblyomma ovale. 3.2. PCR assays for mammal and tick endogenous genes Out of 97 wild boar blood samples, 67 (69.07%) were positive in the PCR for the endogenous mammalian gapdh gene. All the 184 tick DNA samples were positive in the PCR for the 16S rRNA tick endogenous gene. 58 3.2.1. Occurrence of Anaplasmataceae agents DNA in wild boars’ blood and tick samples In the PCR-based screening assays for Anaplasmataceae agents based on the 16S rRNA gene, 5.97% (4/67) of wild boars’ blood samples and 50.54% of ticks (93/184 [7 pools of five nymphs and 85 adults of A. sculptum, and one female of A. ovale]) were positive. Additionally, 9.24% (17/184) of the tick samples were positive for Ehrlichia spp. in the dsb-based PCR. None sample was positive in the nPCR based on the 16S rRNA gene of Anaplasma spp. One hundred tick samples positive in the screening PCR assays based on the 16S rRNA and dsb genes were submitted to additional PCR assays for molecular characterization: 24 (24% [24/100]), 27 (27%[27/100]), 60 (60% [60/100]), and 43 (43% [43/100]) were positive in the molecular assays based on the groEL, gltA, 23S-5S intergenic region and sodB genes, respectively. One to two samples positive in the molecular assays for 16S rRNA, dsb, sodB genes and the 23S-5S intergenic region were chosen for sequencing. The samples were chosen according to the intensity of the bands on the agarose gel electrophoresis and the location. Despite attempts, successful sequencing of groEL and gltA amplicons, which showed faint bans in the agarose gel electrophoresis, was not achieved. All the 67 wild boars’ blood and 184 tick samples were negative in the multiplex qPCR assays for Ehrlichia and Anaplasma based on the groEL gene. 59 Table 3 - Parameters (range and mean of efficiency, R2, slope, and y-intercept) of qPCR assays for Anaplasmataceae (groEL gene), haemoplasmas (16S rRNA gene), Bartonella spp. (nuoG gene), and Coxiella burnetii (1S1111 gene) used in this study for screening wild boars’ blood and associated ticks DNA samples for the studied agents Agent (target gene) Samples Analyzed Efficiency R 2 Slope Y- intercept Ehrlichia spp. (groEL) Blood 98.9% 0.098 -3.349 38.273 Ticks 86.5% to 113.4% (mean=102 .88%) 0.872 to 0.998 (mean=0. 972) -3.694 to -3.038 (mean= - 3.279) 36.295 to 39.735 (mean=37. 493) Anaplasma spp. (groEL) Blood 101.2% 0.993 -3.293 37.895 Ticks 91.6 to 101.5% (mean=97 .44%) 0.994 to 0.999 (mean=0 .990) -3.542 to -3.287 (mean=- 3.389) 40.185 to 43.96 (mean=42 .462) Hemoplasmas (16S rRNA) Blood 85.5- 95.0% (mean=90 .25) 0.996- 0.984 (mean=0 .990) -3.448 - - 3.725 (mean=- 3.585) 41.908- 42.390 (mean=42 .149) Ticks 94.8% to 104.1% (mean= 99.17%) 0.914 to 0.999 (mean= 0.97) -3.228 to -3.452 (mean= - 3.344) 38.044 to 40.900 (mean= 39.158) Bartonella spp. (nuoG) Blood 101.8 and 105% (mean=105 .9) 0.999 -3.103 and - 3.28 (mean=- 3.191) 37.17 and 39.01 (mean=37 .17) Ticks 95.5% to 108.7% (mean= 102.3%) 0.998 to 1 (mean= 0.999) -3.129 to -3.335 (mean= - 3.251) 36.624 to 39.007 (mean= 37.11) Coxiella burnetii (1S1111 gene) Blood 100.7% to 104.3% (mean= 102.5%) 0.978 to 0.984 (mean= 0.981) -3.305 to -3.224 (mean= - 3.264) 44.267 to 45.247 (mean= 44.759) Ticks 99% to 109.8% (mean= 104.5%) 0.991 to 0.998 (mean=0. 996) 3.107 to - 3.343 (mean= - 3.222) 38.179 to 39.272 (mean=39. 193) 60 3.2.2. Occurrence of hemoplasmas DNA in wild boars’ blood and tick samples Out of the 67 wild boars’ blood samples analyzed, 88.06% (59/67) were positive in the qPCR for hemoplasmas based on the 16S rRNA gene. The copy number of a fragment of the 16S rRNA gene ranged from 1.66 x 101 to 6.90 x 105, with an average of 1.93 x 104 copies per microliter of DNA. Out of 59 positive blood samples, only 8 (13.55%) showed estimated quantification higher than 104 copies and were submitted to a PCR assay based on the 23S rRNA gene for additional molecular characterization. As a result, 24% (3/8) samples were positive in the abovementioned PCR assay. Considering that the obtained 23S rRNA sequences showed double peaks in electropherogram, they were submitted to cloning using the pGEM T-easy system (Promega). Regarding the tick samples analyzed, 16 (8.69% [16/184]) samples were positive and had their quantification estimated in the qPCR based on the 16S rRNA gene for porcine hemoplasmas. The estimated quantification ranged from 3.41 x 100 to 9.49 x 101, with an average of 2.8 x 101 copies of a fragment of the 16S rRNA gene per microliter of DNA. Furthermore, 69 samples (37.5% [58/184 - 10 pools of five nymphs and 47 adults of A. sculptum, and one adult female of A. ovale]) were also positive, but could not be quantified, due to the low amount of initial DNA of hemoplasmas in the sample tested (Monte Carlo effect) (Bustin et al., 2009). Since no tick samples showed estimated quantification of a fragment of the 16S rRNA gene of porcine hemoplasmas per microliter higher than 101, these samples were not additionally characterized by the PCR assay based on the 23S rRNA gene. The parameters (efficiency, R2, slope and Y-intercept) of 61 qPCR assays for porcine hemoplasmas followed Bustin et al. (2009) and are showed in Table 3. 