Old Agents and Novel Variants of Tick-borne Microorganisms From Angola, 2017 Ana María Palomar San Pedro University Hospital-CIBIR https://orcid.org/0000-0002-5461-5874 Israel Molina Vall d'Hebron Hospital: Hospital Universitari Vall d'Hebron Cristina Bocanegra Vall d'Hebron Hospital: Hospital Universitari Vall d'Hebron Aránzazu Portillo San Pedro University Hospital-CIBIR Fernando Salvador Vall d'Hebron Hospital: Hospital Universitari Vall d'Hebron Milagros Moreno Hospital Nossa Shenora de La Paz Jose A. Oteo (  jaoteo@riojasalud.es ) San Pedro Unniversity Hospital-CIBIR https://orcid.org/0000-0001-7080-6542 Research Article Keywords: Ticks, tick-borne microorganisms, zoonotic agents, Rickettsia, Anaplasmataceae, Coxiella, Borrelia, Spiroplasma, Babesia, Angola Posted Date: December 22nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1153383/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/7 Abstract The study of microorganisms from ticks collected in cattle from Angola is reported herein, demonstrating the circulation of the pathogen R. aeschlimannii and potential novel tick-borne microorganisms with unknown pathogenicity belonging to Ehrlichia, Spiroplasma, Coxiella, Babesia and Francisella spp. and corroborating the presence of Rickettsia africae and Babesia bigemina. Introduction The COVID-19 pandemic and epidemics like EBOLA, Lassa fever, Zika virus disease, Nipah virus infection, avian in uenza, etc. have strengthened the importance of One Health to prevent spillovers. Human and animal health and the environment are interconnected, and factors such as globalization, climate change, changes in land uses, population growth, etc. could trigger new zoonotic outbreaks [1]. Early detection and knowledge of potential zoonotic agents, including vector-borne microorganisms, are relevant to implement containment measures and prevent related infectious diseases. Thus, surveillance systems of vectors and their microorganisms are required. Zoonotic agents, often underdiagnosed due to lack of diagnostic resources, are a known major cause of disease in Sub-Saharan Africa, and studies have raised the need of improving protocols for fever of unknown origin (FUO) management [2]. Tick-borne relapsing fever, rickettsiosis and babesiosis have been reported from southern Africa [2–3], but tick-borne diseases from Angola are hardly known. Angolan livestock population is increasing (https://www.fao.org/faostat/en/ # data/QCL), mainly based on cattle production, and the expansion of livestock industry is linked to the incidence of zoonosis [4]. Therefore, we report the study of selected microorganisms in ticks from Angolan cattle. Materials And Methods Ticks were collected from cattle in a slaughterhouse of Cubal (Benguela Province, Angola) from 1-8 July 2017, and preserved in ethanol 70%. Specimens were classi ed using a taxonomic key [5]. Selected individuals (at least two specimens from each morphologically classi ed species and those doubtful according to morphological features) were genetically characterized by PCR of mitochondrial genes (Additional le: Table S) using individual DNAs from legs subjected to ammonium extraction [6]. Furthermore, tick halves were pooled (1-9 specimens) according to species and developmental stages. DNA from pools was extracted using DNeasy Blood & Tissue kit (Qiagen), following manufacturer’s recommendations with overnight lysis. Mitochondrial 16S rRNA PCRs were performed as controls of pool extractions (Additional le: Table S). Bacteria (Rickettsia, Anaplasmataceae, Borrelia, Coxiella and Spiroplasma) and protozoa (Theileria and Babesia) were screened using speci c PCR assays. Pan-bacterial 16S rRNA PCR was also performed (Additional le: Table S). Nucleotide sequences were analyzed, compared with those available in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and submitted to GenBank, when different. Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used for multiple sequence alignment. Phylogenetic analyses were conducted with MEGA X (http://www.megasoftware.net) using maximum likelihood method including all sites. Con dence values for individual branches of resulting trees were determined by bootstrap analysis (500 replicates). Results A total of 124 ticks ( ve nymphs, 28 males and 91 females) were collected and morphologically classi ed as six Amblyomma variegatum, six Hyalomma truncatum, 107 Rhipicephalus decoloratus and ve Rhipicephalus spp. Whenever performed, genetic characterization con rmed morphological identi cation, and also allowed to identify three Rhipicephalus duttoni and one Rhipicephalus evertsi mimeticus (Tables 1-2) among those Rhipicephalus spp. Table 1 Comparison (% identity) of the studied Angolan tick mitochondrial amplicons with available GenBank sequences. Tick species % identity (bp)-GenBank accession No. (No. of analysed amplicons) 16S RNA 12S RNA COI A. variegatum 99.0 (404/408)-L34312 (3) 99.4 (339/341)-HQ856466 (3) 99.3 (560/564)-MK648415 (1) H. truncatum 99.8 (401/403)-LC634545 (2) 100 (341/341)-AF150031 (2) 99.2-99.4 (617/622-670/674)-KY457529 (2) R. decoloratus 99.5-99.8 (399-400/401)- KY457525 (4) 99.7 (343/344)-NC_052828 (4) 99.4-99.1 (616/620-652/658)-NC_052828 (3) R. evertsi mimeticus 99.7 (370/371)-MF425975 (1) 100 (318/318)- AF031862 (1) NA R. duttoni 99.7 (352/353)-MW080164 (3) 98.7 (310/314)-MF425966 (1) NA Rhipicephalus sp. 97.0 (393/405)-LC634554† (1) 98.2 (333/339)-KY4575421 (1) NA bp: base pairs; A.: Amblyomma; H.: Hyalomma; R.: Rhipicephalus; NA: Not ampli ed; †Rhipicephalus simus Page 2/7 Table 2 Microorganisms ampli ed in this study. Data show the species names and the highest identity with public sequences (%; GenBank accession number) followed by the number of pools in which they have been detected and, in brackets, the number of ticks from each pool. Microorganisms Rickettsia spp. Target gene ompA Amblyomma variegatum Hyalomma truncatum Rhipicephalus decoloratus Rhipicephalus duttoni (2: 5N, 1M)† (3: 1M, 5F)† (16: 24M, 83F)† (2: 1M, 2F)† R. africae R. aeschlimannii (100;HQ335157) R. africae - - - - - - NP NP NP NP NP NP (100;CP001612) 2(5N, 1M) Anaplasma/ Neoehrlichia/ Ehrlichia spp. groESL - 3(1M, 5F) - Rhipicephalus evertsi mimeticus (1: 1F)† Rhipicephalus sp. (1: 1M)† (100;CP001612) 1(9M) Ehrlichia spp. (100;MW054557) 6(44F) Ehrlichia spp. gltA NP NP Ehrlichia spp. (96.9-97.0; KX987353)‡ 6(44F) 16S rRNA§ NP NP Ehrlichia sp. (99.9;AF497581) 1(9F)¶ Borrelia spp. aB - - - - - - glpQ - - - - - - Coxiella burnetii IS1111 - - - - - - Coxiella/ Francisella spp. rpoB Coxiella spp. SNC Coxiella spp. Coxiella sp. Coxiella sp. Coxiella sp. (100;KP985329) (95.9; KP985337) (99.1;KP985331) (97.8;KP985337) 1 (1F) 1 (1M) (98.9-99.2; KP985305) 16 (24M,38F) 2(5N, 1M) groEL 16S rRNA§ Spiroplasma spp. rpoB 2 (1M, 2F) Coxiella spp. Francisella sp. Coxiella spp. Coxiella sp. Coxiella sp. Coxiella sp. (99.5; KP985486) (96.8;CP013022, CP012505)†† (100;KP985510) (97.3;KY678195) (98.2;KY678195) (98.3;CP011126) 2 (5N, 1M) 1 (4F) 16 (24M,38H) 2 (1M, 2F) 1 (1F) 1 (1M) NP Francisella sp. Coxiella sp. NP NP NP (99.6;AB001522) (99.4;JQ480818) - - - NP NP NP - - - - 1 (4F)‡‡ - 1(5H)¶ Spiroplasma spp. (99.4;KP967687)§§ 3 (24M) 16S rRNA NP NP Spiroplasma spp. (98.7-100; KP967685)¶¶ 3 (24M) Theileria spp./ Babesia spp. 18S rRNA Babesia spp. - B. bigemina (91.4;AB734390) (100;KF606863) 2 (5N, 1M) 2 (10F) Page 3/7 Microorganisms Babesia spp. Target gene ITS 1 ITS 2 Amblyomma variegatum Hyalomma truncatum Rhipicephalus decoloratus Rhipicephalus duttoni (2: 5N, 1M)† (3: 1M, 5F)† (16: 24M, 83F)† (2: 1M, 2F)† Babesia spp. NP B. bigemina NP NP NP NP NP NP (70.9; LK391709) (98.8-100; EF458251)¶¶ 2(5N, 1M) 2 (10H) Babesia spp. (74.7; EF186914) 2 (5N, 1M) NP B. bigemina Rhipicephalus evertsi mimeticus (1: 1F)† Rhipicephalus sp. (1: 1M)† (99.5;EF458266) 2 (10F) Numbers in brackets indicate (number of