Old Agents and Novel Variants of Tick-borne Microorganisms From Angola, 2017

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 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 Target gene
Amblyomma variegatum   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).

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 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.