3.2.3. Occurrence of Bartonella DNA in wild boars’ blood and tick samples All 67 wild boars’ blood samples analyzed were negative in the qPCR assay for Bartonella spp. based on the nuoG gene. Out of the 184 tick DNA samples analyzed, 3.8% (7/184 [7 adults of A. sculptum]) were positive in the qPCR assay for Bartonella spp. based on the nuoG gene. Two samples were not amenable to quantification due to the low amount of initial Bartonella DNA in the sample tested (Monte Carlo effect). Copy numbers ranged from 1.60 x 100 to 1.97 x 101, with an average of 1.32 x 101 copies per microliter of DNA. The parameters (efficiency, R2, slope and Y- intercept) of qPCR assays for Bartonella spp. followed Bustin et al. (2009) and are showed in Table 3. Among the seven tick-DNA samples positive in the qPCR for Bartonella spp. based on the nuoG gene, four and one were positive the conventional PCR assays for Bartonella spp. based on the nuoG and gltA genes, respectively. On the other hand, all the seven tick samples were negative in PCR assays based on the rpob and ribC genes and on the 16S-23S rRNA intergenic region (ITS). 3.2.4. Occurrence of Coxiella burnetii DNA in wild boars’ blood and tick samples Out of the 67 blood samples analyzed, all showed to be negative in the qPCR assays for C. burnetii based on the 1S1111 gene. The 184 tick DNA 62 samples analyzed were also negative in qPCR assays for C. burnetii based on the 1S111 gene. The parameters (efficiency, R2, slope and Y-intercept) of qPCR assays for C. burnetii followed Bustin et al. (2009) and are showed in Table 3. 3.2.5. Occurrence of Rickettsia DNA in ticks collected from wild boars Out of the 184 tick samples analyzed, 0.54% (1/184 [a pool of five A. sculptum nymphs]) tested positive in the conventional PCR assay for Rickettsia spp. based on the gltA gene, but negative in the PCR assays based on the ompA, ompB, and 17KDa genes. 3.2.6. Occurrence of piroplasmids in wild boars’ blood and tick samples All 67 wild boars’ blood samples were negative in the nPCR assay for piroplasmids based on the 18S rRNA gene. Out of 184 tick samples analyzed, 1.6% (3/184 [1 adult and 2 pools of five A. sculptum nymphs]) were positive in the 18S rRNA gene-based nPCR assay for Piroplasmida. Based on the intensity of the bands obtained on the agarose gel electrophoresis, only one sample was submitted for sequencing. 3.3. Co-positivity for vector-borne agents in wild boars’ blood and tick samples None of the wild boars’ samples were positive for both Anaplasmataceae agent and hemotropic mycoplasmas simultaneously. On the other hand, A. sculptum ticks showed the following co-positivities: 1.08% (1/92 [1 adult]) for Anaplasmataceae/Piroplasmida; 55.43% (51/92 [47 adults and 4 nymphs]) 63 Anaplasmataceae/hemoplasmas; 1.08% (1/92 [1 adult]) Anaplamastaceae/Bartonella spp. ; 3.26% (3/92 [1 adult and 2 pools of five nymphs]) hemoplasmas/Piroplasmida; 2.17% (2/92 [2 adults]) hemoplasmas/Bartonella spp.; 1.08% (1/92 [1 pool of five nymphs]) hemoplasmas/Rickettsia spp.; 2.17% (2/92 [2 adults]) Anaplasmataceae/hemoplasmas/Piroplasmida; 2.17% (2/92 [2 adults]) Anaplasmataceae/hemoplasmas/Bartonella spp. The female of A. ovale was positive for both Anaplasmataceae and hemoplasmas. 3.4. BLASTn analyses The BLASTn results for all agents and target genes are shown in Table 4. 64 Table 4. Percentage of BLASTn-associated identity of sequences of Anaplasmataceae, Rickettsiaceae, Bartonellaceae, hemoplasmas and piroplasmids detected in wild boars and associted ticks sampled in southeastern Brazil. Species /ID Target Agents Targe t gene Query Lengh t (pb) Query- coverag e (%) E- valu e Identit y (%) GenBank accession numbers Sus scrofa/ 19 Anaplasmatace ae 16S rRNA 235 100 1X10 -112 98.72 A. phagocytophilu m detected in Haemophysalis longicornis in China (KX810088) A. sculptu m/ 29B Anaplasmatace ae 16S rRNA 230 100 7X10 -115 100 Ehrlichia sp. detected in A. sculptum in Brazil (MT514732) A. sculptu m/ 25K Anaplasmatace ae 16S rRNA 318 100 7X10 -163 100 Ehrlichia sp. detected in A. sculptum in Brazil (MT514732) A. sculptu m/ 34D Ehrlichia sp. dsb 347 89 1X10 -159 100 Ehrlichia sp. detected in Amblyomma sp. in Brazil (HQ388287) A. sculptu m/ 35O Ehrlichia sp. dsb 346 89 1X10 -159 100 Ehrlichia sp. detected in Amblyomma sp. in Brazil (HQ388287) A. sculptu m/ 1D Ehrlichia sp. ITS- 23S- 5S 390 100 0 95,69 E. chaffeensis detected in a human in the USA (CP007480) A. sculptu m/ 26D Ehrlichia sp. ITS- 23S- 5S 401 100 0 96,78 E. chaffeensis detected in a human in the USA (CP007480) A. ovale/ 24C Ehrlichia sp. ITS- 23S- 5S 304 100 2 X10- 136 96,09 E. chaffeensis detected in a human in the USA (CP007480) A. sculptu m/ 34L Bartonella sp. nuoG 257 100 1 X10- 124 98,84 