pools: number of ticks and developmental stage); ‡Two genetic variants were identi ed; §Pan-bacterial PCR assay; ¶PCR assay performed to four samples but, because this is a pan-bacterial PCR assay (Additional le:Table S), the bacterium was only ampli ed from one sample; ††With 87,6% and 65% query cover, it reached 98.2% and 98.7% identity with Francisella sp. detected in soft and hard ticks, respectively (MW287617 and KY678032); ‡‡With 92% query cover, it reached 99.8% identity with Francisella sp. ampli ed from Hyalomma truncatum (JF290387); §§with 42% query cover, the sequences are identical to available Spiroplasma sequences from Rhipicephalus decoloratus (MK267083-4) but also to those detected in other Rhipicephalus and Ixodes species (MK267073-7,MK267082,MK267085); ¶¶Nucleotide sequences show several ambiguous bases; N: nymphs; M: males; F: females; SNC: Sequences not conclusive; NP: Not performed. † Twenty- ve pools (two A. variegatum, three H. truncatum, 16 R. decoloratus, two R. duttoni, one R. evertsi mimeticus, and one Rhipicephalus sp.) were screened for microorganisms. Rickettsia spp. was found in 6/25 pools. According to ompA, Rickettsia africae was detected in two A. variegatum and one R. decoloratus pools; and R. aeschlimannii, in three H. truncatum pools (Table 2). Ehrlichia spp. was found in 6/25 pools of female R. decoloratus. Analysis of groESL, gltA and 16S rRNA amplicons revealed the highest identities with unclassi ed Ehrlichia (Table 2, Figure), and showed less than 93.5%, 87.6% and 99.2% identity, respectively, with validated species. Other Anaplasmataceae, Borrelia spp. (relapsing fever or Lyme groups) or Coxiella burnetii were not detected. Nevertheless, Coxiella spp. were found in all but H. truncatum pools. For H. truncatum, rpoB sequences showed inconclusive data, whereas groEL and universal 16S rRNA sequences showed the highest similarity (<97% and 99.6%, respectively) with Francisella sp. in one pool. This 16S rRNA amplicon showed 99.8% identity (92% query cover) with Francisella endosymbiont of H. truncatum JF290387 (Table 2). For the remaining tick species, different Coxiella genotypes were found. All but two were identical or closely related to public sequences. Genotypes detected in R. duttoni and Rhipicephalus sp. did not reach >98.3% identity with Coxiella (Table 2, Figure). Spiroplasma sp. was ampli ed from three R. decoloratus male pools (Table 2). According to rpoB, it was closely related to Spiroplasma ixodetis and related strains of hard ticks (Figure). Babesia bigemina was identi ed in two R. decoloratus female pools, and Babesia sp. was detected in two A. variegatum pools, according to 18S rRNA, ITS-1 and ITS-2 analysis (Table 2, Figure). Novel sequences of this study were deposited on GenBank under accession numbers: OK481091-OK481100; OK481107-OK481113; OK491113-OK491116; OK482869-OK482874; OK514711-OK514725. Discussion This study reports the detection of well-known pathogens: R. africae, R. aeschlimannii and B. bigemina, and scarce characterised Ehrlichia, Coxiella, Francisella, Spiroplasma and Babesia species with unknown pathogenicity in ticks from cattle in Angola. Our results corroborate the circulation of R. africae and demonstrate the circulation of R. aeschlimannii in Angola. Although R. aeschlimannii human infection had been reported from South Africa and H. truncatum had been suggested as vector [7–8], this pathogen had not been previously found in Angola. African tick-bite fever is endemic in Sub-Saharan Africa but no cases from Angola have been noti ed [2–3]. This study con rms the recent detection of R. africae in A. variegatum (recognized vector) [9], suggesting that cases could be misdiagnosed. The presence of R. africae in R. decoloratus is known but their role as vector should be investigated [3, 10]. Moreover, our nding in fed ticks could be due to blood meal or cofeeding. Only six Ehrlichia species are currently