Bartonella machadoae sp. nov. isolated 65 from wild rodents in the Pantanal wetland (CP087114.1) A. sculptu m/ 35N Bartonella sp. nuoG 223 100 5 X10- 97 96,41 Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland (CP087114.1) A. sculptu m/ 35R Bartonella sp. nuoG 252 96 2 X10- 101 93,98 Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland (CP087114.1) A. sculptu m/ 35S Bartonella sp. nuoG 258 74 3 X10- 70 92,67 % Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland (CP087114.1) A. sculptu m/ 35S Bartonella sp. gltA 686 100 0 97.96 Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland (CP087114.1) A. sculptu m/ 35S Rickettsia sp. gltA 219 100 1 X10- 108 100 Rickettsia bellii detected in ticks in Colombia (MT174170) A. sculptu m/ 4A Ehrlichia sp. sodB 228 100 2 X10- 110 99,12 Ehrlichia sp. detected in an horse in Nicaragua (KJ434180) Sus scrofa/ 3 Mycoplasma sp. 23S rRNA 721 100 0 88,78 Mycoplasma parvum detected in pigs in Brazil (MT530439) 66 3.5. Phylogenetic analyses 3.5.1. Phylogenetic analyses of the 16S rRNA, dsb, sodB genes and 23S-5S (ITS) sequences of Anaplasma spp. and Ehrlichia spp. The maximum likelihood (ML) phylogenetic analysis based on the 16S rRNA gene of Anaplasmataceae family (335 bp) and TIM2+I+G evolutionary model positioned the sequence detected in a wild boar blood sample (ID #19 - OM842840) in a single clade, close to the other clades comprising Anaplasma species, with a clade support of 70%. On the other hand, the two sequences obtained from a male (25K - OM842839) and a female (29B - OM842838) A. sculptum were positioned into different Ehrlichia clades. While the sequence 29B was closely positioned to E. ewingii, the sequence 25K was closely related to Ehrlichia sp. detected in A. sculptum collected from a horse in the Pantanal wetland, state of Mato Grosso do Sul, and E. ruminantium (Figure 3). Sus scrofa/ 6 Mycoplasma sp. 23S rRNA 722 100 0 99,86 Mycoplasma parvum detected in pigs in Brazil (MT232831) Sus scrofa/ 80- clone1 Mycoplasma sp. 23S rRNA 559 100 0 99.64 M. suis detectado detected in pigs in USA (NR_103970) Sus scrofa/ 80- clone2 Mycoplasma sp. 23S rRNA 550 97 0 99,63 Mycoplasma parvum detected in pigs in Brazil (MT530439) A. sculptu m/ 36A Piroplasmida 18S rRNA 733 100 0 99,86 Cytauxzoon sp. detected in cats in USA (MT904037) 67 Figure 3. Phylogenetic analysis of 16S rRNA sequences (335 bp alignment) of Anaplasmataceae agents inferred by Maximum Likelihood method and TIM2+I+G evolutionary model. Brucella melitensis was used as an outgroup. The ML phylogenetic analysis based on the dsb gene of Ehrlichia spp. (339 bp) and evolutionary model TN+F+I grouped two sequences obtained from two males of A. sculptum (34D-OM863958 and 35O-OM863959) into a clade that showed to be sister to the that one comprising Ehrlichia genotypes previously detected in Amblyomma sp. nymphs collected from a jaguar (Panthera onca) from Mato Grosso do Sul State, central-western Brazil, with a clade support of 96%. These sequences were separated from those previously detected in horses from the state of Paraná (Southern Brazil) and peccaries (Tayassu pecari) from the state of Pará (northern Brazil), with 81% and 66% support (Figure 4). 68 Figure 4. Phylogenetic analysis of dsb sequences (339 bp alignment) of Ehrlichia spp. inferred by Maximum Likelihood method and TN+F+I evolutionary model. Ehrlichia ruminantium was used as an outgroup. The ML phylogenetic analysis using the evolutionary model HKY+G for the 23S-5S intergenic region (406 bp) grouped two Ehrlichia sequences detected in A. sculptum (1D - OM863955 and 26D - OM863956) in a clade that showed to be a sister clade to that one comprising an Ehrlichia genotype detected in adult A. ovale (24C - OM863957), with a support of 94% (Figure 5). These two clades were grouped separately from those containing sequences of other Ehrlichia species. 69 Figure 5. Phylogenetic analysis of 23S-5S sequences (406 bp alignment) of Ehrlichia spp. inferred by Maximum Likelihood method and HKY+G evolutionary model. Anaplasma platys, Anaplasma phagocytophilum and Anaplasma marginale were used as an outgroup. The ML phylogenetic inference based on the sodB gene (534 bp) and the TPM3+G as evolutionary model positioned the Ehrlichia sequences detected in A. ovale (OM863960) and A. sculptum (OM863961) in the same clade, together with Ehrlichia genotypes previously detected in horses from the state of Paraná and Nicaragua, in a clade that showed to be sister to that one comprising E. ruminantium, with a clade support of 95% (Figure 6). Figure 6. Phylogenetic analysis of sodB sequences (534 bp alignment) of Ehrlichia spp. inferred by Maximum Likelihood method and TPM3+G evolutionary model. Anaplasma phagocytophilum was used as an outgroup. 70 3.5.2. Phylogenetic analyses of nuoG and gltA gene sequences of Bartonella spp. Phylogenetic analyses (ML) based on the nuoG (363 bp) (Figure 7) and gltA (712 bp) (Figure 8) genes from Bartonella spp. with evolutionary models TNe+I+G and TPM+I+G, respectively, positioned the sequences detected in A. sculptum specimens collected from wild boars into the same clade containing Bartonella machadoae, a Bartonella species recently described in wild rodents in the Brazilian Pantanal, with clade support rates of 95 and 57%, respectively. Figure 7. Phylogenetic analysis of nuoG sequences (363 bp alignment) of Bartonella spp. inferred by Maximum Likelihood method and TNe+I+G evolutionary model. Brucella abortus was used as an outgroup. 71 Figure 8. Phylogenetic analysis of gltA sequences (712 bp alignment) of Bartonella spp. inferred by Maximum Likelihood method and TPM+I+G evolutionary model. Brucella abortus was used as an outgroup. 3.5.3. Phylogenetic analysis of gltA gene sequence of Rickettsia spp. The Maximum Likelihood (ML) phylogenetic analysis based on the gltA gene (634 bp alignment) of Rickettsia spp. and evolutionary model K3PU+F+G4 72 positioned the sequence 36E detected in an pool of A. sculptum nymphs within the R. bellii clade, with 91% clade support (Figure 9). Figure 9. Phylogenetic analysis of gltA sequences (634 bp alignment) of Rickettsia spp. inferred by Maximum Likelihood method and K3PU+F+G4 evolutionary model. Orientia tsutsugamushi was used as an outgroup. 3.5.4. Phylogenetic analysis of the 23S rRNA gene sequences of hemotropic mycoplasmas The maximum likelihood (ML) phylogenetic analysis based on the 23S rRNA gene (800 bp alignment) of hemotropic Mycoplasma spp. and the TIM+G as evolutionary model positioned all hemoplasma sequences detected in wild boars’ blood samples in the upper two clusters of the dendogram, with clade support of 93% and 50%, respectively, along with the other sequences obtained from wild boars and pigs sampled in Brazil and the United States. While the 73 sequences detected in samples #3 (OM868237) and #6 (OM868238) were grouped into the Mycoplasma parvum clade, sequence #6 was positioned in a sub-clade that showed to be sister to a clade of a hemoplasma sequence detected in a pig from the state of Goiás (central-western Brazil) (MT232828), demonstrating that it is phylogenetically closer to the latter than to sequence #3 obtained in this study. The other sequences of hemotropic Mycoplasma sp. were positioned into the Mycoplasma suis clade of the dendogram. These sequences indeed corresponded to genotypes generated by two clones that were obtained from sample #80, which curiously positioned separated from each other: while the sequence corresponding to clone 1 (OM868239) showed closer phylogenetic proximity with M. suis strain Illinois (NR103970) detected in swine from the United States, clone 2 (OM868240) comprised a sister group to a M. suis sequence detected in swine from Brazil (MT530439) (Figure 10). Figure 10. Phylogenetic analysis of 23S rRNA sequences (800 bp alignment) of hemotropic Mycoplasma spp. inferred by Maximum Likelihood method and TIM+G evolutionary model. Bacillus cereus and Bacillus subtilis was used as an outgroup. 74 3.5.5. Phylogenetic analysis of the 18S rRNA gene sequence of Piroplasmida Phylogenetic analysis based on a piroplasmid 18S rRNA gene alignment (709 bp) and TN+G evolutionary model positionedd the sequence detected in a pool of A. sculptum nymphs (OM842841) close to Cytauxzoon felis sequences detected in cats from the United States (Figure 11). Figure 11. Phylogenetic analysis of 18S rRNA sequences (709 bp alignment) of Cytauxzoon spp. inferred by the Maximum Likelihood method and TN+G evolutionary model. Babesia bigemina, Babesia bovis and Babesia canis were used as an outgroup. 4. Discussion The present study showed low molecular occurrence for Anaplasmataceae agents in wild boars sampled in the southeastern region of Brazil. Out of 67 wild boars’ blood samples, 5.97% (4/67) were positive in the molecular assay for Anaplasmataceae agents based on the 16S rRNA gene. On 75 the other hand, ticks collected from these animals showed high occurrence (50.54% [93/184]) for these agents. The Anaplasma sp. sequence detected in a wild boar blood sample in the present study was positioned closely to two Anaplasma sp. sequences detected in A. coelebs ticks collected from coatis (Nasua nasua), in the Iguazu National Park, Paraná, southern region of Brazil. On the other hand, in Peninsular Malaysia, Koh et al. (2016), using a PCR assay based on the 16S rRNA gene, detected Anaplasma bovis in 7/10 boars. According to Galindo et al. (2012), although pigs are susceptible to A. phagocytophilum, such animals are able to control infection through activation of the innate immune response, as well as by processes of phagocytosis and autophagy. Although Anaplasma sp. detected in the present study is a possible new species yet to be described based on isolation and Whole Genome Sequencing techniques, the low prevalence for the agent in question could be related to the control of