recognized and all but one cause ehrlichiosis [11], a disease with human cases reported from southern Africa [3]. Moreover, ‘Candidatus’ have been proposed and Ehrlichia genotypes have been partially characterized. Further studies are needed to determine their taxonomic status and pathogenic potential. Herein, a novel Ehrlichia genotype has been detected in six R. decoloratus pools. Tick diet based on blood is unbalanced, and endosymbionts (e.g. Coxiella-like, Francisella-like…) provide essential nutrients for ticks [12]. Although virulence genes identi ed in pathogenic related species, C. burnetii and Francisella tularensis, could be absent or non-functional in symbionts, Coxiella-like has been considered pathogen [13]. Herein, Coxiella-like was detected in all but H. truncatum pools, and isolates were identical or closely related to those Page 4/7 previously ampli ed in the corresponding tick species, except potential novel Coxiella genotypes of R. duttoni and Rhipicephalus sp. Francisella sp. was detected in 1/3 H. truncatum pools, showing a sequence genetically related with a Francisella sp. endosymbiont amplicon of this species. Spiroplasma spp. have been found in several hard tick species, and the role of this genus as pathogen has been suggested [14]. Herein, Spiroplasma sp. closely related to S. ixodetis was detected in 3/16 R. decoloratus pools. Spiroplasma sp. was previously detected in this species according to a short rpoB sequence (Table 2), and this study provides a wider genetic identi cation. Babesia bigemina, responsible for babesiosis, is prevalent in Angolan cattle [15]. Our study demonstrates its presence in R. decoloratus (competent vector) in Angola. Moreover, a potential novel Babesia species is circulating in Angolan A. variegatum. These results should be considered to elaborate protocols for FUO patients’ management in Angola. Surveillance of ticks and tick-borne microorganisms is needed to evaluate the risk of tick-borne diseases in Angola. Declarations Acknowledgments Partial results of this study were presented at XXIII National Congress SEIMC (Madrid-Spain, 2019). Funding This work has been partially funded by European Regional Development Funds (FEDER). Competing interests The authors have no competing interests to declare. Ethics approval and consent to participate Not application Authors’ contributions Palomar AM: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Molina I: Conceptualization, Resources, Formal analysis, Writing - review & editing. Bocanegra C: Conceptualization, Investigation, Writing - review & editing. Portillo A: Resources, Methodology, Formal analysis, Writing - review & editing. Salvador F: Conceptualization, Investigation, Supervision, Writing - review & editing. Moreno M: Investigation, Writing - review & editing. Oteo JAO: Conceptualization, Resources, Formal analysis, Funding acquisition, Writing - original draft, Writing - review & editing. References 1. Otu A, Effa E, Meseko C, Cadmus S, Ochu C, Athingo R, et al. Africa needs to prioritize One Health approaches that focus on the environment, animal health and human health. Nat Med. 2021;27:943-6. doi: 10.1038/s41591-021-01375-w. 2. Maze MJ, Bassat Q, Feasey NA, Mandomando I, Musicha P, Crump JA. The epidemiology of febrile illness in sub-Saharan Africa: implications for diagnosis and management. Clinical Microbiology and Infection. 2018;24:808-14. doi: 10.1016/j.cmi.2018.02.011. 3. Chitanga S, Gaff H, Mukaratirwa S. Tick-borne pathogens of potential zoonotic importance in the southern African Region. J S Afr Vet Assoc. 2014;85:1084. doi: 10.4102/jsava.v85i1.1084. 4. Rohr JR, Barrett CB, Civitello DJ, Craft ME, Delius B, DeLeo GA, et al. Emerging human infectious diseases and the links to global food production. Nat Sustain. 2019;2:445-56. doi: 10.1038/s41893-019-0293-3 5. Walker AR, Bouattour A, Camicas JL, Estrada-Peña A, Horak IG, Latif AA, et al. Ticks of domestic animals in Africa: a guide to identi cation of species. Bioscience reports, Edinburgh. 