Sus scrofa infection for agents of the genus Anaplasma. The detection of Anaplasma sp. in wild boars in the present study (1.49% [1/67]) corroborates previous studies conducted in other parts of the world. Anaplasma phagocytophilum DNA has already been reported in wild boars sampled in Romania (4,48% [39/870] by nPCR based on 16S rRNA gene) (Kiss et al., 2014), Germany (12,5% [3/24] by PCR based on 16S rRNA gene) (Silaghi; Pfister; Overzier, 2014), Belgium (0,97% [5/513] by nPCR based on 16S rRNA gene) (Nahayo et al., 2014); Slovenia (4,03% [10/248] by nPCR based on groESL operon) (Strasek Smrdel et al., 2009), Slovakia (28,2% [11/39] by PCR based on 16S rRNA gene) (Kazimírová et al 2018), and Czech Republic (5,1% [28/550] by nPCR based on the groEL gene) (Hrazdilová et al., 2020). Recently, Hornok et al. (2022), using a qPCR assay based on the groEL gene, detected A. 76 phagocytophilum DNA in 19 ticks collected from boars in Hungary: 0.6% (1/165) in Dermacentor reticulatus, 10.34% (3/29) Haemaphysalis concinna and 1.6% (15/90]) Ixodes ricinus. While Anaplasma sp. was detected only in blood samples from wild boars in the present study, Ehrlichia spp. was detected in specimens of A. sculptum and A. ovale collected from the sampled animals. In the phylogenetic analysis based on the 16S rRNA gene, two genotypes of Ehrlichia spp. were identified in A. sculptum ticks: while one genotype grouped with E. ewingii, the other genotype grouped with Ehrlichia sp. previously detected in horse-associated A. sculptum in the state of Mato Grosso do Sul and E. ruminantium. Interestingly, Ehrlichia dsb sequences detected in A. sculptum adult ticks collected from wild boars in the present study were positioned into a large clade containing Ehrlichia sp. genotypes previously detected in jaguars (Panthera onca)-associated Amblyomma nymphs in the state of Mato Grosso do Sul and an Ehrlichia sp. genotype detected in a peccary in the Pará state (Soares et al., 2017). In the phylogeny based on the dsb gene, Ehrlichia sp. previously detected in horses from Paraná State (Vieira et al., 2016) represented a sister group to the sequences detected herein. The Ehrlichia sodB sequence detected in adults specimens of A. sculptum and A. ovale collected from wild boars in the present study was shown to be phylogenetically related to Ehrlichia sequences previously detected in horses from Brazil and Nicaragua (Vieira et al., 2018), albeit separated from them with 95% clade support. Finally, two sequences of the ITS region (23S-5S) of Ehrlichia obtained from A. sculptum and one from A. ovale collected from wild boars in the present study were positioned into the same clade, separated from the other 77 clades containing other Ehrlichia species. It is important to point out that ITS (23S-5S rRNA) sequences of Ehrlichia genotypes detected in horses, peccaries and jaguar-associated ticks were not available in Genbank at the time this manuscript was written. Interestingly, 16S rRNA genotypes of Ehrlichia detected in A. sculptum collected from horses in the state of Mato Grosso do Sul (Muraro et al., 2021) and from wild boars in the present showed proximity to E. ruminantium. Collectively, the results of phylogenetic analyses based on fragments of the 16S rRNA, dsb, sodB genes and ITS (23S-5S) intergenic region genes suggest the occurrence of Ehrlichia genotypes in Amblyomma species collected from wild boars that are closely related to those previously detected in horses, peccaries, and ticks collected from jaguars. Considering that A. sculptum is a tick species with low host specificity, especially in the larvae and nymph stages, the Ehrlichia genotypes detected in ticks carried by wild boars in the present study might have acquired these genotypes from other hosts, such as wild felids and horses. Even though qPCR assays are considered to show higher sensitivity than conventional PCR assays, all wild boars’ blood and tick samples in this study were negative in the multiplex qPCR assays for Ehrlichia spp. and Anaplasma spp. based on the groEL gene. This result could be explained by the fact that the referred qPCR protocol was designed to catch four Ehrlichia species (E. canis, E. chaffeensis, E. muris, Ehrlichia sp. “Anan”) and six Anaplasma species (A. phagoytophilum, A. platys, A. bovis, A. ovis, A. centrale, and A. marginale). Therefore, the primers and probes used might have not been able to hybridize to groEL gene of the putative novel genotypes of Ehrlichia and Anaplasma found in this body of work. Similar results were found when screening biological samples 78 of armadillos, sloths, anteaters (Calchi et al., 2020) and bats (Ikeda et al., 2021) for Ehrlichia and Anaplasma using this qPCR protocol. On the other hand, Ehrlichia and Anaplasma genotypes were detected in biological samples from wild rodents (Benevenute et al., 2017) and wild birds (Sacchi et al., 2021) using the referred qPCR assay. Regarding the occurrence of hemoplasmas, the qPCR assay for porcine hemoplasmas based on the 16S rRNA gene identified 59 (59/67 [88.05%]) wild boars’ blood samples and 16 (16/184 [8.69%]) ticks positive for hemotropic mycoplasmas. Previously, Dias et al. (2019), using the same qPCR assay, detected hemoplasmas DNA in 50% (7/14) of the wild boars sampled in the state of São Paulo. Fernandes et al. (2021), by using a qPCR based on the 16S rRNA gene, detected DNA of hemotropic mycoplasmas in 58.5% (38/65) of the wild boars sampled in the state of Goiás, central-western Brazil. The high occurrence of hemoplasmas in wild boars in Brazil differs from that found in Slovakia, where Hoelzle et al. (2010) detected, by using a qPCR assay based on the 16S rRNA gene, M. suis DNA in 10% of 359 sampled boars. The mean quantification of a fragment of the 16S rRNA gene of porcine hemoplasmas estimated by qPCR in wild boars’ blood DNA samples (mean =1.93 x104 copies per microliter of DNA) was shown to be higher than the mean obtained from tick DNA samples (mean=2.8x101). Since there are no previous reports of M. parvum and M. suis detection in ticks, hemoplasma DNA detected in ticks could represent residual blood from the ticks hematophagy on the sampled boars. Nevertheless, Shi et al. (2019) demonstrated that Rhipicephalus microplus ticks were able to transmit 'Candidatus Mycoplasma haemobos' from sheep and goats in China to murine models (BALBc). These authors also showed 79 the occurrence of transstadial transmission and transovarian transmission of this hemoplasma species in R. microplus. Thus, the real role of ticks in hemoplasma transmission should be further investigated among free-living boars. In domestic swine, mechanical transmission of hemotrophic mycoplasmas occurs through surgical materials, such as contaminated needles, or directly via excretions and blood from wounds caused through hierarchical fights between animals (Dietz et al., 2014; Henderson et al., 1997). Recently, detection of M. suis in piglets, prior to lactation, indicates that this pathogen can also be transmitted transplacentally (Stadler et al., 2019). Also, the detection of M. suis DNA in Haematopinus suis lice might indicate a possible role of this arthropod as a mechanical vector in the transmission of this agent (Acosta, Ruiz & Sanchez, 2019). Based on the phylogeny for the 23S rRNA gene of Mycoplasma spp. it was possible to infer the occurrence of M. parvum, M. suis and a putative new genotype of hemoplasma (detected in Sus scrofa #3). While most of the sequences detected in the present study showed identity values higher than 99% with M. suis and M. parvum sequences previously detected in Genbank, the 23S rRNA sequence of hemoplasma detected in boar #3 showed 88.7% identity with M. parvum. Recently, Fernandes et al. (2021) also detected 16S rRNA sequences of hemoplasmas in wild boars in the state of Goiás and Paraná with identity values of 91.29% and 92.83% for M. parvum and M. suis, respectively. Due to the low identity values, the authors suggested the occurrence of new hemoplasma species in the studied population of wild boars. On the other hand, hunting dogs were parasitized by M. haemocanis, while hunters were negative for hemoplasmas (Fernandes et al., 2021). Since M. suis has been detected in dogs from Argentina (Mascarelli et al., 2016) and in pig farms workers from China 80 (Yuan et al., 2009), future studies are needed to investigate whether wild boars can act as a source of infection for M. suis for hunting dogs and hunters. Interestingly, 23S rRNA sequences from hemoplasmas referring to clones from sample #8, were positioned separated from each other in the phylogenetic analysis: while the sequence corresponding to clone 1 showed to be closer to M. suis (strain lllinois) previously detected in pigs in the United States, clone 2 formed a sister group with a sequence from M. suis previosuly detected in a swine from Brazil (MT530439). These results suggest the occurrence of more than one hemoplasma genotype in the same wild boar, as previously demonstrated by Sonálio et al. (2020) in commercial swine farms in central-western Brazil. With regard to the detection of Bartonella spp. in wild boars in this study, all blood samples were negative in the nuoG gene-based qPCR assay. On the other hand, Beard et al. (2011) identified sequences from the ITS intergenic region (16S-23S rRNA) of B. henselae (5 sequences), B. koehlerae (7 sequences) and B. vinsonii subsp. berkhoffi (3 sequences) in 19.7% (15/76) of feral pigs in North Carolina, United States. Future studies should be conducted aiming at submitting wild boars’ blood samples to the diagnostic platform that combines BAPGM liquid cultures (Bartonella Alpha Proteobacteria Growth Medium) and solid cultures on blood or chocolate agar, followed by real-time and conventional PCR assays, in order to increase the chances of detection of Bartonella spp., as