2003;201 pp. . Portillo A, Santos AS, Santibáñez S, Pérez-Martínez L, Blanco JR, Ibarra V, et al. Detection of a non-pathogenic variant of Anaplasma phagocytophilum in Ixodes ricinus from La Rioja, Spain. Ann NY Acad Sci. 2005;1063:333–6. Doi:10.1196/annals.1355.053 7. Pretorius AM, Birtles RJ. Rickettsia aeschlimannii: A new pathogenic spotted fever group rickettsia, South Africa. Emerg Infect Dis. 2002;8:874. doi: 10.3201/eid0808.020199. . Mediannikov O, Diatta G, Fenollar F, Sokhna C, Trape JF, Raoult D. Tick-borne rickettsioses, neglected emerging diseases in rural Senegal. PLoS Negl Trop Dis. 2010;4:e821. doi: 10.1371/journal.pntd.0000821. 9. Barradas PF, Mesquita JR, Ferreira P, Gärtner F, Carvalho M, Inácio E, et al. Molecular identi cation and characterization of Rickettsia spp. and other tick-borne pathogens in cattle and their ticks from Huambo, Angola. Ticks Tick Borne Dis. 2021;12:101583. doi: 10.1016/j.ttbdis.2020.101583. 10. Portillo A, Pérez-Martínez L, Santibáñez S, Blanco JR, Ibarra V, Oteo JA. Detection of Rickettsia africae in Rhipicephalus (Boophilus) decoloratus ticks from the Republic of Botswana, South Africa. Am J Trop Med Hyg. 2007;77:376-7. 11. Saito TB, Walker DH. Ehrlichioses: An Important One Health Opportunity. Vet Sci. 2016;3:20. doi: 10.3390/vetsci3030020. Page 5/7 12. Angelakis E, Mediannikov O, Jos SL, Berenger JM, Parola P, Raoult D. Candidatus Coxiella massiliensis Infection. Emerg Infect Dis. 2016;22:285-8. doi: 10.3201/eid2202.150106. 13. Buysse M, Duron O. Evidence that microbes identi ed as tick-borne pathogens are nutritional endosymbionts. Cell. 2021;184:2259-60. doi: 10.1016/j.cell.2021.03.053. 14. Palomar AM, Premchand-Branker S, Alberdi P, Belova OA, Moniuszko-Malinowska A, Kahl O, et al. Isolation of known and potentially pathogenic tickborne microorganisms from European ixodid ticks using tick cell lines. Ticks Tick Borne Dis. 2019;10:628-38. doi: 10.1016/j.ttbdis.2019.02.008. 15. Sili G, Byaruhanga C, Horak I, Steyn H, Chaisi M, Oosthuizen MC, et al. Ticks and tick-borne pathogens infecting livestock and dogs in TchicalaTcholoanga, Huambo Province, Angola. Parasitol Res. 2021;120:1097-102. doi: 10.1007/s00436-020-07009-3. Figures Figure 1 Phylogenetic analysis of the microorganisms detected in this study from ticks collected from cattle in Angola (marked with diamond). The maximum likelihood trees were obtained using the General Time Reversible model, a discrete Gamma-distribution and a proportion of invariable sites (GTR+G+I), nucleotide substitution selected according to the Akaike information criterion implemented in Mega X. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Numbers (>60%) shown at the nodes correspond to bootstrapped percentages (for 500 repetitions). The GenBank accession numbers of the sequences used in these analyses are shown in brackets. A. Ehrlichia phylogeny was based on 23 partial 16S rRNA gene sequences with a total of 1,373 positions in the nal dataset. Candidatus Neoehrlichia mikuresis was used as an outgroup. B. Ehrlichia phylogeny was based on 22 partial groESL gene sequences with a total of 1,232 positions in the nal dataset. Candidatus Neoehrlichia mikuresis was used as an outgroup. C. Coxiella-like phylogeny was based on 51 partial rpoB and groEL concatenated sequences with a total of 1,055 positions in the nal dataset. Rickettsiella sp. was used as an outgroup. D. Phylogeny of Spiroplasma spp. found in ticks based on 18 partial rpoB sequences with a total of 588 positions in the nal dataset. E. Phylogeny of Babesia species based on 18S rRNA analysis. The analysis involved 40 nucleotide sequences and a total of 481 positions in the nal dataset. Plasmodium falciparium was used as outgroup. Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. Page 6/7 Supplementarytable.docx graphicalabstract.jpg Page 7/7