recently demonstrated in blood samples from cats from southeastern Brazil (Furquim et al., 2021) and rodents from central-western Brazil (do Amaral et al., 2022). In contrast, 3.80% (7/184) of the ticks collected from wild boars in the present study were positive for Bartonella spp.. The phylogenetic inferences 81 based on both nuoG and gltA genes positioned the sequences obtained from A. sculptum collected from wild boars in the same clade as Bartonella machadoae, a species recently described by our research group in wild rodents in the Brazilian Pantanal (do Amaral et al., 2022), with a clade support of 95 and 55%, respectively. To the best of authors’ knowledge, this is the first report of Bartonella sp. DNA in A. sculptum ticks. Since Bartonella machadoae has so far been detected only in wild rodents and all wild boars sampled herein were negative for this agent, one would suggest that A. sculptum nymphs might have acquired bartonellae when feeding on wild rodents in surrounding regions, and infection by this Bartonella species has been transstadially transmitted. Future studies aiming at investigating the vector competence of A. sculptum in the transmission of B. machadoae are much needed. Taking into account the low specificity of A. sculptum and parasitism on humans, the role of this tick species in the transmission of Bartonella spp. should be further investigated. Indeed, the role of ticks in the transmission of Bartonella spp. has been investigated in recent years. In this regard, 9.8% (9/92 [three nymphs and six adults]) of I. ricinus ticks collected from vegetation in France were positive for Bartonella spp. by a seminested PCR assay based on the gltA gene. The sequence obtained from an adult tick showed 96% identity with B. schoenbuschensis (Halos et al., 2005). Cotté et al. (2008) demonstrated, by using an artificial membrane feeding system, the ability of infection and transstadial transmission of B. henselae in I. ricinus collected in France. Similar results were found by Wechtaisong et al. (2020) when using Rhipicephalus sanguineus as an experimental model. All blood and tick samples collected from wild boars in the present study were negative in the IS1111 gene-based qPCR assay for C. burnetii. Recently, 82 Lim et al. (2020) reported the presence of Coxiella endosymbionts DNA in 16.6% (5/32) of Haemaphysalis hystricis ticks collected from free-ranging wild boars in Malaysia. Coxiella burnetii is capable of infecting a wide variety of wild and domestic animals. In Brazil, DNA of this agent has already been detected in placenta samples from goats (De Oliveira et al., 2018) and spleens from wild rodents (Rozental et al., 2017) and bats (Ferreira et al., 2018). Serological evidence of exposure to this agent has already been reported in cattle and deer from Brazil (Zanatto et al., 2019a; 2019b; De Souza Ramos et al., 2020). Recently, C. burnetii DNA has been detected in fresh cheese samples in southeastern Brazil (Nascimento et al., 2021), emphasizing the need for further studies about the epidemiology of Q fever in Brazil, where human cases have already been described (Mares-Guia et al., 2016; Rozental et al., 2018; Lemos et al., 2018). Regarding the occurrence of piroplasmids, none wild boar sampled in the present study was positive in the 18S rRNA gene-based molecular assay. An 18S rRNA sequence detected in a pool of A. sculptum nymphs was positioned closely to Cytauxzoon felis sequences detected in cats in the United States. We hypotethized that Amblyomma sculptum nymphs might have been infected in the larval stage when feeding on wild felids. To the best of authors’ knowledge, wild boars are not susceptible hosts for Cytauxzoon sp., which has been detected so far only in domestic (André et al., 2015b) and wild felids (André et al., 2008; De Sousa et al., 2017), bears (Moustafa et al., 2020) and meerkats (Leclaire, Menard and Berry, 2014). In fact, tracks of wild felids (ocelots [Leopardus pardalis] and jaguars [Puma concolor]) were found on the sites where the animals were sampled (personal communication, Prof. Dr. Estevam Guilherme Lux Hoppe; 83 Supplementary material). It is noteworthy that the present study detected, for the first time, the presence of Cytauxzoon sp. DNA in A. sculptum ticks in Brazil. Based on these findings, we suggest that this tick species may play a role as a vector for Cytauxzoon sp. in Brazil. Indeed, Amblyomma sculptum can parasitize wild felids (Widmer et al., 2011), which in turn may play a role as reservoirs of Cytauxzoon sp. in Brazil (André et al., 2018; De Sousa et al., 2017). Apart from the results obtained in the present regarding the occurrence of piroplasmids in wild boars, Hornok et al. (2022) recently detected Babesia canis and Babesia cf. crassa in 4 (2.42% [4/165]) D. reticulatus and one (3.4% [1/29]) H. concinna collected from wild boars in Hungary, respectively, using a 18S rRNA gene-based PCR assay. Herein, out of 184 tick samples submitted to a gltA gene-based PCR assay for Rickettsia spp., only one (0.54% [1/184]) A. sculptum was positive. The sequence obtained in the present study was positioned in the same clade as R. bellii, with a clade support of 91%. The occurrence of R. bellii has been reported in 25 different tick species in America, demonstrating breadth of infection by this Rickettsia species in different tick species (Costa et al., 2017). Although this Rickettsia species has been recognized as a non-pathogenic rickettsial agent, Snellgrove et al. (2021) demosntrated that guinea pigs experimentally infected with R. bellii intra-peritoneally showed fever, orchitis and dermatitis. The detection of co-infection by R. bellii and R. amblyommatis in Amblyomma calcaratum and Amblyomma longirostre ticks, and by R. bellii and R. parkeri-like in Amblyomma sp. (haplotype Nazareth) collected from wild birds in Brazil, draws attention to how underestimated coinfection of ticks by two or more species of 84 Rickettsia is treated, emphasizing the possible role of R. bellii in the bio-ecology of Rickettsia spp. transmitted by arthropod vectors (Abreu et al, 2019). Indeed, the role of wild boars as carriers of ticks infected by Rickettsia spp. has already been demonstrated on the island of Corsica (France) (R. slovaca and R. aeschlimannii) (Defaye et al., 2021), Italy (R. slovaca and R. raoultii) (Sgroi et al., 2020), China ('Candidatus Rickettsia laoensis') (Wang et al., 2020), Japan (Rickettsia japonica, Rickettsia tamurae) (Motoi et al., 2013, Someya et al., 2015), and Spain (Rickettsia massiliae, R. slovaca and R. raoultii) (Castillo-Contreras et al., 2021). In Brazil, Kmetiuk et al. (2019) detected the presence of anti-Rickettsia spp. antibodies (Rickettsia bellii, R. rickettsii, and R. rhipicephali) in 72.5% (58/80) wild boars, 14.1% (24/170) hunting dogs, and 14.7% (5/34) hunters in the southern and central-western region of Brazil, emphasizing the possible role of wild boars as carriers of zoonotic Rickettsia species. Considering that wild boars have a broad geographic dispersion during their lifetime (Hartley et al., 2014), the data obtained in this body of work suggests that the studied population of wild boars may act as spreaders of ticks infected with pathogens, such as Ehrlichia spp., Bartonella spp., hemoplasmas and Cytauxzoon spp.. 5. Conclusions High and low occurrence for hemoplasmas and Anaplasmataceae agents, respectively, was detected in the wild boars sampled in the present study. Wild boars are parasitized, despite the low occurrence, by a putative new genotype of Anaplasma sp. Amblyomma sculptum ticks carried by wild boars were shown to be infected with Ehrlichia genotypes phylogenetically 85 associated with E. ewingii, E. ruminantium, and new Ehrlichia genotypes previously detected in horses, pecaries, and ticks collected from jaguars. Mycoplasma suis, M. parvum and a putative new hemoplasma genotype were detected in wild boars in the southeast region of Brazil. Rickettsia bellii was detected in A. sculptum nymphs collected from wild boars. This is the first molecular detection of hemoplasmas, Bartonella machadoae and Cytauxzoon sp. in A. sculptum. Coinfection by more than one vector-borne agent was reported in ticks collected from wild boars in the present study. Wild boars and associated ticks do not seem to participate in the epidemiological cycle of C. burnetii in the studied region. Wild boars in addition to be susceptible to infection by Anaplasma sp. and hemotropic mycoplasmas, may act as potential dispersers of ticks infected with Ehrlichia spp., Bartonella spp, hemotropic mycoplasmas and Cytauxzoon in Brazil. In a nutshell, the results found herein have important epidemiological implications in the transmission of bartonellosis, erlichiosis, hemoplasmosis and cytauxzoonosis to humans and animals, more specifically to horses, rodents, domesticated pigs and cats. Acknowledgments The authors would like to thank the "Fundação de Amparo à Pesquisa do Estado de São Paulo" for the financial support (FAPESP Processes #2018/02753-0, #2019/26403-0, 2020/07826-5 and #2020/12037-0) and for the master scholarship granted (FAPESP Process number 2019/24726-7). M.R.A. is a Productivity Scholar of the "National Council for Scientific and Technological Development" (CNPq Process number #303701/2021-8). Ethics Statement 86 This study was approved by the “Ethics Committee for Animal Experimentation” of FCAV/UNESP (Faculty of Agricultural and Veterinary Sciences of the São Paulo State University) under protocol number #014836/18. The “Instituto Chico Mendes de Conservação da Biodiversidade (ICMBIO)” provided the required annual permits for the capture of wild boars and collection of blood samples and ticks (#62641-2 and #66626-1). Conflicts of interest The authors declare no conflict of interest. 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